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https://openalex.org/W4200400562	https://www.frontiersin.org/articles/10.3389/fpls.2021.767478/pdf	English	null	Data_Sheet_2.XLSX	null	2,021	cc-by	13,080	Rose JP, Kriebel R, Kahan L, DiNicola A, González-Gallegos JG, Celep F, Lemmon EM, Lemmon AR, Sytsma KJ and Drew BT (2021) Sage Insights Into the Phylogeny of Salvia: Dealing With Sources of Discordance Within and Across Genomes. Front. Plant Sci. 12:767478. doi: 10.3389/fpls.2021.767478 Sage Insights Into the Phylogeny of Salvia: Dealing With Sources of Discordance Within and Across Genomes Jeffrey P. Rose1,2, Ricardo Kriebel2, Larissa Kahan2, Alexa DiNicola2, Jesús G. González-Gallegos3, Ferhat Celep4, Emily M. Lemmon5, Alan R. Lemmon6, Kenneth J. Sytsma2 and Bryan T. Drew1 Jeffrey P. Rose1,2, Ricardo Kriebel2, Larissa Kahan2, Alexa DiNicola2, Jesús G. González-Gallegos3, Ferhat Celep4, Emily M. Lemmon5, Alan R. Lemmon6, Kenneth J. Sytsma2 and Bryan T. Drew1 1 Department of Biology, University of Nebraska at Kearney, Kearney, NE, United States, 2 Department of Botany, University of Wisconsin–Madison, Madison, WI, United States, 3 CONACYT, Instituto Politeìcnico Nacional, CIIDIR – Durango, Durango, Mexico, 4 Department of Biology, Faculty of Arts and Sciences, Kırıkkale University, Yah¸sihan, Turkey, 5 Department of Biological Science, Florida State University, Tallahassee, FL, United States, 6 Department of Scientiﬁc Computing, Florida State University, Tallahassee, FL, United States ORIGINAL RESEARCH published: 24 November 2021 doi: 10.3389/fpls.2021.767478 ORIGINAL RESEARCH published: 24 November 2021 doi: 10.3389/fpls.2021.767478 ORIGINAL RESEARCH published: 24 November 2021 doi: 10.3389/fpls.2021.767478 Edited by: Stefan Wanke, Dresden University of Technology, Germany Next-generation sequencing technologies have facilitated new phylogenomic approaches to help clarify previously intractable relationships while simultaneously highlighting the pervasive nature of incongruence within and among genomes that can complicate deﬁnitive taxonomic conclusions. Salvia L., with ∼1,000 species, makes up nearly 15% of the species diversity in the mint family and has attracted great interest from biologists across subdisciplines. Despite the great progress that has been achieved in discerning the placement of Salvia within Lamiaceae and in clarifying its infrageneric relationships through plastid, nuclear ribosomal, and nuclear single-copy genes, the incomplete resolution has left open major questions regarding the phylogenetic relationships among and within the subgenera, as well as to what extent the infrageneric relationships differ across genomes. We expanded a previously published anchored hybrid enrichment dataset of 35 exemplars of Salvia to 179 terminals. We also reconstructed nearly complete plastomes for these samples from off-target reads. We used these data to examine the concordance and discordance among the nuclear loci and between the nuclear and plastid genomes in detail, elucidating both broad-scale and species-level relationships within Salvia. We found that despite the widespread gene tree discordance, nuclear phylogenies reconstructed using concatenated, coalescent, and network-based approaches recover a common backbone topology. Moreover, all subgenera, except for Audibertia, are strongly supported as monophyletic in all analyses. The plastome genealogy is largely resolved and is congruent with the nuclear backbone. However, multiple analyses suggest that incomplete lineage sorting does not fully explain the gene tree discordance. Instead, horizontal gene ﬂow has been important in both the deep and more recent history of Reviewed by: Itzi Fragoso-Martínez, Instituto de Ecología (INECOL), Mexico Reviewed by: Itzi Fragoso-Martínez, Instituto de Ecología (INECOL), Mexico Roser Vilatersana, Consejo Superior de Investigaciones Cientíﬁcas, Spanish National Research Council (CSIC), Spain *Correspondence: Jeffrey P. Rose rosej@unk.edu Specialty section: This article was submitted to Plant Systematics and Evolution, a section of the journal Frontiers in Plant Science Received: 30 August 2021 Accepted: 22 October 2021 Published: 24 November 2021 INTRODUCTION Robust phylogenetic hypotheses are also crucial for making informed decisions to ensure an accurate and stable taxonomic circumscription, from the species level to higher-level classiﬁcations. Contemporaneous with these computational advances, new sequencing technologies have facilitated relatively easy and cost-eﬀective sequencing of complete organellar genomes and hundreds to thousands of nuclear loci. The conﬂuence of these two areas of biology has made for an exciting time for studies in systematic biology but has also presented challenges as to how to best analyze these datasets. For example, individual loci may have relatively little phylogenetic information and thus, confound analyses that rely on individual gene trees. In addition, the computational ability of many current algorithms are challenged by the number of terminals present in the species tree and especially the phylogenetic network that a researcher wishes to estimate (Hejase and Liu, 2016; Solís-Lemus and Ané, 2016; Rose et al., 2021). Despite these challenges, an ever- increasing proportion of phylogenomic studies employ methods that account for sources of intra-genomic discordance, especially due to ILS. While methods that employ the multispecies coalescent only are relatively fast and tractable on datasets with dozens to hundreds of terminals, it is increasingly clear that hybridization and introgression are important processes at both shallow and deep phylogenetic scales, and this aﬀects all branches of the Tree of Life (Folk et al., 2018). If horizontal gene ﬂow has been operative, the species tree estimated by methods that only account for ILS may diﬀer substantially from the “true” species Sage and its relatives (Salvia L.) comprise ∼1,000 species, with a subcosmopolitan distribution across a diversity of habitat types (Kriebel et al., 2019). It is the largest genus within the mint family (Lamiaceae) and one of the largest genera of plants. There are three broadly deﬁned centers of diversity of Salvia (Walker et al., 2004): East Asia (∼100 spp.; subgenus (subg.) Glutinaria; Hu et al., 2018), the Mediterranean (∼250 spp.; subg. Salvia, Sclarea), and especially, Mexico, Central, and South America (∼580 spp., subg. Calosphace; González-Gallegos et al., 2020). Salvia is not only of interest from an economic perspective, given its culinary use (e.g., chia: S. hispanica L.; rosemary: S. rosmarinus (L.) Spenn.; sage: S. oﬃcinalis L.), but also in its horticultural importance (e.g., blue sage: S. nemorosa L.; pineapple sage: S. elegans Vahl; Russian sage: S. yangii B.T.Drew). Citation: Rose JP, Kriebel R, Kahan L, DiNicola A, González-Gallegos JG, Celep F, Lemmon EM, Lemmon AR, Sytsma KJ and Drew BT (2021) Sage Insights Into the Phylogeny of Salvia: Dealing With Sources of Discordance Within and Across Genomes. Front. Plant Sci. 12:767478. doi: 10.3389/fpls.2021.767478 November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 1 Rose et al. Phylogeny of Salvia Salvia. Our results provide a robust species tree of Salvia across phylogenetic scales and genomes. Future comparative analyses in the genus will need to account for the impacts of hybridization/introgression and incomplete lineage sorting in topology and divergence time estimation. Salvia. Our results provide a robust species tree of Salvia across phylogenetic scales and genomes. Future comparative analyses in the genus will need to account for the impacts of hybridization/introgression and incomplete lineage sorting in topology and divergence time estimation. Keywords: anchored hybrid enrichment, cyto-nuclear discordance, distance metrics, incongruence, Lamiaceae, Robinson–Foulds distance, Salvia Keywords: anchored hybrid enrichment, cyto-nuclear discordance, distance metrics, incongruence, Lamiaceae, Robinson–Foulds distance, Salvia Frontiers in Plant Science \| www.frontiersin.org INTRODUCTION tree not only topologically, but also in branch lengths (Leaché et al., 2014). The misestimation of both properties may impact myriad downstream analyses. It has long been recognized that when generating multilocus nucleotide sequence data, diﬀerent datasets may generate alternative gene tree topologies and, by extension, diﬀering hypotheses of relationships among species (Pamilo and Nei, 1988; Rieseberg and Soltis, 1991; Maddison, 1997). The underlying causes for why such diﬀering gene tree topologies may exist (apart from analytical artifacts) have been well- discussed in the literature, and include gene duplication, incomplete lineage sorting (ILS), lateral gene transfer, and introgression/hybridization (Degnan, 2018). These processes are not mutually exclusive, and the history of one locus may be shaped by multiple processes. For many years, the solution to deal with such discordance was to analyze incongruent datasets separately, attempt to reconcile these topologies into a consensus tree, or concatenate all loci together to generate a “total-evidence” hypothesis of the species relationships (Ané et al., 2007). The concept of genomic concordance, coupled with new methods for estimating species trees while taking into account ILS and/or horizontal gene ﬂow have been important advances in the ﬁeld of systematic biology (Ané et al., 2007; Baum, 2007; Heled and Drummond, 2009; Mirarab et al., 2014; Yu and Nakhleh, 2015; Edwards et al., 2016; Solís-Lemus and Ané, 2016). Apart from discordance among nuclear loci, gene trees may diﬀer among genomes. This phenomenon is well-known and often referred to as “cytonuclear discordance” (Rieseberg and Soltis, 1991). In plants, this is best demonstrated in cases of putative “chloroplast capture” which have been documented for decades (e.g., Smith and Sytsma, 1990). Such discordance has generally been taken as evidence of horizontal gene ﬂow, even though organellar genomes are also susceptible to ILS, albeit with a much faster expected time to coalescence, relative to nuclear loci. Simulation studies have generally conﬁrmed that most cases of chloroplast (technically plastid) capture are indeed best explained by horizontal gene ﬂow, rather than ILS (Folk et al., 2017; Morales-Briones et al., 2018; Rose et al., 2021). Therefore, a better understanding of the evolutionary history of clades requires an assessment of the contribution of each of the multiple processes responsible for the discordance among loci. This assessment is important not only for producing a robust phylogenetic hypothesis, but also for selecting methods, taxa, and loci appropriate for the downstream analyses of trait evolution, historical biogeography, and diversiﬁcation rates. INTRODUCTION Salvia is ﬂorally diverse (Kriebel et al., 2019, 2020) and easily characterized by the presence of two stamens with an elongate (or swollen) anther connective, in addition to several micromorphological synapomorphies (Drew et al., 2017). In many species of Salvia, the connective has been variously modiﬁed – possibly multiple times – into a staminal lever mechanism to facilitate eﬀective pollination Frontiers in Plant Science \| www.frontiersin.org November 2021 \| Volume 12 \| Article 767478 2 Phylogeny of Salvia Rose et al. (Claßen-Bockhoﬀet al., 2003, 2004; Walker and Sytsma, 2007; Wester and Claßen-Bockhoﬀ, 2007; Celep et al., 2020). (Claßen-Bockhoﬀet al., 2003, 2004; Walker and Sytsma, 2007; Wester and Claßen-Bockhoﬀ, 2007; Celep et al., 2020). the monophyly of subg. Audibertia is not fully supported. Third, within subg. Calosphace, section (sect.) Axillares was recovered as a sister to the “Hastatae clade”. instead of sister to all other Calosphace, in conﬂict with several previous studies (Jenks et al., 2013; Drew et al., 2017; Fragoso-Martínez et al., 2018). The placement of sect. Axillares has important implications for understanding character evolution in subg. Calosphace (e.g., Fragoso-Martínez et al., 2018; Kriebel et al., 2019, 2020, 2021). As a result of its practical importance to humans, distribution, size and taxonomic complexity, and unique pollination biology, Salvia has received considerable attention from systematists and pollination biologists. Early in the study of the phylogenetic placement of Salvia, it was realized that the genus was polyphyletic or broadly paraphyletic, with several smaller genera embedded within it (Walker and Sytsma, 2007). Subsequent phylogenetic analyses have conﬁrmed that ﬁve previously recognized small genera (Dorystaechas Boiss. & Heldr., Meriandra Benth., Perovskia Kar., Rosmarinus L., and Zhumeria Rech.f. & Wendelbo) are nested within several clades of Salvia (Walker and Sytsma, 2007; Drew and Sytsma, 2012; Will and Claßen-Bockhoﬀ, 2014, 2017; Drew et al., 2017). To accommodate these small genera, Drew et al. (2017) and Kriebel et al. (2019) presented an expanded concept of Salvia, recognizing a total of 11 subgenera, although their informal circumscription of subg. “Heterosphace” represents a geographically diverse assemblage of lineages. Given the relatively sparse sampling of Salvia diversity from previous phylogenomic analyses, as well as the limited exploration of any discordance surrounding the backbone relationships in Salvia, we aimed to sample the species diversity in Salvia better using AHE to fulﬁll several goals. Species Sampling in Salvia and Outgroups In total, the analyses consisted of 190 samples, including 179 Salvia and all subgenera recognized by Kriebel et al. (2019) except for the small subg. Meriandra (Benth.) J.B.Walker, B.T.Drew & J.G.González. For ease of discussion, we will refer to the “Heterosphace” clade as a subgenus, although we acknowledge that this is not a formally named taxon. Within the subtribe Salviinae, we sampled the clade sister to Salvia (six species of Lepechinia and one species of Melissa: Drew and Sytsma, 2011, 2012, 2013). The ultimate outgroup consisted of samples from all the remaining subtribes of Mentheae, which represents a monophyletic group (Drew and Sytsma, 2012): Lycopinae (Lycopus uniﬂorus Michx.), Menthinae (Clinopodium mexicanum (Benth.) Govaerts), Nepetinae (Agastache pallida (Lindl.) Cory), and Prunellinae (Prunella vulgaris L.). INTRODUCTION (1) Fully resolve the backbone of Salvia and assess the monophyly of the subgenera, quantifying discordance and accounting for both ILS and horizontal gene ﬂow. (2) Generate a species tree for a much broader species-level sampling of Salvia, testing the eﬃcacy of the AHE data for resolving shallow-level relationships. (3) Examine cytonuclear discordance at multiple phylogenetic scales by mining oﬀ-target organellar reads. To date, most phylogenetic studies of Salvia have relied on plastid or nuclear ribosomal external transcribed spacer (ETS) and especially internal transcribed spacer (ITS) sequences (Walker and Sytsma, 2007; Jenks et al., 2013; Will and Claßen- Bockhoﬀ, 2014, 2017; Dizkirici et al., 2015; Walker et al., 2015; Fragoso-Martínez et al., 2018; Hu et al., 2018). The resolution at multiple phylogenetic scales with these markers is variable by clade and, while there has been evidence for several deeper-level clades along the backbone of Salvia, relationships among them have usually either not been resolved or well supported. Discordance among plastid and nuclear ribosomal loci is generally found but not well-discussed or quantiﬁed (but see Walker et al., 2015). In cases where relationships diﬀer across studies and marker sets, it is not clear if the diﬀerences are due to a true discordance in genealogical history or are from errors in the phylogenetic estimation (cf. Will and Claßen- Bockhoﬀ, 2017: Figure 1). Drew et al. (2017) further investigated the backbone relationships in Salvia using two low-copy nuclear loci. While several key nodes remained unresolved and there was a clear conﬂict between the loci, they found an increased resolution for the backbone relationships. More recently, Zhao et al. (2020) used complete plastomes from seven of 11 subgenera and recovered a nearly fully resolved backbone across Salvia except for uncertainty in the placements of subg. Perovskia and Rosmarinus. Frontiers in Plant Science \| www.frontiersin.org Anchored Hybrid Enrichment: Library Preparation, Enrichment, Sequencing, and Nuclear Locus Assembly Total DNA was extracted from the silica gel-dried or fresh leaf tissue using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, United States). The DNA concentrations were veriﬁed using a Qubit R⃝2.0 Fluorometer (Life Technologies, Eugene, OR, United States). We used the AHE method (Lemmon and Lemmon, 2012). As the samples were sequenced across several years of study, we enriched them using slightly diﬀerent probe sets (Supplementary Data Sheet S1). The samples sequenced early on in our studies utilized a generic angiosperm kit that targets 517 loci (Buddenhagen et al., 2016; Mitchell et al., 2017) or the Salvia-speciﬁc probes utilized in Kriebel et al. (2019), designed using the genome skimming of several Salvia Multilocus nuclear datasets from anchored hybrid enrichment (AHE) have been successfully used to resolve deep and shallow level relationships across multiple angiosperm lineages, including Salvia (Fragoso-Martínez et al., 2017; Kriebel et al., 2019). Previously, we presented a species tree of 35 Salvia exemplars from 10 of 11 subgenera based on 316 nuclear loci using concatenation and one coalescent method (Kriebel et al., 2019). While this topology is congruent with the plastome phylogeny of Zhao et al. (2020) in areas where the two studies overlap in subgeneric sampling, several factors bear further consideration in Kriebel et al. (2019). First, the branching order of subg. “Heterosphace”, Salvia, and Sclarea is not fully supported. Second, November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 3 Phylogeny of Salvia Rose et al. FIGURE 1 \| The ASTRAL species tree of Salvia and outgroups. The ingroup branches are colored by subgenus and the subgenera are also labeled to the right. The major clades as discussed in the text are also indicated for subg. Calosphace. Thickened branches denote those with >0.95 ASTRAL local posterior probability. Pies at major nodes summarize the percentage of various phylogenetic signals across 101 gene trees which can be rooted. The numbers at the left of the pies show the total number of gene trees in which the clade is found, followed by the total number of gene trees that conﬂict with that clade. The remainder of the gene trees, if any, do not provide information on that particular relationship. Anchored Hybrid Enrichment: Library Preparation, Enrichment, Sequencing, and Nuclear Locus Assembly The numbers at selected, incompletely supported nodes show the ASTRAL local posterior probability followed by the ASTRAL bootstrap support and the bootstrap support from the concatenated maximum likelihood analysis, summarized on the ASTRAL species tree. For support across all branches, see Supplementary Figures S1, S2, S4. For support on the best-scoring maximum likelihood tree, see Supplementary Figure S3. FIGURE 1 \| The ASTRAL species tree of Salvia and outgroups. The ingroup branches are colored by subgenus and the subgenera are also labeled to the right. The major clades as discussed in the text are also indicated for subg. Calosphace. Thickened branches denote those with >0.95 ASTRAL local posterior probability. Pies at major nodes summarize the percentage of various phylogenetic signals across 101 gene trees which can be rooted. The numbers at the left of the pies show the total number of gene trees in which the clade is found, followed by the total number of gene trees that conﬂict with that clade. The remainder of the gene trees, if any, do not provide information on that particular relationship. The numbers at selected, incompletely supported nodes show the ASTRAL local posterior probability followed by the ASTRAL bootstrap support and the bootstrap support from the concatenated maximum likelihood analysis, summarized on the ASTRAL species tree. For support across all branches, see Supplementary Figures S1, S2, S4. For support on the best-scoring maximum likelihood tree, see Supplementary Figure S3. additional loci were lost during trimming and masking due to excessive missing data, resulting in a ﬁnal dataset of 114 loci. species. The Salvia-speciﬁc probes targeted the same regions as the generic angiosperm kit, only neighboring exons could be combined and target regions extended, resulting in a target of 291 moderately conserved, low copy nuclear loci and their variable ﬂanks. The library preparation, enrichment, assembly, and alignment of nuclear loci were performed at the Florida State University Center for Anchored Phylogenomics1 and are described in detail in Kriebel et al. (2019). Because of the large number of samples, a substantial number of loci were lost during the orthology assessment, resulting in 123 recovered loci. Nine 1www.anchoredphylogeny.com Nuclear Dataset 1: Gene Tree Distances Nuclear Dataset 1: Gene Tree Distances We further examined the patterns of gene tree discordance to test if the observed gene tree discordance across Salvia and its constituent subgenera are consistent with the expectation under ILS alone. To do this, we ﬁrst generated 1,000 gene trees under the multispecies coalescent using the treesim.contained_coalescent function in DendroPy v.4.5.2 (Sukumaran and Holder, 2010) using the ASTRAL species tree as the “true” tree. To compare the observed discordance with what would be expected under ILS, we measured the pairwise tree distance of each gene tree (expected and observed) from the ASTRAL species tree using three metrics, considering branching order alone and ignoring branch lengths: the Robinson–Foulds distance (RF) (Robinson and Foulds, 1981), the method proposed by Nye et al. (2006), and the clustering information (CI) metric proposed by Smith (2020a). Calculations were made on complete gene trees or gene trees pruned to the subgenus of interest, as appropriate. Since some of the observed gene trees were missing terminals, we only used observed gene trees which contained >75% of all terminals in the clade of interest. Gene tree distances were calculated using the “TreeDist” package in R (Smith, 2020b), collapsing all the branches in the observed gene trees with <33% BS. The distances were normalized so that they ranged from 0 to 1, with 0 indicating complete agreement between the gene tree and species tree. We tested for mean diﬀerences observed in the gene tree discordance among clades using a Using the BUCKy CFs and the Quartet MaxCut tree, we calculated a phylogenetic network with the SNaQ function in the Julia package PhyloNetworks (Solís-Lemus and Ané, 2016; Solís-Lemus et al., 2017). This package uses maximum pseudo-likelihood to ﬁt a network while also accounting for ILS. PhyloNetworks considers quartet topologies only and does not take into account information from branch lengths in individual gene trees. Furthermore, PhyloNetworks assumes a level-1 network: a network where each hybrid node only has one lineage transferring genetic material horizontally. We ﬁrst tested the ﬁt of models allowing from 0–5 reticulation events (h) and compared the models using their pseudo-likelihood score. The best network model was selected by examining at which value of h the pseudo-likelihood score plateaus, following the recommendation of Solís-Lemus et al. (2017). For each value of h, we selected the best network over 30 search replicates. Nuclear Dataset 1: Complete Dataset To examine the monophyly of the subgenera (when represented by multiple samples) and assess shallow-level relationships, we assembled species trees using all accessions. First, we concatenated all loci and generated a maximum likelihood species tree in RAxML v.8.2.11 (Stamatakis, 2014) under GTR + 0. We assessed the branch support with 500 rapid bootstrap (BS) replicates. November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 4 Phylogeny of Salvia Rose et al. one-way ANOVA with post hoc testing using the Tukey Test with the aov and glht functions in the “stats” and “multcomp” (Hothorn et al., 2008) R packages, respectively. We tested for diﬀerences in the mean gene tree discordance between observed and expected gene trees using a two-tailed Welch’s t-test. Second, we generated a species tree under the multispecies coalescent using ASTRAL-III (Zhang et al., 2018). Using a batch Perl script, we generated individual gene trees using RAxML under GTR + 0, assessing the branch support for each locus with 100 rapid BS replicates. We analyzed all the maximum likelihood gene trees in ASTRAL, measuring the branch support in two ways: by using the RAxML BS trees as input to ASTRAL with 100 replicates, and also by calculating the ASTRAL local posterior probability (LPP) (Sayyari and Mirarab, 2016) for each quadripartition. Nuclear Dataset 2: Placeholder Dataset Our second nuclear dataset investigated deeper phylogenetic relationships in Salvia, accounting for both ILS and horizontal gene ﬂow. Because the existing methods for inferring phylogenetic networks are computationally demanding for datasets with more than several dozen terminals, we constructed a dataset of one representative for each subgenus. For each subgenus placeholder, we selected the sample with the greatest number of captured loci, and in the case of ties, the total number of aligned bp. We did not allow any missing data, yielding a matrix of 57 loci for 10 species of Salvia plus Lepechinia chamaedryoides (Balb.) Epling as the outgroup. To reconstruct the phylogenetic networks, we ﬁrst generated concordance factors for each possible quartet. Using a batch script, we ran MrBayes v.3.2.6 (Ronquist et al., 2012) to ﬁnd the best gene tree for each locus. The gene trees were inferred under GTR + I + 0 using three runs of three chains each for ﬁve million generations each with sampling every 5,000 generations with a chain temperature of 0.4, swap frequency of 500 generations, and a 30% burnin. Frontiers in Plant Science \| www.frontiersin.org Nuclear Dataset 1: Complete Dataset Following the MrBayes analysis, a Bayesian concordance analysis on the posterior sample of gene trees was conducted in BUCKy v.1.4.4 (Ané et al., 2007; Larget et al., 2010) with 100,000 post-burnin generations and the amount of a piori discordance among loci set to the default of 1. This analysis calculates all possible quartets and prunes on the MrBayes gene trees to all but the four terminals of interest. Then, BUCKy is run on each pruned gene tree to generate a table of all quartet concordance factors (CFs) and their SEs. Using these CFs, we generated a preliminary population tree using Quartet MaxCut (Snir and Rao, 2012). q p To detail the gene tree conﬂict/support for each clade in the species tree, we used Phyparts (Smith et al., 2015). Phyparts takes an estimate of a species tree and set of rooted gene trees and provides four numbers for each clade: the number of loci supporting a clade, the number of loci supporting the main conﬂicting clade, the number of loci supporting all other conﬂicting clades, and the number of loci without information for a relationship. Trees were optimally rooted with our outgroups outside of Salviinae, but in cases where these were missing, we rooted trees with Melissa and/or Lepechinia. Gene trees that contained only Salvia were excluded from the Phyparts analysis. Note that rooting with Salviinae may inﬂate the gene tree support for the monophyly of Salvia and possibly also show misleading support or conﬂict for the relationships among Lepechinia, Melissa, and Salvia. However, we allowed this potentially incorrect rooting because our chief interest was in the relationships within Salvia. To mitigate the eﬀects of uncertainty in the gene tree estimation providing artiﬁcial conﬂict (or support) for clades, we collapsed the branches in each gene tree with <33% BS. Plastome Assembly and Phylogenetic Analysis underlying topology despite some discordance. The monophyly of Salvia is strongly supported by all measures of support (ASTRAL LPP = 1.0/ASTRAL BS = 100/concatenated BS = 100). In addition, the monophyly of each subgenus for which we had multiple samples is strongly supported by BS/LPP and by the vast majority of loci, with two exceptions. First, subg. Heterosphace, while unambiguously supported by measures of statistical support (ASTRAL LPP = 1.0/ASTRAL BS = 100/concatenated BS = 100), has 23/84 (27%) informative loci conﬂicting its monophyly. Second and more strikingly, the monophyly of subg. Audibertia is poorly supported by ASTRAL LPP (0.69) with more loci conﬂicting its monophyly than supporting it (55/73, 75%). However, its monophyly is more strongly supported by the other metrics (ASTRAL BS = 91/concatenated BS > 99), although both sections of subg. Audibertia: sects. Audibertia (S. columbariae Benth., S. mellifera Greene, S. munzii Epling) and Echinosphace (S. californica Brandegee, S. funerea M.E. Jones) are more strongly supported as monophyletic. We assembled the nearly complete plastomes of the Salviinae samples by mapping the oﬀ-target reads to previously published plastomes of Salvia for the ingroup or Melissa for the outgroup Salviinae. The assembly of the plastomes was conducted in Geneious v.10.2.3 (Kearse et al., 2012), following the procedure of Rose et al. (2021). For outgroup Salviinae, we used the whole plastome sequence of Melissa yunnanensis C.Y.Wu & Y.C.Huang (GenBank accession MT634148.1) as a reference. For Salvia, we constructed a “super” reference sequence based on the strict consensus of 18 GenBank plastomes (Supplementary Data Sheet S2) aligned with MAFFT v.7.023b (Katoh and Standley, 2013) under default parameters. We used Geneious to map all the forward and reverse reads from our sequences by ﬁrst trimming all raw reads, and then assembling them to the appropriate reference using an iterative reﬁnement of up to ﬁve times with the default Geneious mapper and medium sensitivity. Consensus sequences were generated using the strict consensus approach. If the coverage for a particular site was <7, the consensus nucleotide was scored as a gap. Unmapped regions were treated as missing data and reads mapped to multiple positions were excluded from consensus calculations. Newly generated plastomes were aligned with the aforementioned GenBank sequences using MAFFT with default parameters. Ambiguously aligned regions were removed manually and were generally distinguished by putative inversions, repeat regions, an abundance of gaps, and/or uncertain base calls. Subgeneric Monophyly and Major Relationships in Salvia We were able to root 101 of the 114 gene trees. Species trees resulting from concatenation and accounting for ILS with ASTRAL are completely congruent in the major backbone relationships in Salvia, although support for these relationships sometimes varies across the approach and support metrics (Figure 1 and Supplementary Figures S1–S4). The ASTRAL normalized quartet score, or proportion of the gene tree quartet trees satisﬁed by the species tree, is 0.91, suggesting a clear Dataset Metrics The aligned locus length for the 114 loci ranged from 105– 3,671 bp, with a mean length of 1,133 bp. The samples contained sequence data for an average of 96.25 loci, with most locus dropout in the non-Salviinae outgroups. We were able to extract the majority of the plastome, with aligned plastomes totaling 157,683 bp. The backbone of the plastome tree is nearly identical to that of the nuclear species trees (Supplementary Figure S5), with all major nodes and monophyly of the subgenera receiving maximal support except for the placement of subg. Glutinaria (BS = 89). The only major topological diﬀerence is that subg. Perovskia is weakly supported as sister to Rosmarinus (BS = 47). Nuclear Dataset 1: Gene Tree Distances We examined the branch support on the best phylogenetic network using the bootsnaq function with 50 runs of 10 replicates each. November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 5 Phylogeny of Salvia Rose et al. Plastome Assembly and Phylogenetic Analysis The earliest divergence in Salvia involves two major clades. First is a clade formed by the most recent common ancestor (MRCA) of subg. Glutinaria and Calosphace (ASTRAL LPP = 1.0/ASTRAL BS = 100/concatenated BS = 100). Subgenus Glutinaria is sister to all remaining subgenera, with a grade formed by the successive sisters of subg. Zhumeria and Dorystaechas, and subg. Audibertia, sister to subg. Calosphace. All of these relationships are strongly supported with the exception of the placement of subg. Glutinaria (ASTRAL LPP = 0.85/ASTRAL BS = 89/concatenated BS > 99; 22/71 informative gene trees). g ) The second major clade is formed by the MRCA of subg. Perovskia and Salvia. The monophyly of this clade is not fully supported (ASTRAL LPP = 0.73/ASTRAL BS = 92/concatenated BS = 100; 25/82 informative gene trees), nor are many of the intersubgeneric relationships within it (Figure 1). Subgenus Perovskia is sister to subg. Rosmarinus + Heterosphace + Salvia + Sclarea (ASTRAL LPP = 0.91/ASTRAL BS = 95/concatenated BS = 100; 22/69 informative gene trees). While the monophyly of subg. Heterosphace + Salvia + Sclarea is fully supported, relationships among the subgenera are slightly less certain, with subg. Salvia sister to Sclarea being the best resolution of relationships (ASTRAL LPP = 0.94/ASTRAL BS = 99/concatenated BS = 100; 26/81 informative gene trees). A plastome tree was inferred in RAxML under GTR + 0 with 500 rapid BS replicates. As described above in Section “Nuclear Dataset 1: Gene Tree Distances”, we measured the tree-to-tree distances between the entire plastome tree and its subclades to the ASTRAL species tree. Infrageneric Relationships, Shallow-Scale Resolution, and Gene Tree Discordance Within subg. Calosphace, our nuclear data suggest that sect. Axillares is sister to the Hastatae clade, but with strongly conﬂicting support (ASTRAL LPP = 0.38/ASTRAL BS = 86/concatenated BS = 92; 20/60 informative gene trees), November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 6 Phylogeny of Salvia Rose et al. while our plastid data place sect. Axillares as sister to all other Calosphace (BS = 100). There is also uncertainty about the deepest divergences in subg. Salvia, with weak support based on the ASTRAL and concatenated analyses. F5,454 = 92.4, p < 0.0001). Post hoc testing suggests that compared with the ASTRAL species tree, subg. Glutinaria and Heterosphace have signiﬁcantly less discordant nuclear gene trees on average than all other subgenera, while subg. Salvia is more discordant than Calosphace for the Nye and CI metrics, and subg. Sclarea is more discordant than Calosphace for CI alone (Table 1). Likewise, although they are only point estimates, the discordance between the plastome and the ASTRAL tree is elevated for subg. Sclarea and especially, Salvia relative to all other subgenera (Table 1). Overall, there is fairly strong support (>90% support across all metrics) for many shallow-scale relationships, but support is notably very low or non-existent for some ASTRAL clades which do not appear in the best tree in the concatenated analysis or are in the low frequency in the BS replicates (Supplementary Figure S6), especially within the radiation of core Calosphace (e.g., relationships among S. chamaedryoides Cav., S. coahuilensis Fern., S. microphylla Kunth, and S. muelleri Epling), subg. Salvia (e.g., if S. oﬃcinalis s.s. is monophyletic or not), and subg. Sclarea (e.g., the placement of S. sclarea L.). There is a much more obvious infrageneric gene tree conﬂict between the nuclear loci and the plastome, with many shallower relationships conﬂicting between the two datasets, especially in subg. Calosphace, Salvia, and Sclarea (Figure 2). Compared with the expectation under ILS alone, the nuclear gene tree discordance in the observed gene trees is generally on par with or is less than what would be expected for under ILS based on RF distance but is greater than what would be expected in subg. Calosphace, Salvia, and Sclarea based on both the Nye and CI metrics (Table 2). Phylogenetic Networks The t-values and associated p-values for each distance metric/clade combination are based on Welch’s t-test with the hypothesis that the mean tree distance for the observed and expected gene trees are equal, or in other words, that the tree distances based on empirical data are what would be expected under incomplete lineage sorting alone. Signiﬁcant t/p-values at α = 0.05 are indicated in bold. TABLE 1 \| Summary of the mean gene tree distances in Salvia and the selected subgenera within and across genomes. The three different distance metrics, Robinson–Foulds (RF), Nye, and clustering information (CI), compare the topology of the gene trees to the ASTRAL species tree, and we considered the branching order alone. Metrics were calculated on the gene trees that can be rooted and have clade occupancy > 75% of sampled tips. The distances were normalized so that they ranged from 0 to 1, with 0 indicating complete agreement between a gene tree and a species tree. The letters denote signiﬁcantly different among-group differences for each metric in mean nuclear gene tree discordance compared to the species tree based on an ANOVA with post hoc testing at α = 0.05. Differences among the plastomes were not tested because they represent one gene tree. TABLE 2 \| Nuclear gene tree distances in Salvia and selected subgenera. Clade RF Nye CI tRF pRF tNYE pNYE tCI pCI mean (SD) mean (SD) Mean (SD) Salvia-wide Observed 0.61 (0.07) 0.41 (0.06) 0.36 (0.06) –4.87 7.82 × 10−6 7.84 7.45 × 10−11 5.08 3.65 × 10−6 Expected 0.65 (0.03) 0.35 (0.02) 0.32 (0.02) subg. Calosphace Observed 0.69 (0.08) 0.52 (0.10) 0.57 (0.11) –2.92 4.39 × 10−3 10.67 < 1.00 × 10−15 8.83 1.12 × 10−13 Expected 0.72 (0.05) 0.40 (0.03) 0.46 (0.04) subg. Glutinaria Observed 0.29 (0.21) 0.20 (0.17) 0.24 (0.18) –1.04 0.30 0.49 0.63 –0.029 0.98 Expected 0.32 (0.17) 0.19 (0.09) 0.24 (0.11) subg. Heterosphace Observed 0.28 (0.17) 0.18 (0.11) 0.22 (0.13) –1.06 0.29 0.98 0.33 –0.11 0.91 Expected 0.30 (0.17) 0.17 (0.09) 0.22 (0.11) subg. Salvia Observed 0.67 (0.17) 0.44 (0.16) 0.50 (0.16) –0.91 0.36 4.70 8.84 × 10−6 2.56 0.012 Expected 0.69 (0.12) 0.36 (0.07) 0.46 (0.09) subg. Phylogenetic Networks The best phylogenetic network contained four reticulation events along the backbone of Salvia (hmax = 4). The major topology (i.e., bifurcating backbone) was identical to that recovered by the ASTRAL and concatenated analysis of all nuclear loci (Figure 3). Quartet CF along the backbone were generally > 0.50, and these edges received full BS support Gene tree distances demonstrate signiﬁcant gene tree conﬂicts within subgenera, both within and across genomic compartments (Table 1), and there are also signiﬁcant diﬀerences in the gene tree distances among the subgenera for all three metrics (RF: F5,454 = 145.7, p < 0.0001; Nye: F5,454 = 98.6, p < 0.0001; CI: FIGURE 2 \| Tanglegram illustrating the cytonuclear discordance in Salvia based on the nuclear ASTRAL species tree (A) and the plastome tree (B). Links connect identical tips, with nodes rotated to minimize the link overlap. Ingroup links are colored by subgenus. Clades that differ between the two trees are indicated by ﬁlled circles on the plastome tree. To minimize the discordance caused by the error in the gene tree estimation, clades in the plastome tree with <75% bootstrap support have been collapsed into polytomies. FIGURE 2 \| Tanglegram illustrating the cytonuclear discordance in Salvia based on the nuclear ASTRAL species tree (A) and the plastome tree (B). Links connect identical tips, with nodes rotated to minimize the link overlap. Ingroup links are colored by subgenus. Clades that differ between the two trees are indicated by ﬁlled circles on the plastome tree. To minimize the discordance caused by the error in the gene tree estimation, clades in the plastome tree with <75% bootstrap support have been collapsed into polytomies. November 2021 \| Volume 12 \| Article 767478 7 Frontiers in Plant Science \| www.frontiersin.org Phylogeny of Salvia Rose et al. with the exception of the placement of subg. Zhumeria (CF = 0.42, BS = 96) and the sister relationship of subg. Salvia and Sclarea (CF = 0.42, BS = 86). While the BS analysis found evidence for horizontal gene ﬂow, there was considerable uncertainty regarding the number and placement of reticulation events, with BS replicates recovering either three (52%) or four reticulation events. Inheritance probabilities (γ, the fraction of the nuclear genome involved in a reticulation event) for the four reticulation events on the best-ﬁtting network ranged from 7 to 36% (Figure 3). The best network recovered gene ﬂow from the stem of subg. Phylogenetic Networks Salvia to Heterosphace (γ = 0.36, BS = 66), stem subg. Rosmarinus to stem MRCA of Calosphace + Glutinaria (γ = 0.26, BS = 66), stem MRCA of subg. Calosphace + Glutinaria to Zhumeria (γ = 0.10, BS = 10), and stem subg. Dorystaechas to Audibertia (γ = 0.07, BS = 34). Alternative reticulation events found in frequency > 10% in the BS replicates involved the stem of subg. Salvia and Sclarea (BS = 22, with alternative relationships among Heterosphace, Salvia, and Sclarea), stem MRCA subg. Rosmarinus + Salvia and MRCA Calosphace + Glutinaria (BS = 28), stem subg. Glutinaria and stem Audibertia + Calosphace TABLE 1 \| Summary of the mean gene tree distances in Salvia and the selected subgenera within and across genomes. clade n-tips nuclear loci plastome RF Nye CI RF Nye CI Salvia-wide 179 0.61b 0.41b 0.36b 0.63 0.37 0.29 subg. Calosphace 93 0.69c 0.52d 0.57d 0.72 0.41 0.45 subg. Glutinaria 10 0.29a 0.20a 0.24a 0.38 0.23 0.25 subg. Heterosphace 10 0.28a 0.18a 0.22a 0.08 0.08 0.07 subg. Salvia 37 0.67bc 0.44bc 0.50c 0.93 0.64 0.74 subg. Sclarea 20 0.70c 0.48cd 0.49c 0.78 0.46 0.51 The three different distance metrics, Robinson–Foulds (RF), Nye, and clustering information (CI), compare the topology of the gene trees to the ASTRAL species tree, and we considered the branching order alone. Metrics were calculated on the gene trees that can be rooted and have clade occupancy > 75% of sampled tips. The distances were normalized so that they ranged from 0 to 1, with 0 indicating complete agreement between a gene tree and a species tree. The letters denote signiﬁcantly different among-group differences for each metric in mean nuclear gene tree discordance compared to the species tree based on an ANOVA with post hoc testing at α = 0.05. Differences among the plastomes were not tested because they represent one gene tree. TABLE 1 \| Summary of the mean gene tree distances in Salvia and the selected subgenera within and across genomes. clade n-tips nuclear loci plastome RF Nye CI RF Nye CI Salvia-wide 179 0.61b 0.41b 0.36b 0.63 0.37 0.29 subg. Calosphace 93 0.69c 0.52d 0.57d 0.72 0.41 0.45 subg. Glutinaria 10 0.29a 0.20a 0.24a 0.38 0.23 0.25 subg. Heterosphace 10 0.28a 0.18a 0.22a 0.08 0.08 0.07 subg. Salvia 37 0.67bc 0.44bc 0.50c 0.93 0.64 0.74 subg. Phylogenetic Networks Sclarea 20 0.70c 0.48cd 0.49c 0.78 0.46 0.51 The three different distance metrics, Robinson–Foulds (RF), Nye, and clustering information (CI), compare the topology of the gene trees to the ASTRAL species tree, and we considered the branching order alone. Metrics were calculated on the gene trees that can be rooted and have clade occupancy > 75% of sampled tips. The distances were normalized so that they ranged from 0 to 1, with 0 indicating complete agreement between a gene tree and a species tree. The letters denote signiﬁcantly different among-group differences for each metric in mean nuclear gene tree discordance compared to the species tree based on an ANOVA with post hoc testing at α = 0.05. Differences among the plastomes were not tested because they represent one gene tree. TABLE 2 \| Nuclear gene tree distances in Salvia and selected subgenera. Clade RF Nye CI tRF pRF tNYE pNYE tCI pCI mean (SD) mean (SD) Mean (SD) Salvia-wide Observed 0.61 (0.07) 0.41 (0.06) 0.36 (0.06) –4.87 7.82 × 10−6 7.84 7.45 × 10−11 5.08 3.65 × 10−6 Expected 0.65 (0.03) 0.35 (0.02) 0.32 (0.02) subg. Calosphace Observed 0.69 (0.08) 0.52 (0.10) 0.57 (0.11) –2.92 4.39 × 10−3 10.67 < 1.00 × 10−15 8.83 1.12 × 10−13 Expected 0.72 (0.05) 0.40 (0.03) 0.46 (0.04) subg. Glutinaria Observed 0.29 (0.21) 0.20 (0.17) 0.24 (0.18) –1.04 0.30 0.49 0.63 –0.029 0.98 Expected 0.32 (0.17) 0.19 (0.09) 0.24 (0.11) subg. Heterosphace Observed 0.28 (0.17) 0.18 (0.11) 0.22 (0.13) –1.06 0.29 0.98 0.33 –0.11 0.91 Expected 0.30 (0.17) 0.17 (0.09) 0.22 (0.11) subg. Salvia Observed 0.67 (0.17) 0.44 (0.16) 0.50 (0.16) –0.91 0.36 4.70 8.84 × 10−6 2.56 0.012 Expected 0.69 (0.12) 0.36 (0.07) 0.46 (0.09) subg. Sclarea Observed 0.70 (0.10) 0.48 (0.10) 0.49 (0.11) –3.10 2.62 × 10−3 8.35 1.14 × 10−12 4.37 3.51 × 10−5 Expected 0.74 (0.07) 0.38 (0.04) 0.43 (0.05) The three different distance metrics, RF, Nye, and CI, compare the topology of the gene trees to the ASTRAL species tree, and we considered the branching order alone. The gene trees are either empirical trees that can be rooted and have clade occupancy > 75% of sampled tips (observed) or 1,000 gene trees simulated under the multispecies coalescent using the ASTRAL species tree (expected). The distances were normalized so that they ranged from 0 to 1, with 0 indicating complete agreement between a gene tree and the species tree. Phylogenetic Networks Sclarea Observed 0.70 (0.10) 0.48 (0.10) 0.49 (0.11) –3.10 2.62 × 10−3 8.35 1.14 × 10−12 4.37 3.51 × 10−5 Expected 0.74 (0.07) 0.38 (0.04) 0.43 (0.05) The three different distance metrics, RF, Nye, and CI, compare the topology of the gene trees to the ASTRAL species tree, and we considered the branching order alone. The gene trees are either empirical trees that can be rooted and have clade occupancy > 75% of sampled tips (observed) or 1,000 gene trees simulated under the multispecies coalescent using the ASTRAL species tree (expected). The distances were normalized so that they ranged from 0 to 1, with 0 indicating complete agreement between a gene tree and the species tree. The t-values and associated p-values for each distance metric/clade combination are based on Welch’s t-test with the hypothesis that the mean tree distance for the observed and expected gene trees are equal, or in other words, that the tree distances based on empirical data are what would be expected under incomplete lineage sorting alone. Signiﬁcant t/p-values at α = 0.05 are indicated in bold. TABLE 2 \| Nuclear gene tree distances in Salvia and selected subgenera. The three different distance metrics, RF, Nye, and CI, compare the topology of the gene trees to the ASTRAL species tree, and we considered the branching order alone. The gene trees are either empirical trees that can be rooted and have clade occupancy > 75% of sampled tips (observed) or 1,000 gene trees simulated under the multispecies coalescent using the ASTRAL species tree (expected). The distances were normalized so that they ranged from 0 to 1, with 0 indicating complete agreement between a gene tree and the species tree. The t-values and associated p-values for each distance metric/clade combination are based on Welch’s t-test with the hypothesis that the mean tree distance for the observed and expected gene trees are equal, or in other words, that the tree distances based on empirical data are what would be expected under incomplete lineage sorting alone. Signiﬁcant t/p-values at α = 0.05 are indicated in bold. November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 8 Frontiers in Plant Science \| www.frontiersin.org Phylogeny of Salvia Rose et al. FIGURE 3 \| Phylogenetic network based on 57 nuclear genes depicting backbone relationships in Salvia with one exemplar per subgenus, with internal branch lengths in coalescent units. Phylogenetic Networks The best-ﬁtting network has four reticulation events. Inheritance probabilities (γ) are indicated next to the lineage which is inferred to have received genetic material. The numbers above the branches on the bifurcating major topology are quartet concordance factors or the proportion of the genome supporting each quartet. The numbers below the major topology branches and to the right of hybrid branches are bootstrap support values. Ingroup tips are colored by subgenus. FIGURE 3 \| Phylogenetic network based on 57 nuclear genes depicting backbone relationships in Salvia with one exemplar per subgenus, with internal branch lengths in coalescent units. The best-ﬁtting network has four reticulation events. Inheritance probabilities (γ) are indicated next to the lineage which is inferred to have received genetic material. The numbers above the branches on the bifurcating major topology are quartet concordance factors or the proportion of the genome supporting each quartet. The numbers below the major topology branches and to the right of hybrid branches are bootstrap support values. Ingroup tips are colored by subgenus. FIGURE 3 \| Phylogenetic network based on 57 nuclear genes depicting backbone relationships in Salvia with one exemplar per subgenus, with internal branch lengths in coalescent units. The best-ﬁtting network has four reticulation events. Inheritance probabilities (γ) are indicated next to the lineage which is inferred to have received genetic material. The numbers above the branches on the bifurcating major topology are quartet concordance factors or the proportion of the genome supporting each quartet. The numbers below the major topology branches and to the right of hybrid branches are bootstrap support values. Ingroup tips are colored by subgenus (BS = 40), and stem subg. Glutinaria and stem MRCA Calosphace + Dorystaechas (BS = 40). regarding the intersubgeneric relationships, where supported. Discordance among nuclear loci can largely be reconciled by invoking ILS and horizontal gene ﬂow. Our results largely corroborate our previous analysis involving a larger number of loci with fewer terminals (Kriebel et al., 2019). Despite the fewer loci examined in this study, the average locus length is nearly twice as long (623 vs. 1,133 bp), presumably increasing the accuracy in the gene tree estimation. Additionally, compared with Kriebel et al. (2019) we found increased ASTRAL BS support for the monophyly of subg. Audibertia (>0.99 vs. 0.79) and for the sister relationship of subg. Salvia and Sclarea (0.99 vs. 0.79). However, we did not ﬁnd convincing support for A Robust Phylogenetic Hypothesis for Salvia Heterosphace and Salvia as a better explanation for the discordance (BS = 66), an alternative resolution of the relationships with the gene ﬂow between subg. Salvia and Sclarea is also possible (BS = 22). Given this result, it is also possible that the constraint of the level-1 network is not an appropriate model, and subg. Salvia may be a hybrid between subg. Heterosphace and Sclarea. This hypothesis requires further testing. On the other hand, we found poor support for the remaining two inferred horizontal gene ﬂow events on our best network, especially for the gene ﬂow involving subg. Zhumeria. While slightly better supported, the horizontal gene ﬂow between subg. Audibertia and Dorystaechas seem implausible since it necessitates the gene ﬂow between their ancestors in North America and Southwest Asia (Kriebel et al., 2019). Despite its absence from the best network, the BS replicates suggest a strong possibility of gene ﬂow involving subg. Glutinaria, especially with the MRCA of Audibertia + Calosphace (possibly extended to Dorystaechas), which would be more consistent with our current understanding of the historical biogeography of Salvia. While the backbone topology of the plastome of Salvia does not contradict that of the nuclear tree, it is still incompletely supported. However, it is unclear how much more information about the major relationships in Salvia, especially the relationships of subg. Perovskia and Rosmarinus, can be garnered from it. Analyses incorporating complete or nearly complete plastomes have failed to recover a fully supported backbone (Zhao et al., 2020; Supplementary Figure S5). Therefore, adding the portions of the plastome that we excluded does not seem to provide a viable solution. Mitogenomes, possibly combined with plastomes, are a possible avenue of research for a fully supported organellar phylogeny. Apart from a level-1 network possibly being an unreasonable restriction to our dataset, the uncertainty in the placement of horizontal gene ﬂow may be due to the relatively few loci employed here. For example, SNAq may recover false positive hybridization events in the datasets with < 100 loci (Solís-Lemus and Ané, 2016). Overall, our results highlight that it is essential to complement searches for best-ﬁtting networks with BS analyses. A ﬁnal open question regarding the deeper-level phylogeny of Salvia is the relationship of the unsampled subg. Meriandra. We expect this subgenus to be closely related to subg. A Robust Phylogenetic Hypothesis for Salvia Our results demonstrate that despite the gene tree discordance, the backbone relationships of Salvia are identical using nuclear and plastid data. These genomes support the monophyly of all currently recognized subgenera and are largely concordant November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 9 Phylogeny of Salvia Rose et al. considerable uncertainty regarding which clades were involved in some of the horizontal gene ﬂow events. We are, however, fairly certain that one gene ﬂow event involved the stem MRCA of subg. Glutinaria and Calosphace, with the gene ﬂow involving either the ancestor of subg. Rosmarinus alone (BS = 66) or the MRCA of subg. Rosmarinus and Salvia (BS = 28), and this likely explains the uncertainty regarding the placement of subg. Perovskia and Rosmarinus. subg. Audibertia based on the ASTRAL LPP, and the monophyly for this subgenus based on the nuclear data has been somewhat unclear (Walker et al., 2004, 2015; Walker and Sytsma, 2007; Drew et al., 2017; Will and Claßen-Bockhoﬀ, 2017), although it is clearly monophyletic based on the datasets with a good sampling of plastid loci (Walker et al., 2015; Supplementary Figure S5). Unexpectedly, we found some uncertainty regarding the placements of subg. Perovskia, Rosmarinus, and Zhumeria, which were previously placed with BS = 100 in Kriebel et al. (2019). Nevertheless, the placement of all these subgenera, especially Perovskia and Rosmarinus, has varied widely across previous molecular studies incorporating low-copy nuclear loci (Drew et al., 2017), transcriptomes (Mint Evolutionary Genomics Consortium, 2018), nuclear ribosomal ITS/ETS (Drew and Sytsma, 2012; Will and Claßen-Bockhoﬀ, 2017; Kriebel et al., 2019), and plastid data (Walker and Sytsma, 2007; Drew and Sytsma, 2012; Drew et al., 2017; Will and Claßen-Bockhoﬀ, 2017; Zhao et al., 2020, 2021). The varying placement of subg. Rosmarinus across studies–and indeed across the loci examined in this study–can be explained by a combination of ILS and ancient horizontal gene ﬂow (see below). This may be true for subg. Zhumeria as well, although it is less likely that horizontal gene ﬂow has been involved in that case given the low BS support for any such gene ﬂow. Likewise, it seems probable that the uncertainty regarding the branching order of subg. Heterosphace, Salvia, and Sclarea is the result of the gene ﬂow. Although our phylogenetic network favors horizontal gene ﬂow between subg. A Robust Phylogenetic Hypothesis for Salvia Dorystaechas and possibly even sister to it based on previous molecular results (Walker and Sytsma, 2007; Drew and Sytsma, 2012; Will and Claßen-Bockhoﬀ, 2017; Kriebel et al., 2019). The placement of subg. Meriandra has important implications, not only for the historical biogeography of the genus but also implications for the timing and geographic location of any gene ﬂow between the ancestors of subg. Audibertia and Dorystaechas, if present (see below). Support for Key Infrageneric Structure Our AHE data provides good support for many shallow-scale relationships in Salvia (Figure 1 and Supplementary Figures S1–S3). We clarify the placement of sect. Axillares within subg. Calosphace, with the nuclear evidence slightly favoring a hypothesis of the sister relationship of sect. Axillares and the Hastatae clade (sects. Blakea, Hastatae, and Standleyana), although the support based on ASTRAL LPP is noticeably weak. This relationship is identical to that suggested by the nuclear ribosomal DNA (Fragoso-Martínez et al., 2018; Kriebel et al., 2019, Appendix S7), but not the plastid data (Drew et al., 2017; Will and Claßen-Bockhoﬀ, 2017; Fragoso-Martínez et al., 2018) or one low copy nuclear marker (PPR: Drew et al., 2017), which instead placed sect. Axillares as sister to the remainder of subg. Calosphace. It is still unclear if the uncertainty in the placement is due to ILS or gene ﬂow, which should be investigated in future studies focused on subg. Calosphace, Evidence for Gene Flow in the Backbone of Salvia: But Where? While the major topology of our phylogenetic network in Salvia is clear and strongly recovers the same bifurcating backbone found across other analyses, each with diﬀerent assumptions, it also suggests that such a relatively simple tree may not be the best model of the phylogenetic history of Salvia (Figure 3). While all of our BS trees recovered at least three gene ﬂow events, there is November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 10 Phylogeny of Salvia Rose et al. especially since the placement of sect. Axillares has important macroevolutionary implications. combination with much younger crown ages for the MRCAs of what this study samples in subg. Sclarea (13.4 My) and Salvia (<7.8 My) relative to Calosphace (20.1 My) (Kriebel et al., 2019). However, based on the excess of the nuclear gene tree discordance in the aforementioned clades relative to the expectation under the multispecies coalescent, we suggest that in these clades, especially in subg. Salvia and Sclarea, the multispecies coalescent does not provide an ideal model of phylogenetic relationships. Instead, a model with horizontal gene ﬂow in these lineages is likely a better explanation for the excess of gene tree discordance observed in our data. While another possibility for this pattern is that error in the gene tree estimation adds artiﬁcial discordance, we reject this as a major complicating factor given that we collapsed very poorly supported edges in observed gene trees. Where our sampling of the Old World lineages permits, we corroborated relationships among deep subgeneric splits which were strongly supported in previous studies (subg. Glutinaria: Hu et al., 2018, subg. Heterosphace: Will and Claßen-Bockhoﬀ, 2014, 2017). Within the other Old World subgenera, the support for relationships in previous studies has not been robust enough to warrant a discussion of the major relationships (Will and Claßen-Bockhoﬀ, 2014, 2017), and thus our results, where it is well supported, are novel. From a taxonomic perspective, it is encouraging that for the few species for which we have multiple accessions, morphologically deﬁned species are monophyletic, paraphyletic due to the inclusion of only one other species, or essentially form a polytomy with other morphologically similar species, rather than polyphyletic and/or found in large polytomies (Figure 1 and Supplementary Figures S1–S3). This suggests that our AHE dataset has the power to not only resolve deeper relationships in Salvia but also to provide information pertinent to species delimitation. Evidence for Gene Flow in the Backbone of Salvia: But Where? The stark discordance present between the ASTRAL tree and the plastome tree at many shallow nodes in subg. Calosphace, Salvia, and Sclarea, with especially large distances between the nuclear and plastid trees in subg. Salvia, is also highly suggestive of an important contribution from horizontal gene ﬂow, either hybridization or introgression (Figure 2). However, depending on the amount of past backcrossing, a signal for past gene ﬂow may be absent from the nuclear genome in some cases. While we did not test it here, we do not think ILS is a likely explanation for the intergenomic gene tree conﬂict given the results from other angiosperm systems (Folk et al., 2017; Morales-Briones et al., 2018; Lee-Yaw et al., 2019; Rose et al., 2021), although error in the species tree estimation is a possible explanation. In future studies, we expect that relatively under-sampled subgenera should show increasing levels of cytonuclear discordance as we increase species sampling, especially within the subg. Glutinaria (Hu et al., 2018). The potentially confounding eﬀects of polyploidy and whole-genome duplications (WGD) were not evaluated here but are being investigated. Infrageneric Structure: Incomplete Lineage Sorting and Horizontal Gene Flow Explain Strong Gene Tree Discordance While phylogenomic datasets show great promise to resolve relationships in previously intractable angiosperm lineages, the irony is that many of these groups have undergone rapid radiations (Larson et al., 2020; Shee et al., 2020; Rose et al., 2021; Thomas et al., 2021), which increases the chance of gene tree heterogeneity due to ILS (Pamilo and Nei, 1988; Maddison, 1997; Oliver, 2013). Rampant gene tree discordance need not mean that species trees are poorly supported, provided that the discordance is consistent with the underlying model used to generate the species tree. Indeed, our analysis suggests that much of the gene tree discordance is at least consistent with ILS, given the high support for many infrageneric relationships based on our ASTRAL analysis (Figure 1 and Supplementary Figures S1, S2). Conversely, the low support for relationships in approaches that only take ILS into account may be due to either the lack of information about a given relationship in the underlying sequence data, or a more complex model of relationships (i.e., one involving horizontal gene ﬂow). Our results demonstrate that while the average discordance of nuclear gene trees is consistent with what would be expected under ILS alone in the relatively under-sampled subg. Glutinaria and Heterosphace, it exceeds what would be expected under ILS in subg. Calosphace, Salvia, and Sclarea (Table 2). More strikingly, since that under ILS gene tree discordance should increase simply as a function of taxon sampling, it is notable that subg. Salvia and Sclarea have observed mean nuclear gene tree discordance on par with or slightly lower than that for subg. Calosphace, despite being represented by many fewer tips (Table 1). Finally, it is worth a brief note concerning why we found that the mean RF tree distance often conﬂicts with the other distance metrics by demonstrating that the mean discordance is either on par with expectations under the multispecies coalescent or observed gene trees are, in some cases, less discordant. Despite being a widely used metric, RF distance is probably too conservative in penalizing against relatively minor topological diﬀerences (Smith, 2020a), as the movement of a single tip may result in maximum tree-to-tree distances even though all other tips show the same branching pattern. Thus, collapsing poorly supported edges of observed gene trees into polytomies downplays discordance, while any minor topological diﬀerences in the fully-resolved expected gene trees are penalized. Frontiers in Plant Science \| www.frontiersin.org ACKNOWLEDGMENTS We gratefully acknowledge Holly Forbes from the UC-Berkeley Botanical Garden, and Cindy Newlander and Mike Kintgen from the Denver Botanical Garden for granting permission to collect garden specimens and assisting us. We are grateful to Jay Walker, N. Ivalú Cacho, Eleftherios Dariotis, and Rolando Uría for their help in collecting the specimen. Several issues still need clariﬁcation, especially in the placement of subg. Meriandra and in the monophyly of subg. Audibertia. We are conﬁdent that future analyses using this same or expanded set of loci, in concert with an evaluation of polyploidy and WGD processes, will resolve these issues. Additionally, targeted analyses of clades or further methodological advances will allow us to tease apart horizontal gene ﬂow at shallower scales. Our phylogenetic hypothesis, as well as future, time-calibrated phylogenetic hypotheses of the entire genus Salvia, its constituent subgenera, and targeted clades, will provide an invaluable framework for which to conduct multiple comparative analyses in this fascinating genus. DATA AVAILABILITY STATEMENT Supplementary Figure S2 \| The ASTRAL species tree of Salvia and outgroups, with bootstrap support on branches. Ingroup branches are colored by subgenus. The data presented in the study are deposited in the NCBI Sequence Read Archive (SRA) as BioProject PRJNA773953. Supplementary Figure S3 \| The RAxML maximum likelihood tree of Salvia and outgroups based on the concatenated nuclear matrix, with bootstrap support on branches. Ingroup branches are colored by subgenus. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. 767478/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. 767478/full#supplementary-material Supplementary Figure S1 \| The ASTRAL species tree of Salvia and outgroups, with local posterior probabilities on branches. Ingroup branches are colored by subgenus. Supplementary Figure S1 \| The ASTRAL species tree of Salvia and outgroups, with local posterior probabilities on branches. Ingroup branches are colored by subgenus. CONCLUSION Our updated AHE dataset provides evidence for a well- supported backbone of Salvia and indicates that there is an emerging consensus of relationships in the genus that extends across genomic compartments. Past diﬃculty in inferring relationships has likely been caused by a combination of uninformative markers, ILS, and horizontal gene ﬂow. To One possible explanation for this is that the increased ILS results from the very rapid radiations of these clades, in November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 11 Phylogeny of Salvia Rose et al. ﬁnancial support provided by CONACYT by means of the project CB-2015-01-255165. ﬁnancial support provided by CONACYT by means of the project CB-2015-01-255165. ﬁnancial support provided by CONACYT by means of the project CB-2015-01-255165. ﬁnancial support provided by CONACYT by means of the project CB-2015-01-255165. the latter point, while our dataset clearly shows evidence of horizontal gene ﬂow at deep and shallow scales in Salvia, we are presently unable to conﬁdently demonstrate how many ancient gene ﬂow events occurred and where they are placed. This highlights the importance of assessing the support for the best-ﬁtting phylogenetic network, rather than only presenting the best network. FUNDING This manuscript was funded in part by the University of Wisconsin Botany Department Hofmeister Endowment, NSF- DEB collaborative grant to KS and BD (DEB-1655606 and DEB-1655611), and TUBITAK project number 2219 to FC for postdoctoral studies in the United States. JG-G appreciates the Supplementary Figure S6 \| Tanglegram illustrating the disagreement between the ASTRAL (A) and concatenated maximum likelihood (B) species trees based on nuclear data. Links connect identical tips, with nodes rotated to minimize link overlap. Ingroup branches are colored by subgenus. Clades that differ between the two trees are indicated by ﬁlled circles on the concatenated maximum likelihood tree. AUTHOR CONTRIBUTIONS Supplementary Figure S4 \| Phyparts summary of gene trees. Pies at major nodes summarize the percentage of various phylogenetic signals across 101 gene trees which can be rooted. The numbers at the left of the pies show the total number of gene trees in which the clade is found, followed by the total number of gene trees that conﬂict with that clade. The remainder of the gene trees, if any, do not provide information on that particular relationship. Ingroup branches are colored by subgenus. Supplementary Figure S4 \| Phyparts summary of gene trees. Pies at major nodes summarize the percentage of various phylogenetic signals across 101 gene trees which can be rooted. The numbers at the left of the pies show the total number of gene trees in which the clade is found, followed by the total number of gene trees that conﬂict with that clade. The remainder of the gene trees, if any, do not provide information on that particular relationship. Ingroup branches are colored by subgenus. JR, RK, BD, and KS conceived and undertook the project. JR, RK, LK, AD, JG-G, FC, EL, AL, and BD assisted with the data collection. JR analyzed the data and led the writing with contributions from all authors. All the authors contributed to the article and approved the submitted version. Supplementary Figure S5 \| The RAxML maximum likelihood tree of Salvia and outgroups based on entire plastomes, with bootstrap support on branches. GenBank accessions are removed so that the plastome tree matches the nuclear tree in the tip composition. Ingroup branches are colored by subgenus. REFERENCES Gene trees in species trees. Syst. Biol. 46, 523–536. Mint Evolutionary Genomics Consortium (2018). Phylogenomic mining of the mints reveals multiple mechanisms contributing to the evolution of chemical diversity in Lamiaceae. Mol. Plant 11, 1084–1096. doi: 10.1016/j.molp.2018. 06.002 Fragoso-Martínez, I., Martínez-Gordillo, M., Salazar, G. A., Sazatornil, F., Jenks, A. A., García-Peña, M. D. R., et al. (2018). 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Frontiers in Plant Science \| www.frontiersin.org November 2021 \| Volume 12 \| Article 767478 REFERENCES Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Sukumaran, J., and Holder, M. T. (2010). DendroPy: a Python library for phylogenetic computing. Bioinformatics 26, 1569–1571. Thomas, A. E., Igea, J., Meudt, H. M., Albach, D. C., Lee, W. G., and Tanentzap, A. J. (2021). Using target sequence capture to improve the phylogenetic resolution of a rapid radiation in New Zealand Veronica. Am. J. Bot. 108, 1289–1306. 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. Walker, J. B., Drew, B. T., and Sytsma, K. J. (2015). Unravelling species relationships and diversiﬁcation within the iconic California Floristic Province sages (Salvia subgenus Audibertia, Lamiaceae). Syst. Bot. 40, 826–844. Walker, J. B., and Sytsma, K. J. (2007). Staminal evolution in the genus Salvia (Lamiaceae): molecular phylogenetic evidence for multiple origins of the staminal lever. Annals Bot. 100, 375–391. Copyright © 2021 Rose, Kriebel, Kahan, DiNicola, González-Gallegos, Celep, Lemmon, Lemmon, Sytsma and Drew. 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. Walker, J. B., Sytsma, K. J., Treutlein, J., and Wink, M. (2004). Salvia (Lamiaceae) is not monophyletic: implications for the systematics, radiation, and ecological specializations of Salvia and tribe Mentheae. Am. J. Bot. 91, 1115–1125. Wester, P., and Claßen-Bockhoﬀ, R. (2007). Floral diversity and pollen transfer mechanisms in bird-pollinated Salvia species. Ann. Bot. 100, 401–421. November 2021 \| Volume 12 \| Article 767478 Frontiers in Plant Science \| www.frontiersin.org 14
https://openalex.org/W3110064398	https://nottingham-repository.worktribe.com/preview/5461681/MedAll%20International%20Virtual%20Medical%20Conference.pdf	English	null	Perceptions and Experiences of the University of Nottingham Pilot SARS-CoV-2 Asymptomatic Testing Service: A Mixed-Methods Study	International journal of environmental research and public health/International journal of environmental research and public health	2,020	cc-by	418	Poster presentation Blake, H.; Corner, J.; Cirelli, C.; Hassard, J.; Briggs, L.; Daly, J.M.; Bennett, M.; Chappell, J.G.; Fairclough, L.; McClure, C.P.; Tarr, A.; Tighe, P.; Favier, A.; Irving, W.; Ball, J. Perceptions and Experiences of the University of Nottingham Pilot SARS-CoV-2 Asymptomatic Testing Service: A Mixed- Methods Study. Blake, H.; Corner, J.; Cirelli, C.; Hassard, J.; Briggs, L.; Daly, J.M.; Bennett, M.; Chappell, J.G.; Fairclough, L.; McClure, C.P.; Tarr, A.; Tighe, P.; Favier, A.; Irving, W.; Ball, J. Perceptions and Experiences of the University of Nottingham Pilot SARS-CoV-2 Asymptomatic Testing Service: A Mixed- Methods Study. MedAll IVMC: Sat 13th March 2021 MedAll International Virtual Medical Conference https://medall.org/conference (abstract accepted 25.02.21) MedAll IVMC: Sat 13th March 2021 MedAll IVMC: Sat 13th March 2021 Abstract We aimed to explore student and staff perceptions and experiences of a pilot SARS-CoV-2 asymptomatic testing service (P-ATS) in a UK university campus setting. This was a mixed-method study comprised of an online survey, and thematic analysis of qualitative data from interviews and focus groups conducted at the mid-point and end of the 12-week P-ATS programme. Ninety-nine students (84.8% female, 70% first year; 93.9% P-ATS participants) completed an online survey, 41 individuals attended interviews or focus groups, including 31 students (21 first year; 10 final year) and 10 staff. All types of testing and logistics were highly acceptable (virus: swab, saliva; antibody: finger prick) and 94.9% would participate again. Reported adherence to weekly virus testing was high (92.4% completed ≥6 tests; 70.8% submitted all 10 swabs; 89.2% completed ≥1 saliva sample) and 76.9% submitted ≥3 blood samples. Students tested to “keep campus safe”, “contribute to national efforts to control COVID-19”, and “protect others”. In total, 31.3% had high anxiety as measured by the Generalized Anxiety Disorder scale (GAD-7) (27.1% of first year). Students with lower levels of anxiety and greater satisfaction with university communications around P-ATS were more likely to adhere to virus and antibody tests. Increased adherence to testing was associated with higher perceived risk of COVID-19 to self and others. Qualitative findings revealed 5 themes and 13 sub-themes: “emotional responses to COVID-19”, “university life during COVID-19”, “influences on testing participation”, “testing physical and logistical factors” and “testing effects on mental wellbeing”. Asymptomatic COVID-19 testing (SARS-CoV-2 virus/antibodies) is highly acceptable to students and staff in a university campus setting. Clear communications and strategies to reduce anxiety are likely to be important for testing uptake and adherence. Strategies are needed to facilitate social connections and mitigate the mental health impacts of COVID-19 and self-isolation.
W2333536134.txt	https://zenodo.org/records/2423135/files/article.pdf	de		Ueber die Verseifbarkeit einiger Säureimide (Diamide) und Aminsäuren durch Fermente	Pflügers Archiv für die gesamte Physiologie des Menschen und der Tiere/Pflügers Archiv	1,903	public-domain	6,180	278 M. Gonnermann: Ueber die Verseifbarkeit einiger Sgtureimide (Diamide) und Aminsfiuren dutch Fermente. Von Dr. H . G o n n e r m a n n . Den in diesem Archiv Bd. 89 S. 493 ver0ffentlichten Untersuchungen iiber die Verseifbarkeit einiger Saure-Amide und -Anilide durch Fermente reihen sich die nachstehenden als Erganzung an; es kann daher yon der Wiedergabe der allgemeinen Methode des Yerfahrens abgesehen und auf die erste Arbeit verwiesen werden; nur die Abweichungen, welche die Imide (Diamide) bei den einzelnen Versuchen bedingen, sollten Erwi~hnung finden; bemerkt soll aber hier auch sogleich werden, dass ich die UnlOslichkeit des Fluornatriums in 55 % igem Alkohol, auf die ich in meiner ersten Yersuchsreihe noch nicht aufmerksam geworden war, jetzt wiederholt zur Entfernung dieses oft stSrend wirkenden Stoffes aus den Versuchsgemischen benutzt habe. Die verwendeten Fermente sind dieselben geblieben wie bei der ersten Versuchsreihe; die Organe - - Leber und Niere - - stammten gleichfalls yore Schaf. Die zur Prufung gelangten Imide und Amins~uren waren folgende: O x a m i n s i ~ u r e , S u c c i n i m i d , S u c c i n aminsi~ure, Dibenzamid, Disalicylamid, Phtalimid, denen sich Parabansi~ure anschloss. u mit D i a c e t a m i d und O x i m i d habe ich nicht ausgeftihrt, weil die Darstellung dieser beiden Pri~parate zu umsti~ndlich ist; bei dem Diacetamid ist zudem ohne Weiteres nach meinen Versuchsresultaten anzunehmen, dass dasselbe durch diejenigen Fermente zerlegt wird, die das Acetamid spalten 1). An Stelle des Oximids andererseits glaubte ich Oxamin1) Wi~hrend diese Arbeit druckfertig war, kommtmir durch Prof. N as s e ein Abdruck eines Vortrages yon F. IIofmeister: Ueber den Bau des Eiweissmolekuls, gehalten auf der 74. Versammlung deutscher l~aturforscherund Aerzte in Karlsbad (Naturwissensch. Rundschau Bd. 17 Nr. 42 u. 43. 1902), zur Hand, Ueber die Verseifbarkeit einiger Saureimide (Diamide) u. Amlns~uren etc. 279 saure setzen und im Falle der ~ichtzersetzung durch Fermente und tlistozyme auf ein 81eiches Verhalten des Oximids selbst schliessen zu durfen. Versuche tiber die Zersetzung der hier in Frage kommenden Imide im ThierkSrper sind nur yon Fr. K o e h n e l ) angestellt worden; bei dem Succinimid, I)ibenzamid und Phthalimid wird der yon K o e h n e erhaltenen Resultate gedacht werden. Succinimid und Phthalimid waren yon K a h l b a u m - B e r l i n bezogen; die ubrigen Praparate habe ich selbst dargestellt. Oxamins~ure. Der Darstellung der Oxaminsi~ure lag zunachst die Arbeit von Ost und M e n t e 2) zu Grunde. Nach B a l a r d a) soll das saure Ammoniumoxalat im Oelbad auf 220o--230 o erhitzt werden, die Masse wird hierbei anfangs fiussig, dann teigig und blaht sich schliesslich unter Entwicklung yon Ammoncarbonat auf. O s t und M e n t e geben die Zersetzungstemperatur bei 140 o an, bis die Masse zi~he wird, - - es ist mir nicht gelungen, bei dieser Temperatur eine solche Masse zu erlan~en, sondern dleser Zustand trat erst bei 180~ ein. Nach dieser Vorschrift arbeitend, musste bald auffallen, dass das oxaminsaure Baryum in L5sung bleiben sol], wi~hrend zur Darstellung dieses Salzes oxaminsaures Ammonium durch Baryumchlorid umgesetzt wird, - - d. h. oxaminsaures Baryum fallt aus 4), wie dean auch die meisten Salze der Oxaminsaure sehr schwer in Wasser 15slich sind. m welchem es heisst: ,Das uuf Eiwelss so krahlg wirkende Trypsin ist mcht im Stande, H~ppursaure zu spalten oder auch nut aus sonst so labllen Suureamiden wle Acetamid und Asparagin den Ammoniakrest abzutrennen." Ich habe daraufhin meine Versuche wiederholt und land dm Resultate der ersten Versuchsreihe v o l l b e s t u t l g t : T r y p s i n v e r s e i f t A c e t a m i d u n t e r A b s p a l t u n g yon Essigsaure; es entwickelte sich sogar beim Eindampfen des Hauptversuches Ammoniak, welches rothes Lackmuspapier blaute, wahrend lm Controlversuch Acetamld sich verfiuchtigt und blaues Lackmuspapier durch die Dumpfe schwach gerothet wird. 1) Ueber dus Verhalten einiger Saureimide lm thierischen Organismus. Aus dem Institut fur physiol. Chemie und Pharmukologie iu Rostock (Director Prof. Dr. O. Masse). Inaug.-D~ssert. Rostock 1894. 2) Benchte d. chem. Gesellsch. Bd. 19 S. 3229. 3) Annul. f. Chem. Bd. 42 S. 197. 4) E n g s t r o m , Annal. f. Chem. 1856 S. 453. 280 M. Gonnermann: Die Ausbeute war ubri~ens sehr gering. Viel schne]ler und ganz glatt im Verlauf erhiilt man die Oxaminsiiure nach T o u s s a i n t 1) aus Oxamid durch Kochen mit Ammoniak; nach viersti~ndigem Kochen war das Oxamid vollst~ndig gelSst und, da beim Erkalten der LSsung keine Ausscheidung erfolgte, in Oxamins~ture ubergegangen; die heisse LSsun~ wurde filtriert und so lange eingedampft, bis beim Herausnehmen einer Probe dieselbe sofort erstarrte; dann wurde starke Salzsaure his zur stark sauren Reaction unter Umrtihren zugeffigt und das Gemisch 24 Stunden stehen gelassen. Auf Zusatz der Salzs~ure schied sich die Oxaminsi~ure sofort als weisses Pulver aus; die uberstehende Fltissigkeit wurde abgegossen, der Niederschlag mit a]koholhaltigem Wasser, welche zwar Salmiak, jedoch nicht Oxaminsaure lbst, au%estisst und schliesslich auf dem Saugfilter mit verdunntem Alkohol ausgewaschen, bis Silber]osung keine Trt~bung bewirkte. Eine Probe des getrockneten, weissen Pulvers in heissem Wasser gelSst gab auf Zusatz yon etwas Ammoniak durch Calciumchlorid keine Trt~bung; ebenso wurde Permanganatl~sung bei Ge~enwart yon Schwefelsiiure nach fi~nf Minuten nicht entfiirbt~ so dass ich wohl annehmen kann, dass die Oxaminsaure vSllig frei von Oxalsliure war. Die nun folgenden Versuche mit Fermenten lehnen sich ganz denen mit Oxamid an: yon den Filtraten wurden 50 ccm, entsprechend 0,25 Oxaminsi~ure, abgenommen, durch Ammoniak alkalisch gemacht aufgekocht, nochmals filtrirt, das Filtrat siedendheiss mit Essigsaure tibersi~ttigt und noch heiss mit 10 % iger Calciumchloridlbsung versetzt, ein entstehender bliederschlag mit heissem Wasser ausgewaschen, his Ammoniumoxalat keine Trabung mehr gab, und auf Oxalsliure in stark schwefelsaurer L(isung dutch Permanganat gepruft. Versuche mit thierischen Fermenten. Pepsin. Es wurden nut 1,6 ccm Permanganatl0sung verbraucht, und blieb die Farbe eine halbe Stunde unver~indert, ganz gleich wie bei den Versuchen mit Oxamid; Oxaminsaure wird dutch Pepsin nicht in Oxalsi~ure ubergefuhrt. T r y p s i n . Die zugesetzten 25 ccm SodalSsung wurden dutch die Oxaminsiiure vi~llig neutralisirt, so dass sich ein weiterer Zusatz 1) Annal. f. Chem. Bd. 120 S. 237. Ueber die u einlger S~ureimide (Diamide) u. Amins~turen etc. 281 yon 25 ccm nothig machte; eine Umsetzung in Oxals~ure fand nicht statt. P t y a l i n ist ohne Einwirkung. Versuche mit Histozymen. L e b e r wirkt nicht auf Oxamins~ture ein, wie daraus hervorgeht, class der Verbrauch an Permanganat sehr gering und in beiden Versuchen ziemlich das Gleiche war; es mussten bei einer Umsetzung in Oxalsaure 0,3 Oxaminsaure - - ich nehme an, dass ein Theil in dem Pressr,lckstand geblieben ist - - 76 ccm 1/lo PermanganatlSsung benSthigt haben. N i e r e war gleichfalls wirkungslos. Versuche mit pflanzlichen Fermenten. M a 1t i n und I n v e r t i n sind unwirksam. Emulsin. Das Emulsin 15ste sich im Hauptversuch nicht auf, jedenfalls in Folge der sauren Reaction des Gemisches; nach zweit~tgigem Stehen nahm ich die Prufung auf Oxals~ture vor, land jedoch keine; ich fugte somit einem neuen Versuch 30 ccm Sodah~sung hinzu, - - das Emulsin blieb in truber LSsung, und auch in diesem Versuch war Oxals~ture nicht nachzuweisen. Aus meinen Versuchen mit den einfachen Oxals~ure-Amiden geht hervor, dass dieselben durch alle angewandten Enzyme nicht gespalten werden, auch unzersetzt und, im chemischen Sinn, unsch~tdlich den ThierkSrper passiren, wie ich als sicher angenommen habe und durch K o b e r t ' s neue Versuche mit r e i n em Oxamid, wie er mir mittheilte, best;ttigt worden ist. Anders dagegen steht es mit den complicirten Verbindunoen - - insbesondere Parabans~ure, Alloxan und Alloxantin - - , die nach den Versuehen yon K o e h n e im Thierk6rper fast vollkommen zerstSrt werden. Durch welche Krafte dies geschieht, ergeben die anschliessenden Versuche; was das W i e angeht, so ist wohl mit Sicherheit anzunebmen, dass aus den genannten K0rpern zunachst Oxals~tfire abgespalten wird - - bei der Parabansaure wahrscheinlich nach vorghngiger Umwandlung in Oxalursaure - - , die denn, wie auch K o eh n e aus dem ~Nichtauftreten yon Oxalursaure im Harn schliesst, sofort welter zerfMlt. Der Vollstandigkeit wegen fuhrte ich einige Versuche mit P a r a b a n s ~ u r e H(co-co \CO NH aus; diese geht sehr leicht unter Aufnahme yon 282 M. Gonnermann: Wassermolekulen in Oxalursi~ure und Oxals~ure fiber; es ist demnach eine Umsetzung durch Fermente leicht zu erkennen, wenn im Reactionsgemisch sich Oxalsi~ure nachweisen liisst. K o e h n e gibt als charakteristische Reaction far Parabansaure an - - und bezieht sich auf ,,Harnanalyse" yon N e u b a u e r und V o g e I und auch B ei 1stein --, dass ammoniakalische Chlorcalciumlosung in der Ki~lte keinen Niederschlag gibt; in der ,Harnanalyse"') finde ich jedoch folgende Angabe. die sich nicht auf Parabansaure, sondern auf Oxalurs~ure bezieht: ,,Versetzt man eine wassrige, mi~ssig verdunnte Li)sung yon oxalursaurem Ammon mit Chlorcalcium und Ammoniak, so entsteht kein Niederschlag; die Fhissigkeit bleibt vollkommen klar; erwi~rmt man dagegen die Mischung, so tritt sehr bald, noch weit vor d(~r Siedhitze, Triibung ein, und oxalsaurer Kalk scheidet sich massenhaft aus. Dieses Verhalten ist unzweifelhaft die empfindlichste Reaction, mit welcher sich noch ungtaublich kleine Mengen von Oxalursaure erkennen lassen, sobald man das Mikroskop zu Hulfe nimmt." - Nach diesen Angaben N e u b a u e r ' s wt~rde also, falls in den Versuchsgemischen sich oxalsaurer Kalk ausscheidet, die Parsbaasi~ure bereits durch Einwirkung yon Fermenten in Oxalursiiure glbergegangen sein. Wie ausserst schnell diese Umsetzung eintritt, ergeben die nachfolgenden Versuche. Um nun aber sicher zu sein, dass die eventuell nachgewiesene Oxalsi~ure nur durch Einwirkung der Fermente entstanden ist, dampite ich eine schwach durch Soda alkalisch gemachte L0sung yon Parabausiiure ( 0 , 5 : 1 0 0 ) K a h l b a u m auf dem Wasserbad zur Trockne ein, nahm das blendend weisse Pulver im Wasser auf, ubers~ttigte die L6sung mit Salzsi~ure, fugte Ammoniak und Chlorcalciumlosung zu und beobachtete, d a s s s o f o r t ein s t a r k e r N ~ e d e r s c h l a g e i n t r a t , welcher unter dem Mikroskop krystallinische Gebilde schwer erkennen liess; jedoch erschienen die charakteristischen Formen des Calciumoxalates, als der Niederscblag in etwas Salzshure und Wasser gel0st und Ammoniak vorsichtig t~bergeschichtet wurde~ -- bis sich die Fliissigkeit vollst~indig geklart hatte. Nach diesem uberraschenden Resultat leitete ich folgende Versuche ein : 1. eine LSsung yon Chlorcalcium, mit Ammoniak versetzt, wurde erwi~rmt und bei Seite gestellt; 1) Neubauer und Vogel S. 34. Ueber die Verseifbarkeit einiger S~ureimide(Diamide)u. Aminsaaren etc. 283 2. eine LSsung yon Parabans~ture mit Ammoniak neutralisirt und s o f o r t mit Chlorcalcium versetzt; 3. die gleiche LSsung drei Stunden stehen gelassen und dann mit Chlorcalcium versetzt; 4. die gleiche LSsung auf dem Wasserbad erwi~rmt, abgekiihlt und derselben Chlorcalcium zugegeben; 5. eine LSsung von Parabansaure im Wasserbad yon 60 o 1/~ Stunde erwi~rmt, dann Ammoniak und Chlorcalcium zugefiigt und beobachtet, dass bei 1. keine Veri~nderung eintrat, da selbstverst~ndlich; 2. nicht sofort ein l~iederschlag entstand, jedoch nach schwachem Erwi~rmen; 3. ein ~iederschlag nach einigen Secunden sich bildete; 4. der Niedersehlag sofort eintrat; 5. nicht sofort, jedoch nach kurzem Stehen bei Zimmertemperatur ein bIiederschlag entstand. Die Niederschli~ge bestanden aus Calciumoxalat. Nach diesen Versuchen wird somit die Parabansi~ure bei Gegenwart yon Soda und hmmoniak selbst bei niedri~erer Temperatur, als sie beim Eindampfen auf dem Wasserbad erzielt wird, sowie bereits beim einfachen Erwi~rmen einer wassrigen L6sung derselben auf 60 o C. sehon in Oxalshure umaesetzt; da nun auch eine ammoniakalische Parabanshurel6sung far sich bereits nach einigen Stunden verandert wird und mit Chlorcalciuml6sung einen bTiederschlag yon Calciumoxalat gibt, si~mmtliche Versuchsfiussigkeiten aber, besonders diejenigen mit thierischen Organen, nm die Eiweissstoffe zu entfernen, erwgtrmt oder aufgekocht werden mussen, so kSnnen die Resultate der Versuche uber die Einwirkung yon Fermenten auf Parabans~ture nur sehr zweifelhafter Natur sein, und habe ich aus diesem Grunde solche nieht ausgefuhrt; es lasst sich also leider experimentell nicht nachweisen, in welchen Organen die Bildung yon Oxalurs~ture oder Oxalsaure aus Parabansaure vor sich geht, vielleicht schon im Magen durch die Salzsaure oder im alkalischen Dunndarm ? Succinimid. Bei diesen Versuchen machte ich anfangs die Erfahrung, dass bei allen Aetheraussch~ttelungen der Haupt- und Controlversuche 284 M. Gonnermann: stets ein stickstoffhaltiger Rtmkstand erhalten wurde; es musste mich dies befremden, da bei dem Zusatz yon Phosphorsaure etwa gebildete Succinaminsaure in den Aether nieht tlbergeben konnte, weil diese in Aether und Alkohol unloslich ist, und die erhaltenen Krystalle die rhombischen T~tfeln des Succinimids zeigten. Es lag also die MOglichkeit nahe, dass das Suecinimid sich schwerer in Aether und A]kohol 10st, als in der Literatur angegeben ist, und daher der Verdampfungsrackstand nicht genugend mit Aetheralkohol ausgeschiittelt worden war. Um die Gegenwart der Bernsteinsaure nachzuweisen, benutzte ich die Fallbarkeit yon EisenoxydlOsungen dutch ihre Neutralsalze in der Siedhitze; den vom Aetheralkohol ungel6sten, von Succinimid vollig befreiten, getroekneten Ruckstand extrahirte ich mit 55 %igem Alkohol, welcher naeh meinen bereits erwi~hnten u kaum eine Spur des sehr stOrend wirkenden Fluornatriums aufnimmt, dagegen Natriumsuceinat leicht 10st, verdampfte alas Filtrat zum Theil, neutralisirte und prtffte mit Eisenehloridli~sung: eine beim Erhitzen auftretende gallertartige Ausscheidung konnte nunmehr nur dutch die Gegenwart yon Bernsteinsi~ure bedingt sein, d e n n S u c e i n a m i n saute farbt zwar EisenchloridlOsung dunkel blutroth, lasst jedoch kein basisches Salz beim Erhitzen ausf a l l e n , und S u c c i n i m i d g i b t m i t E i s e n l 0 s u n g a b e r h a u p t keine Farbenreaction. Versuehe mit thierischen Fermenten. P e p s i n. Das Versuchsgemisch wurde unter Zusatz yon etwas NatriumbicarbonaV zur Troekne eingedampft. Bernsteinsaure war nicht nachzuweisen, wohl aber trat eine Dunkelfarbung der EisenchloridlOsung ein, so dass Succinimid durch Pepsin nut in Succinaminsaure ubergefuhrt wird. T r y p s i n . Der alkoholische Verdampfungsrilekstand, mit sehr wenig Wasser aufgenommen, wurde durch verdunnte Salzsaure genau neutralisirt; auf Zusatz yon Eisenchlorid und Erwarmung entstand eine tief braunrothe Flilssigkeit, welche beim Kochen keine Ausscheidung gab; eine andere Probe entwickelte mit Kalilauge Ammoniak, so dass ich annehmen mOehte, dass auch durch Trypsin keine Abspaltung yon Bernsteins~ure aus Suceinimid bewirkt, sondern nur Succinaminsaure gebildet wird. P t y a l i n ist ohne Einwirkung. Ueber die Verselfb~rkelt eimger Saureimide (J)iamlde) u. Aminsauren etc. 285 u mit Histozymen. Der filtrirte, unter Zusatz yon etwas Natriumbicarbonat eingedampfte Digestionsauszug wurde eingedampft, der Rt~ckstand dutch Aetheralkohol ~om Succinimid befreit, das Ungeloste nach Verdampfen der Flussigkeit mit 55 % igem Alkohol ausgezogen und nach Neutralisation der LSsung auf die Umsetzungsproducte weitergepruft, wie bei den Enzymen angegeben. Leider st~rte der Extractionsfarbstoff in der L6sung die Farbenreaction etwas. Leber. Die mbglichst verdannte LSsung wurde nach dem genauen Neutralisiren mit EisenchloridlSsung versetzt und bis zum Sieden erhitzt; es entstand hierbei ein dunkelrother~ gelatin6ser Niederschlag, und da eine Probe, mit starker Kalilauge gekocht, kein Ammoniak entwickelte, durch die entwickelten Dampfe weder Curcumapapier gebr;tunt noch rothes Lackmuspapier geblaut wurde, so kann auf Abwesenheit yon Succinimid und Succinaminshure sicher geschlossen werden: es ist durch das Leberhistozym aus Suecinimid Bernsteins~ure abgespalten worden, welche auch andererseits beim Ausschutteln der mit Phosphors~ure ~bers~ttigten L6sung mit Aether und Verdampfen desselben in Krystallen erhalten wurde. N i e r e. Die mit Aether und 55 % igem Alkoho! ausgezogenen Ruckst~mde wurden in Wasser gel6st und mit Eisenchloridl6sung versetzt: Succinaminsaure und Bernsteins~ure waren nicht nachzuweisen; Nierenhistozym ist ohne Wirkung. Aus den erw~hnten Versuchen yon K o e h n e ergibt sich Mar, dass bei Futterungsversuchen mit Hunden nur ein sehr geringer Theil des in den KSrper eingebrachten Materials denselben unzersetzt passirL der gr6sste Tell dageaen im ThierkSrper in Bernsteinsaure umgewanclelt wird und diese selbst dann, wie die Untersuchungen yon v. L o n g o l ) und E r d m a n n g ) zeigen, in demselben vollkommen zerfallt. Versuche mit pflanzliehen Maltin, Invertin Succinimid. uud E m u l s i n 1) Zeitschr. f. physiol. Chemie Bd. 1 S. 214. 2) Ebenda~ Anmerkung. Fermenten. sind ohne Einwirkung auf 286 N. Gonnermann: Suecinaminslture. Durch die soeben mitgetheilten, mit Succinimid angestellten Yersuche ist erwiesen, dass die meisten Fermente wirkungslos sind, die wirksamen aber, Pepsin und Trypsin, dieses Imid zwar spalten, aber auch nut bis zur Bildung yon Succinamins~ure. So sind denn Prtifungen mit diesen beiden zuletzt genannten Fermenten naturgemi~ss t~berfli~ssig; die mit den ubrigen waren voraussichtlich erfolglos, doch habe ich sie der Vollstandigkeit wegen noch angestellt. Ebenso habe ich auch noeh Versuche mit Leberhistozym gemacht, um festzustellen, ob Succinamins'~ture nicht allein im Entstehungszustande, sondern auch als geschlossenes Molektil zerlegt wird. Zur Darstellung der Succinamins~ture hat sich mir am meisten folgendes Verfahren bewahrt: Ein Gemisch yon 30,0 Succinimid, 48,5 -- die ~iquivalente M e n g e - Barythydrat und 200,0 friseh ausgekoehten destillirten Wassers wurde im E r 1e n m a y e r- Kolben mit eingesenktem Thermometer bis 75 o 24 Stunden erwi~rmt; da bei dieser Temperatur sich kein Ammoniak entwiekelt, so ist die Einwirkung des Baryumhydroxyds auf Succinami~si~ure weniger energisch, als bei Anwendung yon Calciumhydroxyd beobachtet. Vortheilhaft ist es, sich erst Barytwasser frisch darzustellen~ da sich immer wieder etwas Baryumcarbonat ausseheidet, und in das Filtrat bei 75 o C. das Sueeinimid einzutragen; dasselbe lost sich sofort, und eine spatere schwache Trubung yon Baryumcarbonat kann man unberiieksiehtigt lassen. Zum Abseheiden der S~iure aus dem succinaminsauren Baryum soll elne geringe Menge des Salzes un~ zerlegt bleiben; ich entnahm daher der alkalischen LSsung 30 ccm, fNlte die ubrige Menge mit verdunnter Schwefelsi~ure genau aus und fugte die zuruckbehaltene Menge zu; alas Filtrat wurde vorsichtig concentrirt, in eine grosse Menge mit etwas Aether vermischten Alkohols unter stetem Ruhren in dunnem Strahl einge~.oossen und die Ausscheidung einige Tage zur volligen Kli~rung beiseitegestellt. Das mit Aetheralkohol ausgewasehene feine weisse Pulver gab in Wasser gelSst auf Zusatz yon Eisenchloridl6sung beim Erwi~rmen eine fief dunkle Farbung, beim Kochen jedoeh keine Ausscheidung, so dass das Praparat frei von Bernsteinsaure war. Diese Reaction benutzte ich zur Erkennung der Succinamins~ure fur sich wie auch neben Bernsteinsaure; eine dunkelgefi~rbte Flussigkeit mit einem gallertigen Niederschlag beim Kochen enthfilt beide Sauren; U eber die Verseifbarkeit einiger S~ureimide (Diamide)u. Aminsfiuren etc. 287 ist die Fliissigkeit dagegen ~ber dem b~iederschlag farblos, so war nur Bernsteinsi~ure zuge~en: denn Succinimid gibt mit Eisenchlorid keine Reaction. Da sich bernsteinsaures Natrium, wie mich Versuche ]ehrten, leicht in 55 % igem Alkohol aufl0st, Fluornatrium dagegen wie schon erwi~hnt, nicht, so bedarf es demgemliss nur des Ausschuttelns tier bei den Versuchen erhaltenen Verdampfungsrtmkstiinde mit verdunntem Alkohol~ um in dieser LSsung nach genauer bTeutralisation direct auf Bernsteinsi~ure prufen zu kSnnen, ausserdem verdampfte ich die alkoholische LSsung auf dem Wasserbad, nahm den Rilckstand mit wenig Wasser auf und versetzte mit viel Alkohol, filtrirte die Ausscheidung ab, wusch mit Alkohol nach, 15ste in Wasser und prufte weiter. Versuche mit thierischen Ueber P e p s i n und T r y p s i n g]eichen. P t y a 1i n war wirkungslos. Versuche Fermenten. ist das oben Gesagte zu ver- mit Histozymen. Hier kam es darauf an, in Folge des stark gef~rbten Digestionsauszuges, die Eisenreactlon deutlich zu machen; es wurde daher der alkalische Verdampfungsrt~ckstand zunachst mit Aether ausgeschiittelt, um Fett zu beseiti~en; den Ruckstand zog ich mit 55 %igem Alkohol aus, um bTatriumsuccinat in L0sung zu bekommen, und nach mehrmaligem WiederlSsen, Schutteln mit Aether u. s. w. blieb schliesslieh ein schwach gelblichgefarbter Ruckstand, welcher, in Wasser gelSst und neutralisirt, mit EisenlSsung gepruft werden konnte. Leber. Es trat beim Aufkochen der Probeflt~ssigkeit mit Eisenlosung eine starke Dunkelfi~rbung ein, jedoch keine Fi~llung, so dass ich annehmen muss, dass ein Theil der Succinaminsaure noch unzersetzt vorhanden ist, ein Theil derselben jedoch in Bernsteinsi~ure ilbergefuhrt, diese abet zweifellos weiterzerlegt ist, weil Succinimid durch Leber in Bernsteinsa.ure ubergefuhrt wird. N i e r e. Beim Erhitzen mit Eisenchloridlosung entstand keine gelatinSse Ausscheidung~ so dass eine Bildung yon Bernsteinsi~ure nicht anzunehmen ist. Versuche mit pflanzlichen Die pflanzlichen Fermente I n v e r t i n , sind wirkun~slos auf Succinammsi~ure. Fermenten. M a l t i n und E m u l s i n 288 M Gonnermann: Dibenzamid. Dieses Pr~parat musste erst dargestellt werden, und sollen nach B a r t h und S e n h o fe r 1) in ein Gemisch von 7 Theilen rauchender SchwefelMure und 4 Theilen wasserfreier Phosphorsaure allm~thlich 7 Theile Benzonitril eingetragen, die Masse durcbgeschuttelt und nach einigen Stunden mit viel Wasser versetzt werden. Die Krystalle krystallisirt man aus Alkohol um. Nach dieser einfach erscheinenden Vorschriff kommt man nicht zum Ziel, denn dem dick-61igen Sauregemisch sind die vorgeschriebenen Mengen Benzonitril ,,allm~thlich" - - d. h. doch nach und nach oder in kurzen Pausen - - n i c h t zuzufugen, weil bereits nach der Zusetzung der H~lfte an Benzonitril die Gesammtmischung steinhart wurde. Den zweiten Versuch mit 50,6 Benzonitril flihrte ich mit besserem Erfolg so aus, dass ich das Sauregemisch in einen starken Porzellanm~rser gab und unter stetem Ruhren ununterbrochen in dfinnem Strahl die vSllige Menge Benzonitril zufagte~ hierbei trat nattirlich eine Erw~rmung ein~ welche etwas Benzonitril verfluchtigte, allein das Gemisch konnte his zuletzt, wenn auch schwierig durch seine steife Consistenz, gut durchgerahrt werden, - - e i n ,,DurchscMtteln", wie ich es zuerst nach tier Originalvorschrift im Kolben vornshmen wollte, ist geradezu unm6glich. Die Masse war bald steinhart geworden, blieb bis zum nachsten Tag stehen, liess sich sehr schwer klei~stuckenweis aus dem MSrser bringen, -- Porzellanschalen gehen hierbei sicher entzwei; die Stucke zerrieb ich rein, fugte wenig Wasser hinzu und erzielte dadurch nach einiger Zeit ein gleichm~ssiges, syrupOses Gemisch, welches, noch etwas mehr verdunnt, unter Ruhren in die 300fache Menge Wassers an Benzonitril eingegossen, schnell eine milchige Flilssigkeit gab, aus sehr feinen Fl~ckchen yon Dibenzamid bestehend. Die vollstandige Ausscheidung des Productes dauert mindestens drei Tage; der abfiltrirte, ausgesusste und abgepresste Niederschlag wurde in heissem Alkohol gelost, auf dem Wasserbad mit warmem Wasser bis zur eintretenden Trtibung versetzt und die Flussigkeit zur Krystallisation beiseitegestellt; die Mutterlaugen wurden weiterverarbeitet, als noch farblose, geruchfreie Krystalle sich ausschieden. 1) Berichte d. chem. Gesellsch. Bd. 9 S. 975. Ueber dm Velseffba~keltem,ger Samelm~de(Dlamlde) u. Aminsauren etc. 289 Die Ausbeute betrug gegen 16 % des Benzonitrils, -- immer eine ~eringe. UnlSslich in Wasser, leicht 10slich in Alkohol, zeigt-das Benzdiamid unter dem Mikroskop die der Benzo~s~ure ahnlichen Fahnengruppen wie auch grosse Nadeln~ so dass der sichere :Nachweis der Saure erschwert ist, wenn nicht die rhombischen Platten mit den abgesetzten Lfingsseiten vorhanden sind. Alle Versuchsgemische wurden mit etwas Natriumbicarbonat eingedampft~ der Rt~ckstand mit Wasser wieder aufgenommen, das Flltrat eingeengt, mit Phosphorsaure ubers~ttigt, mit Aether 5fter ausgeschuttelt, diese L6sung vorsichtig verdampft: das mikroskopische Bild sowie die Eisenreaction gaben mir die Sicherheit yon der Gegenwart der Benzo~shure oder Hippursaure. Versuche mit thierischen Fermenten. P e p s i n . Beim Verdampfen des atherischen Auszuges auf dem Deckglas zeigten sich federfahnenartige Gebilde, die wohl auf Benzc~s~ure schliessen liessenl; nach dem vSlligen Eindampfen im SchMchen resultirten neben den grossen, balkenf6rmigen Krystallen des Dibenzamids~ auch die Tafeln der Benzo~shure; es wird sonach das Dibenzamid dutch Pepsin zum Theil in Benzo~s~ture ubergefuhrt. T r y p s i n ; eine Verseifung des I)ibenzamids war nicht eingetreten. P t y a 1i n ist ohne Einwirkung. Versuche mit Histozymen. L e b e r. Der atherische Auszug ~ erhalten aus dem mit Phosphorshure angesauerten R~ckstand~ gab beim Verdampfen tafelf~rmige Krystalle, die als Benzo~saure angesehen werden konnten; beim Kochen mit starker Lauge entwickelte sich jedoch auch Ammoniak, welches rothes Lackmuspapier blaute; desshalb wurde der u dampfungsruckstand mit verd0nnter SodalSsung l~ngere Zeit digeri~t, die Flussigkeit abfiltrirt, mit Phosphorsaure abers~ttigt, mit Aether ausgesch~lttelt und dieser verdampft: es erschienen nunmehr die charakteristischen Krystalle der Benzo~saure mit ihren abgesetzten L~ngsseiten wie auch gekreuzte Complexe der Hippursaure. Ein anderer Theil der Flussigkeit wurde mit einem Tropfen Eisenchlorid15sung versetzt und erhitzt: es entstand eine tier blutrothe Farbung, welche meiner Ansicht nach in diesem Fall nur auf die Gegenwart yon Benzo~saure zurt'lckzufuhren ist; eine isabellfarbene Ausscheidung yon basischem hippursaurem Eisen trat allerdings nicht ein. E. P f l ~ . g e r , Archly ffir Physiologte. Bd 95. ~0 290 M. Gonnermann: Es wirkt also das Leberhistozym auf Dibenzamid in gleicher Weise verseifend ein wie auf Benzamid. N i e r e . Durch das Nierenhistozym wird Dibenzamid gleiehfalls in Benzo~saure ubergefuhrt. Aus den Ffitterungsversuchen yon K o e h n e geht hervor, dass das Dibenzamid im ThierkSrper unter Bildung yon Benzo~saure zersetzt und diese als Hippursiiure ausgeschieden wird. Versuche mit p f l a n z l i c h e n Fermenten. Bei den Einwirkungen yon I n v e r t i n , M a l t i n und E m u l s i n waren Verseifungsproducte nicht nachzuweisen. Disalicylamid. Dieses Pri~parat stellte ich nach der Vorschrift yon S c h u l e r u d 1) dar durch Ueberleiten von Salzsi~uregas t~ber in einem Verbrennungsrohr vertheiltes und langsam erhitztes Salicylamid. Das erstarrte Umsetzungsproduct, eine gelbliche Masse, wurde mit warmem Wasser so lanze verrieben und abgeschwemmt, bis eine Probe mit sehr verdtinnter, fast farbloser, dutch einige Tropfen Salzsi~ure angesi~uerter t~isenchloridlOsung keine Blaufarbung mehr gab. Disalicylamid ist in Wasser unlbslieh~ sehwer l(islich in Aether, ziemlich leicht in heissem Alkohol; die alkoholische LSsung wird durch Eisenchlorid roth gefarbt. Zusatz yon Eisenchlorid zu in Wasser vertheiltem Disalieylamid ruft keine Fi~rbung hervor~). Den Nachweis yon gegebildeter Salicyls~ure konnte ich auf gleiche Weise wie bei Salicylamid ausftihren, da auch das Disalicylamid in concentrirter Ammoniumaeetatlosung nicht 16slich ist; doch ich zog vor, die Versuchsgemische zum Theil nach benothigter Zugabe yon Natriumcarbonat einzudampfen, das Natriumsalicylat dutch Wasser auszuziehen, die filtrirte LOsung mit Phosphorsi~ure zu ubersiittigen und den Verdampfungsruckstand auf S~lieyls~mre zu prtffen. Versuche mit thierischen Fermenten. P e p sin. Der Aetherruckstand wurde rein zerrieben und mit concentrirter AmmonaeetatlSsung unter Zusatz einiger Tropfen Salz1) Journal f. l'r. Chemie Bd. 2 H. 2 S. 289. 2) B e l l s t e i ~ l Bd. 2 S. 1499. Ueber die Verseifbarkeiteiniger Saureimide(Diamide)u. Aminsaurenetc. 291 saure vermiseht, diese LSsung mit Wasser verdiinnt, das Filtrat mit Eisenchlorid g e p r u f t in diesem Falle konnte allein Salicylsaure zugegen ~sein - - ; es entstand eine tiefblaue Farbenreaction, so dass ich annehmen musste, dass das Disalicylamid durch Pepsin in Salicylsaure tlbergefuhrt worden ist. Allein, der Controlversuch mit gekochtem Pepsin gab bei gleichem Verfahren die gleiche, wenn auch scheinbar schwachere Farbenreactioni Nach dieser auffallenden Erscheinung schien es mir mSglich, dass die - - wenn auch sehr geringe -- Menge der in beiden Fallen zugesetzten Salzsaure bereits Salicylsaure abgespaltet haben ki~nnte. Nun hatte ich in den gleichzeitig anoestellten T r y p s i n v e r s u c h e n beobachtet, dass in beiden Fallen, also auch bei Verwendung yon gekochtem Trypsin, Salieylsaure gebildet wurde. Das fi~hrte mich denn zu der Vermuthung, die Spaltung werde durch das kohlensaure Natrium veranlasst, welches ja den Trypsin-Versuchsgemischen yon vornherein zugesetzt, in den Pepsinversuchen aber in gewohnter Weise zum Neutralisiren benutzt worden war, in dem letzteren Falle nicht ohne Vermeiden eines kleinen Ueberschusses yon Soda. In der That zeigte sich, dass auch ganz schwache SodalSsung allein das Disalicylamid spaltet, wenn auch, wie dies bei der UnlOslichkeit des Amids begreiflich ist, nur zu emem kleinen Theil. Ob das Pepsin gar nicht auf Disalicylamid einwirkt, war nun freilich auch nicht entschieden; ich mSchte aber die Einwirkung schliesslich doch als n i c h t ~orhanden bezeichnen, (ta Aether aus den sauer gebliebenen Versuchsgemischen keinen Eisenchlorid bli~uenden Stoff aufnahm. Mit T r y p s i n sind nach dem oben Mitgetheilten Versuche unmSglich. P t y a l i n war ohne Einwirkung, weil die Eisenreaction direct vorgenommen wurde und ausblieb. Versuche mit Histozymen. Bei der Empfindlichkeit des Disalicylamids Mkalien gegent~ber musste ich zunachst feststellen, ob nicht auch das alkalisch reagirende Fluornatrium bereits einwirken kbnnte. Das ist aber auch bei langerer Digestion offenbar nicht der Fall, ich brauchte also nicht auf die Verwendung des Fluornatriums in meinen wiederholten Versuchen zu verzichten; nur musste der Zusatz yon Soda bei den bTieren. versuchen foltbleiben; da in den Hauptversuchsgemischen und in den Controlversuchen mit gekochten Organen sich Salicylsi~ure nachweiscl~ 20* a 292 M. Gonnermann: liess, fuhre ich diese auffallende Erscheinung darauf zurfick, dass nach meiner Beobachtung sich das Disalicylamid bereits in 50 %igem Alkohol 16st und demgemftss Eisenchlorid eine Blaufarbung entstehen lassen musste. Die Gemische der neuen Versuchsweise wurden nach tier Digestion aufgekocht, das Filtrat bis zum Syrup eingedampft, diesem etwas hmmonacetat zugeftigt, die LSsung mit Wasser verdunnt und filtrirt: Disalicylamid muss als unlOslich zurilckbleiben, wahrend, eventuell unter Zusatz yon etwas Salzs~iure, Eisenchloridl6sung ~m Filtrat Salicylshure erkennen lassen wurde. L e b e r. Die Filtrate gab ich in sehr verd~innte, mit einigen Tropfen Salzsi~ure versetzte Eisenchloridl0sung: in beiden Versuchen trat nunmehr keine Blauung oder die Rothfi~rbung der Salicylreaction ein, so dass ich als erwiesen annehmen muss, dass das Leberhistozym auf Disalicylamid nicht verseffend einwirkt. ~iere. Diese Versuche waren also ohne Zusatz von Soda ausgefuhrt, und schien mir daher der Erfolg zweifelhaft. Desshalb iiberraschte es reich, dass bereits das Filtrat vom Hauptversuch mit Eisenchlorid eine kirschrothe Farbung gab, wiihrend bei dem Controlversuch diese Reaction nicht eintrat. Im weiteren Verlauf trat die Reaction im Hauptversuch sehr deutlich auf, und nehme ich als erwiesen an, dass Nierenhistozym aus Disalicylamid Salicylsiiure abspaltet, jedoch wohl in geringerem Maasse als bei Gegenwart von Soda. Versuche mit pflanzlichen I n v e r t i n , M a l t i n und E m u l s i n Soda wirkungslos. Fermenten. sind bei Abwesenheit von Phtalimid. Auch bei diesen Versuchen traten bedeutende Schwierigkeiten ein, da Phtalimid sowie die Phtalsi~ure fast gleiche LSsungsverhaltnisse und Krystallform zeigen. Die eingetretene Verseifnng sollte constatirt werden, wenn Phtalsiture sicher nachgewiesen worden ist; diese wfirde sich in den Versuchsl6sungen an Natrium gebunden vorfinden, wenn auch die bisher angewendete Methode eingehalten wird. L a n d s b e r g 1) gibt an, dass die Phtalimidsalze beim Behandeln mit Sauren Phtalimid wieder abscheiden; dagegen o 1) Liebig's Annalen Bd. 215 S. 181 Ueber die Verselfbarkelt einiger Saureimide (Diamide) u. Aminsauren etc. 293 hat K o e h n e 1) gefunden, dass aus einer wi~ssrigen PhtalimidnatriumlSsung -- dieses konnte sich wohl beim Eindampfen der VersuchslSsung mit Soda bilden - - auf Zusatz von Salzsi~ure bei Zimmertemperatur in 11/2 bis 2 Tagen, sogar nach ganz kurzer Zeit nicht Phtahmid, sondern Phtals~ure ausgeschieden wird. Nach dieser Beobachtung K o e h n e ' s ware es also absolut unmOglich, genau nachzuweisen, ob die gefundene Menge Phtalsaure wi~hrend der Einwirkung der Fermente und Histozyme aus Phtalimid entstand oder bereits beim Zerlegen des Phtalimidnatriums durch Phosphorsi~ure abgeschieden worden ist; aber es bildet sich, wie ein besonderer u zeigte, beim Digeriren und Eindampfen des Phtalimids mit Soda die erwfihnte Natriumverbindung nichtl es schied sich aus dem Aetherauszug der mit Phosphorsi~ure stark angesauerten Rucksti~nde immer wieder nur Phtalimid aus. Es stand also nichts im Wege, bei den Versuchen selbst oder nachtraglich Soda zu verwenden. So wurden denn die Gemische unter Zusatz "con etwas Natriumbicarbonat auf dem Wasserbad eingedampft~ mit 93% ioem Alkohol, welcher auch phtalsaures Natrium 15ste, ver. rieben und mit einer grSsseren Menge 24 Stunden unter haufigem Umschutteln digerirt, das Filtrat eingedampft, der Rt~ckstand eventuell in Wasser gelSst, das Filtrat mit Phosphorsi~ure tlbersattigt, durch Aether ausgeschuttelt und dieser verdampft: der Ruckstand konnte nur Phtalsi~ure sein. Versuche mit thierischen Fermenten. P e p s i n. Beim Verreiben des Verdampfungsrt~ckstandes mit 93 % igem Alkohol entstand bei dem Hauptversuch eine milchige Trubung, aus welcher sich dann eine weisse Ausscheidung absetzte; bei dem Controlversuch trat diese Erscheinung nicht ein, sondern es blieb bei geringer Trubung des Alkohols ein sandiger, sich schnell absetzender Ruckstand. Die auf dem Wasserbad eingedampften Filtrate gaben einen weingelb gefarbten Ruckstand; in wenig Wasser aufgenommen, mit etwas Phosphorsiiure versetzt, entstand bei dem I-Iauptversuch eine sandige Ausscheidung, wi~hrend bei dem Controlversuch mit gekochtem Ferment die L~sung klar blieb. Der beim Yerdampfen des ersten Aetherauszuges erhaltene Rt~ckstand wurde nochmals in Aetheralkohol gel6st, und aus dieser LSsung schieden 1) Dissertation. Rostock 1894. 294 1~. Gonnermann: sich sternfiirmig gruppirte, faserige Krystalle wie auch lange Nadeln aus, welche denen entsprachen, die aus einer alkobolischen LSsung yon reiner Phtalsiiure erbalten wurden. Pepsin filhrt Phtalimid in Phtalsi~ure fiber. T r y p s i n. Die erhaltenen Krystalle bestanden theils aus Phtalimid -- es entwickelte sich beim Kochen mit Lauge Ammoniak--, theils aus Phtalsiiure, well eben ersteres etwas in Wasser 15slich ist. Im Controlversuch land sich nur etwas Pbtalimid; auch Trypsin wirkt verseifend. P t y a 1i n war ohne Einwirkung. Versuche mit Histozymen. Den Versuchsgemischen wurde nach dem Erkalten, wenn ni~thig etwas ~atriumbicarbonat bis zur schwach alkaliscben Reaction zugeftigt und die Flussigkeit sechs Stunden unter iifterem Schfitteln stehen gelassen; das weitere Verfahren war wie bei den Fermenten. L e b e r . Der Verdampfungsrtlckstand war gef~rbt und wurde nochmals mit Alkohol ausgezogen; beim langsamen Verdunsten resultirten vorherrschend die langen Nadeln der Phtalsaure; da jedoch beim Kocben mit Lauge sich Ammoniak entwickelte, so ist es zweifellos, dass auch Phtalimid zugegen war und durch das Leberhistozym Phtalimid zum grbssten Theil in Phtals~ure fibergefi~hrt wird. ~iere. Der i~therische Verdampfungsruckstand war i~usserst gering; in dem verbliebenen gelblichen Syrup waren nur sparliche Phtalsi~urenadeln zu finden, auch bei erneutem Aufl0sen in heissem Alkohol und Verdunsten desselben; ich mSchte daher annehmen, dass die in der Leber bereits entstandene Phtals~ture wi~hrend des Passirens der hmieren vSllig zerstSrt wird. Vergleicht man diese Erscheinungen mit den Resultaten, welche K o e h n e bei seinen Futterungsversuchen erhielt, sowie mit den Berichten J u v al t a ' s 1) uber die Zerstbrbarkeit der Phtalsaure nn ThierkSrper, so wird meine Ansicht uber die Wirkm~g des bTierenhistozyms Besti~tigung finden. 1) Zeitschr. f phys. Chemie Bd. 12 S. 26. U e b e r die Y e r s e i f b a r k e i t e i n i g e r S a u r e l m i d e ( D i a m i d e ) u. A m i n s a u r e n etc. Versuche mit pflanzlichen 295 Fermenten. I n v e r t i n und M a l t i n sind wirkungslos. E m u l s i n. Die beiden Versuehe unterschieden sich bereits beim hufl6sen der ~ttherischen Verdampfungsrilcksti~nde in Wasser; bei dem Hauptversuch entstand eine Mare, gelbliche LSsung, withrend bei dem Controlversuch mit gekochtem Emulsin eine milehige Trgtbung eintrat; beim Ansauern mit Phosphorsaure und Ausscht~tteln mit Aether waren wieder getrennte, Mare Schichten vorhanden. Bei dem Kontrolversuch war Phtalimid unverkennbar zu constatiren, wi~hrend aus dem Hauptversuch sich erst nach einigen Tagen Phtalsi~urekrystalle ausschieden; es wirkte also das Emulsin i~hnlich wie auf Acetamid und Formanilid verseifend auf Phtalimid ein. - Zum Schluss lasse ich noch einmal eine Gesammtubersicht der Resultate aus meinen beiden Versuchsreihen folgen. In den horizontalen Reihen fallen sammtliche Oxals~mrederivate aus, wie an einer anderen Stelle bereits hervorgehoben. In den verticalen Reihen fallen die Enzyme Ptyalin, Invertin und Maltin aus, u n d e s ist wohl mit der griissten Wahrscheinliehkeit anzunehmen, dass auch die nicht untersuchten Enzyme dieser Gruppe die Siiureimide nicht zerlegen. Das verschiedene Verhalten der anderen Enzyme lasst sich einstweilen wohl noeh nicht in ein einfaches Bild zusammenfassen; der Wirkungsbezirk eines jeden Fermentes wird sich erst S a u r e a m i d e und -Anilide, Imlde und Aminsauren J Formamid Acetamid . . . . . . + Oxamid . . . . . . Succinamid . . . . . Benzamid . . . . . . Sahcylamid . . . . . + Formanilid . . . . . Acetanilid . . . . . . Oxanilid . . . . . Benzanilid . . . . . Oxammsaore . . . Succinimid .... Succinaminsaure... Dibenzamid . . . . . Disalicylamid .... Phtalimid . . . . . + ~ T P a r a b a n s a u r e : Bel d i e s e r V e r b i n d u n g sicher bestimmen. T - + ,7 + + < + < + T T l a s s t sich i das versei ende -Organ + nieht 296 )/i. Gonnermann: Ueber die Verseifbarkeit einiger Shureimide etc. nach PrUfung an den verschiedenartigsten KSrpern bekannter Constitution feststellen lassenl); hervorgehoben kOnnte wohl die Leistungsfi~higkeit der Leber werden; ihr kommt die der Niere sehr nahe, doch ist Beides im Allgemeinen bekannt aus frtiheren Untersuchungen. 1) Vgl. hierzu die Arbeiten yon Fischer, Zeitschr. f. phys. Chemm Bd. 26 S. 71. 1898. - - Chem. Ber. Bd. 27 S. 2992.
https://openalex.org/W2952234160	https://europepmc.org/articles/pmc6587676?pdf=render	English	null	A Predominance of Clade 17 Candida albicans Isolated From Hemocultures in a Tertiary Care Hospital in Thailand	Frontiers in microbiology	2,019	cc-by	8,260	A Predominance of Clade 17 Candida albicans Isolated From Hemocultures in a Tertiary Care Hospital in Thailand Linh Thi Truc Pham, Sujiraphong Pharkjaksu, Piriyaporn Chongtrakool, Kamol Suwannakarn and Popchai Ngamskulrungroj* Linh Thi Truc Pham, Sujiraphong Pharkjaksu, Piriyaporn Chongtrakool, Kamol Suwannakarn and Popchai Ngamskulrungroj* Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok Noi, Thailand Candida albicans is one of the most common human fungal pathogens. Candidemia has signiﬁcant mortality globally. No epidemiological study of C. albicans based on multilocus sequence typing (MLST) has been conducted in Thailand. Therefore, MLST was used to study the molecular epidemiology of C. albicans blood strains in a large Thai teaching hospital. In vitro virulence phenotypes and antifungal susceptibility testing by broth microdilution were also conducted. Forty-six C. albicans blood strains from 37 patients were collected from the Department of Microbiology, Siriraj Hospital, in 2016 and 2017. Most patients (71.8%) were more than 60 years old, and the case fatality rate was 54.8%. The male-to-female ratio was 5:3. Thirty-four diploid sequence types (DSTs), including six new DSTs, were identiﬁed, with DST2514 (8.7%) and DST2876 (8.7%) as the most common DSTs. Strains were clustered into nine clades. Unlike other studies of C. albicans blood strains in Asia, clade 17 was the most common (13 strains, 28.3%). Sequential allelic changes were evident in sequential strains from one patient. All strains produced phospholipase and hemolysin, while none produced proteinase. The ability to form bioﬁlm was found in 82.6% of the strains. Clade 17 strains showed signiﬁcantly stronger hemolytic activity than non–clade 17 strains (69.2% versus 27.3%; p = 0.022). However, no signiﬁcant association existed between clades and patient mortalities. All were susceptible or wild type to anidulafungin (MIC range = 0.015– 0.12 and GM = 0.030), micafungin (MIC range = ≤0.008–0.015 and GM = 0.008), caspofungin (MIC range = 0.008–0.12 and GM = 0.036), and amphotericin B (MIC range = 0.25–0.5 and GM = 0.381). Only one strain was resistant to voriconazole (MIC range = ≤0.008 to ≥8 and GM = 0.010) and ﬂuconazole (MIC range = 0.12– 16 and GM = 0.398). In conclusion, a high prevalence of clade 17 C. albicans blood strains was found in Thailand, in contrast to other Asian countries. This unique ﬁnding might be explained by the strong hemolytic activity that is required for bloodstream infection of C. albicans. Reviewed by: Reviewed by: Hamid Badali, Mazandaran University of Medical Sciences, Iran Peiying Feng, Sun Yat-sen University, China *Correspondence: Popchai Ngamskulrungroj popchai.nga@mahidol.ac.th Specialty section: This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology Fungi and Their Interactions, a section of the journal Frontiers in Microbiology Received: 03 January 2019 Accepted: 13 May 2019 Published: 14 June 2019 Keywords: Candida albicans, clade, multilocus sequence typing, virulence factors, antifungal susceptibility, candidemia ORIGINAL RESEARCH published: 14 June 2019 doi: 10.3389/fmicb.2019.01194 Edited by: Kin-Ming (Clement) Tsui, Weill Cornell Medicine – Qatar, Qatar Kin-Ming (Clement) Tsui, Weill Cornell Medicine – Qatar, Qatar MLST Analysis S a ys s The seven housekeeping genes including AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b were used for MLST according to a previously published method (Bougnoux et al., 2002). Brieﬂy, PCR mixtures were prepared in 50 µl reaction volumes containing DNase–RNase free water, 10 × buﬀer, 25 mM MgCl2, 2.5 mM dNTPs, 10 µM for each of the forward and reverse primers of each gene, 0.25 µl of 5 U/µl Taq DNA polymerase (Thermo Scientiﬁc, United States), and genomic DNA. The condition was set up with a denaturation step at 95◦C for 5 min, followed by 35 cycles of 95◦C for 40 s, annealing at 50◦C for 40 s, extension at 72◦C for 1 min, and a ﬁnal extension step at 72◦C for 4 min. Approximately 50 ng of DNA was sequenced using each forward and reverse primer on a DNA analyzer (First Base Company, Singapore). The generated sequences were manually edited using MEGA6 software and aligned using Clustal W. The allelic proﬁle (allele number) for each gene and allele combination (diploid sequence type, DST) for the seven loci of each isolate was assigned, or new DST numbers were obtained from the C. albicans MLST database1. For cluster analysis, because the base at each polymorphic site can be homozygous or heterozygous, the nucleotide bases were manually edited according to a previous publication (Tavanti et al., 2005). The bases of each taxon were doubled when the site was homozygous and written in pairs when the site was heterozygous. Relationships among concatenated sequences of the seven loci of each strain were determined using cluster analysis using the unweighted pair group method using their arithmetic averages (UPGMA) and the neighbor joining (NJ) method determined by p distance with pairwise deletion of the MEGA 6 software. A bootstrap of 1,000 replications was used for the construction (Shin et al., 2011; Wu et al., 2017). Bootstrap Several genotyping methods have been used to study the molecular epidemiology of C. albicans. Multilocus sequence typing (MLST) has been widely used to study the molecular characterization of C. albicans based on the sequences of seven housekeeping genes (AAT1a, ACC1, ADP1, MPI1b, SYA1, VPS13, and ZWF1b) (Bougnoux et al., 2003; Afsarian et al., 2015). This method has identiﬁed 19 clades worldwide (Shin et al., 2011; Gong et al., 2018). A 2007 global study of 1,391 C. MLST Analysis albicans strains showed that clade 1 was the most common (33.6%) (Odds et al., 2007). In Asia, the second and third most common clades were clades 4 (8.8%) and 17 (8.2%) (Odds et al., 2007). When considering only blood strains, a 2016 Korean study of 149 C. albicans blood strains showed that clade 18 was the most common (18.8%), followed by clade 4 (15.4%) and clade 1 (14.8%). So far, no epidemiological study of C. albicans based on MLST has been conducted in Thailand. Therefore, MLST was used to study the molecular epidemiology of C. albicans strains of candidemia patients at Siriraj Hospital, including virulence factors and antifungal susceptibility proﬁles. Citation: Pham LTT, Pharkjaksu S, Chongtrakool P, Suwannakarn K and Ngamskulrungroj P (2019) A Predominance of Clade 17 Candida albicans Isolated From Hemocultures in a Tertiary Care Hospital in Thailand. Front. Microbiol. 10:1194. doi: 10.3389/fmicb.2019.01194 June 2019 \| Volume 10 \| Article 1194 Frontiers in Microbiology \| www.frontiersin.org 1 Thai Candida albicans Molecular Epidemiology Pham et al. of Microbiology, Siriraj Hospital, between April 2016 and November 2017. This study was approved by the Siriraj Institutional Review Board, no. SI 091/2016. Informed consent was not required in this work. All strains, collected from the same patients, were considered sequentially collected, as blood samples were taken from diﬀerent body sites at diﬀerent time points. Forty-six strains from 37 individual candidemia patients were collected. Available patient records were retrieved from the hospital registry. A healthcare-associated infection was deﬁned as an onset of candidemia symptoms and/or signs after 48 h of admission. As the hospital is a tertiary care referral center, patient origins varied. Most cases (81.25%; 26 of the 32 available patient records) were from central Thailand; 6.25, 6.25, and 6.25% of cases were from Eastern, Northeastern, and Western Thailand, respectively. The case fatality rate was calculated as the ratio of patient deaths to all candidemia cases in this study. All strains were identiﬁed using CHROMagar Candida chromogenic media (Oxoid, United Kingdom) and RapIDTM YEAST PLUS System (Thermo Scientiﬁc, United States) followed by single- colony subculture on Sabouraud dextrose agar (SDA; Oxoid, United Kingdom) to prepare pure cultures before performing any further analysis. 1https://pubmlst.org/calbicans/ INTRODUCTION Candida albicans is a member of the human ﬂora and is commonly colonized in the human digestive tract. C. albicans is an important cause of candidiasis (Bougnoux et al., 2006). It can cause infections that range from superﬁcial infections to life-threatening systemic infections. Approximately 75% of all women suﬀer from vulvovaginal candidiasis at one point in their life span, and C. albicans accounts for nearly 90% of such cases (Yazdanparast et al., 2015). C. albicans and other Candida species including C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei can travel through the bloodstream and together are causative agents in 90% of invasive candidiasis cases (Vaezi et al., 2017). Many risk factors contributing to candidiasis were reported, including host-related factors such as immunosuppressive disorders, neutropenia, age, or debilitating diseases, and healthcare-associated factors such as catheter use, surgical interventions, transplantation, or the use of antimicrobial drugs (Yapar, 2014; Diba et al., 2018). Virulence is deﬁned as the ability of a microbe to damage its host (Casadevall and Pirofski, 1999), and C. albicans possesses a wide range of virulence factors, including phenotype switching, adhesin expression, thigmotropism, bioﬁlm formation, and extracellular hydrolytic enzyme secretion, to facilitate tissue invasion and ultimately produce the diseases (Mayer et al., 2013). A recent trend showed increased resistance of non-albicans Candida to azole-based antifungal agents, echinocandins, and multiple classes of drugs (Wiederhold, 2017). However, C. albicans remains highly susceptible to most antifungal drugs (Bustamante et al., 2014; Posteraro et al., 2015; Doi et al., 2016; Tadec et al., 2016; Tan et al., 2016; Chapman et al., 2017). MATERIALS AND METHODS Study Sites and Subject Selection In vitro Virulence Study The test for C. albicans phospholipase activity was performed on egg yolk medium according to a previous study FIGURE 1 \| Genetic diversity inferred by UPGMA. Bootstrap support values above 75 for UPGMA (bold) and NJ method (italic bold) were indicated. Ref; reference DST, pt; patient number. Candida albicans strains’ proteinase activity was analyzed on bovine serum albumin medium by the modiﬁed Staib method (Staib, 1965; Sachin et al., 2012). Five microliters of a yeast suspension (108 cells/ml) of the tested and control strains was dropped on a medium containing 1.17% yeast carbon base (Becton Dickinson, United States), 0.2% bovine serum albumin (GoldBio, United States), 2% agar (Oxoid, United Kingdom), and 0.01% yeast extract (HiMedia, India) and then incubated at 37◦C for 2 days. The presence of a clear halo zone around the colony was recorded as evidence of proteinase activity. To measure hemolytic activity, a medium was prepared by adding 7 ml fresh sheep blood (Clinag, Thailand) to 100 ml SDA supplemented with 3% of enriched glucose (HiMedia, India) (Luo et al., 2001). Five microliters of a yeast suspension (108 cells/ml) of the tested and control strains was spotted onto the medium. The plate was then incubated at 37◦C for 48 h. The presence of a distinct translucent halo indicates positive hemolytic activity. The ratios of the diameter of the yeast colony to the total diameter of the colony plus the white precipitation zone, the clear halo zone, and the translucent halo zone were used to represent the level of phospholipase, proteinase, and hemolytic activity, respectively (Sachin et al., 2012). Bioﬁlm formation was determined using the 2,3- bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox anilide (XTT; Thermo Scientiﬁc, United States) reduction assay using 96-well microplates as indicated in previous studies (Yigit et al., 2011; Oz et al., 2012). Brieﬂy, yeast cells were adjusted to a 0.5 McFarland standard with yeast extract–peptone–dextrose medium. One hundred microliters of yeast suspension was seeded in 96-well plates and incubated at 37◦C for 48 h. The cells were then washed twice with phosphate-buﬀered saline to remove non-adhering cells. A 50-µl mixture of 1 mg/ml XTT and 10 µl of activation reagent, phenazine methosulfate (HiMedia, India), was added to the wells and further incubated in the dark for 2 h. Study Sites and Subject Selection Study Sites and Subject Selection All C. albicans strains were collected from positive hemocultures from a diagnostic microbiology laboratory of the Department All C. albicans strains were collected from positive hemocultures from a diagnostic microbiology laboratory of the Department June 2019 \| Volume 10 \| Article 1194 Frontiers in Microbiology \| www.frontiersin.org 2 Thai Candida albicans Molecular Epidemiology Pham et al. (Price et al., 1982). Strains were grown on SDA for 24 h before adjusting to a 0.5 McFarland standard (108 cells/ml). One hundred milliliters of medium containing 13.0 g SDA, 11.7 g NaCl (Merck, Germany) SDA, 11.7 g NaCl (Merck, Germany), 1.48 g CaCl2.2H2O (Merck, Germany), 2% agar (Oxoid, United Kingdom), and 10% sterile egg yolk (Merck, Germany) was prepared to determine extracellular phospholipase activity (Tsang et al., 2007). Five microliters of a yeast suspension (108 cells/ml) of the tested and control strains was dropped onto the surface of the egg yolk–containing plate and incubated at 37◦C for 48 h to measure a precipitation zone around the colony. Extracellular phospholipase activity of C. albicans was considered positive when a white precipitation zone was visible around the colony on the plate (Macfarlane and Knight, 1941). values of ≥75% were deﬁned as statistically signiﬁcant. Finally, clade numbers were assigned according to previous publications (Odds et al., 2007; Tsai et al., 2015; Wu et al., 2015; Gong et al., 2018) or by using the eBURST method as described previously (Odds et al., 2007). DSTs from clade numbers that were not presented in our strains were also included as reference DSTs for UPGMA and NJ analysis (Figure 1). Antifungal Susceptibility Testing Antifungal Susceptibility Testing Antifungal susceptibility testing of the C. albicans strains was conducted using a commercial microdilution method, Sensititre R⃝YeastOne R⃝part YO10 (SYO; Thermo Scientiﬁc, United States). The concentrations of antifungal drugs were: anidulafungin 0.015–8 µg/ml, micafungin 0.008– 8 µg/ml, caspofungin 0.008–8 µg/ml, amphotericin B 0.12–8 µg/ml, 5-ﬂuorocytosine 0.06–64 µg/ml, posaconazole 0.008–8 µg/ml, voriconazole 0.008–8 µg/ml, itraconazole 0.015–16 µg/ml, and ﬂuconazole 0.12–256 µg/ml, according to the manufacturer’s protocol. C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as reference strains. Brieﬂy, 20 µl of yeast suspension McFarland standard no. 0.5 (approximately 106 cells/ml) was suspended in 11 ml of YeastOne R⃝inoculum broth to give a ﬁnal working yeast suspension of approximately 1.5–2.5 × 103 cfu/ml. Then, 100 µl of working yeast suspension was added to each well using a multichannel pipetting device. The SYO panels were covered with adhesive seals and incubated at 35◦C for 24 h. If the positive control showed no yeast growth, the plate was incubated for an additional 24 h. Minimum inhibitory concentration (MIC) results for all test agents were read according to the manufacturer’s instructions. The criteria used to interpret the epidemiological cutoﬀvalues (ECVs) and clinical break points (CBPs) were based on Clinical and Laboratory Standards Institute (CLSI) guidelines: ECVs of anidulafungin = 0.12, micafungin = 0.03, and amphotericin B = 2; CBPs of anidulafungin, micafungin, and caspofungin: S ≤ 0.25, I = 0.5, R ≥1; voriconazole: S ≤0.12, SDD = 0.25– 0.5, R ≥1; and ﬂuconazole: S ≤2, SDD = 4, R ≥8 (Clinical Laboratory Standards Institute, 2012, 2016). RESULTS Kantarcioglu and Yucel, 2002; Li et al., 2003; Tsang et al., 2007; Hasan et al., 2009) as follows: zone of activity of phospholipase and proteinase (Price et al., 1982; Kantarcioglu and Yucel, 2002; Tsang et al., 2007): ≤0.69 = very strong, 0.70–0.79 = strong, 0.80–0.89 = medium, 0.90–0.99 = weak, and 1.00 = negative; hemolytic activity (Rossoni et al., 2013): < 0.64 = strong positive, 0.64 ≤to < 1 = positive, and 1.00 = negative; and bioﬁlm formation (Li et al., 2003; Hasan et al., 2009): O.D. value > GM (geometric mean) = high bioﬁlm formation, O.D. value ≤GM = low bioﬁlm formation, and O.D. value < 0.10 = negative bioﬁlm formation. In vitro Virulence Study The bioﬁlms were quantiﬁed at A490 and deﬁned as low bioﬁlm formation and high bioﬁlm formation by comparing the XTT activity of each strain to the geometrical mean as described previously (Li et al., 2003; Hasan et al., 2009). FIGURE 1 \| Genetic diversity inferred by UPGMA. Bootstrap support values above 75 for UPGMA (bold) and NJ method (italic bold) were indicated. Ref; reference DST, pt; patient number. Each tested virulence phenotype’s expression levels were classiﬁed according to their reference methods (Price et al., 1982; June 2019 \| Volume 10 \| Article 1194 Frontiers in Microbiology \| www.frontiersin.org 3 Thai Candida albicans Molecular Epidemiology Pham et al. Most Candidemia Cases Occurred as Hospital-Associated Infections and in Older Patients Clinical data were available from 32 of the 37 patients. All patients (100%) had one or more immunocompromised conditions, including receiving immunosuppressants or chemotherapy (10 patients, 31.3%), having diabetes mellitus (10 patients, 31.3%), being a newborn (2 patients, 6.2%), and old age (>60 years old) (23 patients, 71.8%). The case fatality rate was 54.8%. The male- to-female ratio was 5:3 (Supplementary Table S1). Most cases (31 patients, 96.9%) were hospital-associated infections except one who had Candida sepsis as a primary diagnosis. A primary source of candidemia was not found in patients in this study. MLST Clade 17 and Clade 1 Were Predominant in C. albicans Blood Strains in Thailand The most common allele types of the AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b genes were allele types 59 (26.1%), 5 (28.3%), 21 (39.1%), 2 (30.4%), 80 (23.9%), 108 (19.6%), and 15 (32.6%), respectively (Supplementary Table S1). The 46 strains yielded 34 distinct DSTs. Forty strains (86.9%) belonged to previously described types (28 DSTs), whereas the remaining six strains (13.0%) belonged to novel DSTs (DST3485, DST3486, DST3487, DST3488, DST3489, and DST3490) that had not previously been identiﬁed (Supplementary Table S1). Among the 34 DSTs, the most common DSTs were DST2514 and DST2876 (8.7% for each DST), followed by DST443 (6.5%) and DST1627, DST2477, DST2904, and DST445 (4.4% for each DST). Based on the UPGMA and eBURST data, nine clades and singleton strains were identiﬁed. Clade 17 (13 strains, 28.3%) and clade 1 (8 strains, 17.4%) were the most and second most common, respectively (Figure 1 and Supplementary Table S1). Sequential Blood Strains Showed Evidence of Sequential Allelic Changes Five of the 37 patients had sequential strains isolated from their blood. Interestingly, three of these ﬁve patients were infected with more than one DST at the same time. Two patients had mixed infections with two DSTs from blood cultures collected on the same date (patient 12 and patient 34; Figure 1 and Supplementary Table S1). Furthermore, C. albicans isolates from patient 8 had three diﬀerent DSTs identiﬁed from sequential blood cultures on days 0, 3, and 5 as DST1710, DST2924, and DST2527, respectively (Supplementary Table S1). Further analysis of diploid allele types (DATs) of the three sequential strains showed sequential changes of only one DAT at a time of the two later-collected strains (Table 1). Speciﬁcally, DAT7 of the VPS13 gene of the S29 strain collected on day 0 changed to DAT100 of the S30 strain collected on day 3. Then, DAT5 of the ADP1 gene of the S29 strain changed to DAT6 of the S1 strain collected on day 5. The proﬁles of the allelic genes from the patients in whom these Clade 17 Strains Showed Signiﬁcantly Stronger Hemolytic Activity As the clade 17 predominance in C. albicans blood strains is uncommon, the virulence phenotype and antifungal susceptibility testing results of clade 17 and the other clades were determined and compared. Four virulence phenotypes were tested. First, the phospholipase zone values of the C. albicans strains ranged from 0.50 to 0.89. All C. albicans strains were phospholipase producers (Table 2 and Supplementary Table S1). Most C. albicans strains showed very strong phospholipase activity (30 strains, 65.2%). No weak phospholipase producer was observed. The mean phospholipase activity of all strains was 0.675 (±0.072). Second, none of the C. albicans produced proteinase (Supplementary Table S1). Third, hemolytic activity was detected in all strains (Table 2 and Supplementary Table S1). The hemolytic zone values ranged from 0.50 to 0.95. Most strains had positive activity (60.9%). The mean hemolytic activity of the C. albicans strains was 0.666 (±0.089). Finally, most C. albicans strains (54.3%) were highly able to form a bioﬁlm layer on the bottom surface of each well (Table 2 and Supplementary Table S2). Association analysis using the chi-square test Statistical Analysis albicans strains showed very strong phospholipase i i ( i 6 %) N k h h li d revealed that clade 17 strains showed signiﬁcantly stronger hemolytic activity than non–clade 17 strains (69.2% versus 37.5% and 24%; p = 0.025). There was no signiﬁcant association between the MLST clades and other virulence phenotypes (p > 0.05) (Table 2). Clade 17 Showed No Signiﬁcant Difference in Antifungal Susceptibility Pattern All strains were susceptible to anidulafungin, micafungin, and caspofungin, and wild type to amphotericin B. Only one isolate (2.2%) was resistant to both voriconazole and ﬂuconazole. The MIC50, MIC90, and geometric mean of all nine drugs are shown TABLE 1 \| Genetic positions of polymorphism on allelic genes of the sequential strains from the three patients. diﬀerent DSTs were detected (patients 8, 12, and 34) are shown in Table 1. diﬀerent DSTs were detected (patients 8, 12, and 34) are shown in Table 1. revealed that clade 17 strains showed signiﬁcantly stronger hemolytic activity than non–clade 17 strains (69.2% versus 37.5% and 24%; p = 0.025). There was no signiﬁcant association between the MLST clades and other virulence phenotypes (p > 0.05) (Table 2). Clade 17 Showed No Signiﬁcant Difference in Antifungal Susceptibility Pattern All strains were susceptible to anidulafungin, micafungin, and caspofungin, and wild type to amphotericin B. Only one isolate (2.2%) was resistant to both voriconazole and ﬂuconazole. The MIC50, MIC90, and geometric mean of all nine drugs are shown in Table 3. There was no signiﬁcant correlation between clade and antifungal susceptibility. Further association analysis revealed that the clade 17 strains did not cause signiﬁcantly higher case fatality rates than clade 1 and the other clades (75% versus 42.9% and 55%; p = 0.340) (Supplementary Table S2). ∗Clade 3 (n = 3), clade 5 (n = 4), clade 7 (n = 2), clade 8 (n = 6), clade 11 (n = 4), clade 12 (n = 1), clade 18 (n = 2), singleton (n = 3). ∗∗Because mixed infections by different DSTs occurred and some strains with identical DSTs from the same patients had variations in virulence phenotypes, all strains from the same patients were considered as different strains and analyzed separately. Statistical Analysis y PASW Statistic 18 (IBM Corporation Armonk, NY, United States) was used for data analysis. To estimate frequencies, descriptive statistical tools were used. GM for bioﬁlm formation was calculated using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, United States). Statistical signiﬁcance of an association between the MLST clusters and virulence phenotype was determined using chi-square or Fisher’s exact test (PASW version 18, IBM, Bangkok, Thailand); p ≤0.05 was considered statistically signiﬁcant. June 2019 \| Volume 10 \| Article 1194 Frontiers in Microbiology \| www.frontiersin.org 4 Thai Candida albicans Molecular Epidemiology Pham et al. TABLE 1 \| Genetic positions of polymorphism on allelic genes of the sequential strains from the three patients. Patient No. Sample No. Collection Date DSTs ADP1 SYA1 VPS13 815 820 826 889 905 946 985 1005 1012 1623 2359 8 S29 Day 0 1710 A/G A S30 Day 3 2924 A/G A/T S1 Day 5 2527 A A/T 12 S36 Day 0 2459 C S37 Day 0 1902 A/C 34 S43/S44 Day 0 1627 C C A A G G T C S45 Day 0 1705 C/T C/T A/G A/G A/G A/G A/T C/T diﬀerent DSTs were detected (patients 8, 12, and 34) are shown in Table 1. revealed that clade 17 strains showed signiﬁcantly stronger hemolytic activity than non–clade 17 strains (69.2% versus TABLE 1 \| Genetic positions of polymorphism on allelic genes of the sequential strains from the three patients. Patient No. Sample No. Collection Date DSTs ADP1 SYA1 VPS13 815 820 826 889 905 946 985 1005 1012 1623 2359 8 S29 Day 0 1710 A/G A S30 Day 3 2924 A/G A/T S1 Day 5 2527 A A/T 12 S36 Day 0 2459 C S37 Day 0 1902 A/C 34 S43/S44 Day 0 1627 C C A A G G T C S45 Day 0 1705 C/T C/T A/G A/G A/G A/G A/T C/T diﬀerent DSTs were detected (patients 8, 12, and 34) are shown in Table 1. Clade 17 Strains Showed Signiﬁcantly Stronger Hemolytic Activity As the clade 17 predominance in C. albicans blood strains is uncommon, the virulence phenotype and antifungal susceptibility testing results of clade 17 and the other clades were determined and compared. Four virulence phenotypes were tested. First, the phospholipase zone values of the C. albicans strains ranged from 0.50 to 0.89. All C. albicans strains were phospholipase producers (Table 2 and Supplementary Table S1). Most C. DISCUSSION In developing countries, candidemia case fatality rates of more than 50% have been reported, which is similar to TABLE 2 \| Number of strains in each MLST cluster in correlation to their phenotypic virulence factors. Bioﬁlm formation (%) Phospholipase activity (%) Hemolytic activity (%) Total Patient outcome∗∗(%) Total Clades High Low None Medium Strong Very strong Positive Strong positive Cure Dead 1 4 (50) 1 (12.5) 3 (37.5) 8 (100) 2 (25) 6 (75) 5 (62.5) 3 (37.5) 8 (100) 4 (57.1) 3 (42.9) 7 (100) 17 6 (46.2) 4 (30.8) 3 (23) 13 (100) 5 (38.5) 8 (61.5) 4 (30.8) 9 (69.2) 13 (100) 3 (25) 9 (75) 12 (100) Other∗ 15 (60) 8 (32) 2 (8) 25 (100) 6 (12) 16 (24) 19 (76) 6 (24) 25 (100) 9 (45) 11 (55) 20 (100) All strains 25 (54.3) 13 (28.3) 8 (17.4) 46 (100) 13 (28.3) 30 (65.2) 28 (60.9) 18 (39.1) 46 (100) 16 (41) 23 (59) 39 (100) P value 0.335 0.503 0.025 − 0.340 − ∗Clade 3 (n = 3), clade 5 (n = 4), clade 7 (n = 2), clade 8 (n = 6), clade 11 (n = 4), clade 12 (n = 1), clade 18 (n = 2), singleton (n = 3). ∗∗Because mixed infections ∗Clade 3 (n = 3), clade 5 (n = 4), clade 7 (n = 2), clade 8 (n = 6), clade 11 (n = 4), clade 12 (n = 1), clade 18 (n = 2), singleton (n = 3). ∗∗Because mixed infections by different DSTs occurred and some strains with identical DSTs from the same patients had variations in virulence phenotypes, all strains from the same patients were considered as different strains and analyzed separately. June 2019 \| Volume 10 \| Article 1194 Frontiers in Microbiology \| www.frontiersin.org 5 Thai Candida albicans Molecular Epidemiology Pham et al. TABLE 3 \| In vitro antifungal susceptibilities of C. albicans strains from hemoculturea,b. Drug MIC (µ/ml) No. (%) of strains interpreted by CBPs No. DISCUSSION (%) of strains interpreted by ECVs Range MIC50 MIC90 GM S SDD I R WT Non-WT AFG 0.015–0.12 0.015 0.12 0.030 46 (100%) N/A 0 0 46 (100%) 0 MFG ≤0.008–0.015 ≤0.008 0.008 0.008 46 (100%) N/A 0 0 46 (100%) 0 CFS 0.008–0.12 0.03 0.06 0.036 46 (100%) N/A 0 0 N/A N/A AmB 0.25–0.5 0.5 0.5 0.381 N/A N/A N/A N/A 46 (100%) 0 5-FC ≤0.06–0.25 ≤0.06 0.06 0.065 N/A N/A N/A N/A N/A N/A PSO 0.015–≥8 0.015 0.03 0.020 N/A N/A N/A N/A N/A N/A VRC ≤0.008–≥8 0.008 0.015 0.010 45 (97.83%) 0 N/A 1 (2.17%) N/A N/A ITC 0.03–≥16 0.06 0.06 0.052 N/A N/A N/A N/A N/A N/A FLU 0.12–16 0.25 1 0.398 45 (97.83%) 0 N/A 1 (2.17%) N/A N/A aN/A, not applicable; GM, geometric mean; CBP, clinical breakpoint; ECV, epidemiology cut-off value; S, susceptible; SDD, susceptible dose dependent; I, intermediate; R, resistant; WT, wild-type strain; AFG, anidulafungin; MFG, micafungin; CFG, caspofungin; AmB, amphotericin B; 5-FC, 5-ﬂucytosine; PSO, posaconazole; VRC, voriconazole; ITC, itraconazole; FLU, ﬂuconazolea. bInterpretation (CBP and ECV: ug/ml) of susceptibility testing were in accordance with the recommendation of the CLSI for C. albicans; CBPs: Anidulafungin, Micafungin and Caspofungin: S < 25, 1 = 0.5, R > 1; Voriconazole: S < 0.12, SDD = 0.25-0.5, R > 1; Fluconazole: S < 2, SDD = 4, R > 8; ECVs, Anidulafungin = 0.12; Micafungin = 0.03; Amphotericin B = 2. TABLE 3 \| In vitro antifungal susceptibilities of C. albicans strains from hemoculturea,b. aN/A, not applicable; GM, geometric mean; CBP, clinical breakpoint; ECV, epidemiology cut-off value; S, susceptible; SDD, susceptible dose dependent; I, intermediate; R, resistant; WT, wild-type strain; AFG, anidulafungin; MFG, micafungin; CFG, caspofungin; AmB, amphotericin B; 5-FC, 5-ﬂucytosine; PSO, posaconazole; VRC, voriconazole; ITC, itraconazole; FLU, ﬂuconazolea. bInterpretation (CBP and ECV: ug/ml) of susceptibility testing were in accordance with the recommendation of the CLSI for C. albicans; CBPs: Anidulafungin, Micafungin and Caspofungin: S < 25, 1 = 0.5, R > 1; Voriconazole: S < 0.12, SDD = 0.25-0.5, R > 1; Fluconazole: S < 2, SDD = 4, R > 8; ECVs, Anidulafungin = 0.12; Micafungin = 0.03; Amphotericin B = 2. High diversity among sequence types of C. albicans was reported in several previous studies (Odds et al., 2007; Shin et al., 2011; Alastruey-Izquierdo et al., 2013; Asadzadeh et al., 2017). DISCUSSION Among 46 clinical strains in this study, high sequence variation was also observed with 34 distinct DSTs. DST254 was derived from the parental DST69, the most common DST found using MLST worldwide (Odds et al., 2007). However, DST69 was not found in this study. Additionally, DST443 and DST693, found in 6.5 and 2.2% of cases in this study, respectively, were the most prevalent DSTs in Taiwan (4.6 and 6.7%, respectively) and China (DST443, 7.7%) (Hu et al., 2015; Wang et al., 2015). DST443, DST457, and DST445 are particular sequence types found in Asia (Wang et al., 2015) that were also present in this study. the 54.8% case fatality rate found in this study (Kaur and Chakrabarti, 2017). Antibiotic resistance is generally one of the common causes of treatment failure of bloodstream infections that leads to patient fatality. However, such resistance is very rare for C. albicans blood strains. A comprehensive 2016 study of C. albicans bloodstream infections in East and Southeast Asia in found that just 1 out of 309 C. albicans blood strains resisted ﬂuconazole, and all strains were sensitive to echinocandins (Tan et al., 2016). Similarly, C. albicans blood strains in our study were also highly susceptible to both echinocandins and azoles. This suggests that antifungal resistance is not a cause of treatment failure for C. albicans bloodstream infections. Rather, candidemia mortality is associated with patients’ underlying conditions or management such as neutropenia from chemotherapy, septic shock, or ICU admission (Boonyasiri et al., 2013). However, comparisons of these factors for C. albicans and non-albicans Candida are lacking. In general, clade 17 is more common in Asia (8.2%) than other continents (0–6.2%) (Odds et al., 2007). Moreover, a 2015 Chinese study also reported clade 17 as the second most common clade identiﬁed from 40 C. albicans non-blood strains (Wu et al., 2015). However, when considering only blood strains, clade 17 was rare (3.8%) in the globally collected C. albicans samples, and clade 1 was the most common (27.8%) (Odds et al., 2007). Furthermore, no clade 17 strains were found in 149 C. albicans blood strains collected in a recent study from Korea (Jung et al., 2016). Only 10.9 and 15% of C. albicans blood strains were identiﬁed as clade 17 in Taiwan (Wang et al., 2015) and China (Wu et al., 2015), respectively. DISCUSSION Therefore, virulence studies were conducted to determine whether the Thai clade 17 isolates were more virulent than isolates from the other clades. The unusually high clade 17 prevalence of the Thai C. albicans blood strains might be explained by the stronger hemolytic activity that is required for C. albicans bloodstream infection (Furlaneto et al., 2018). However, clade 17 strains did not cause a signiﬁcantly higher case fatality rate than the other strains. This suggests that many other factors also aﬀect pathogenesis and patient mortality, especially host Candidemia risk factors are either healthcare related or host related (Kaur and Chakrabarti, 2017). In this study, almost all patients had both types of risk factors, speciﬁcally healthcare-related infections and immunocompromising conditions. In fact, none of the 32 patients whose clinical data were available had no risk factors. However, in- depth details of the candidemia risk factors, including speciﬁc healthcare-related factors (such as use of a central vascular catheter, total parenteral nutrition, urinary catheter) and previous colonization of C. albicans (Kaur and Chakrabarti, 2017), were not available in our database. With the absence of primary sources regarding candidemia in Thailand, as evidenced by this study (Boonyasiri et al., 2013), determining C. albicans colonization is crucial, but it is not common practice in Thailand. Further collection and comprehensive analysis of such data would give an insight into speciﬁc risk factors for candidemia in Thailand. June 2019 \| Volume 10 \| Article 1194 Frontiers in Microbiology \| www.frontiersin.org 6 Thai Candida albicans Molecular Epidemiology Pham et al. and environmental factors (Boonyasiri et al., 2013; Kaur and Chakrabarti, 2017). and environmental factors (Boonyasiri et al., 2013; Kaur and Chakrabarti, 2017). Posteraro et al., 2015; Tan et al., 2016; Chapman et al., 2017). These data conﬁrm echinocandins, ﬂuconazole, and amphotericin B as the drugs of choice for treating patients with candidemia (Pappas et al., 2016). Moreover, a recent study showed that a combination of echinocandin and triazole drugs had potent activity for Candida spp., and triazole drugs had potent activity for Candida spp. (Fakhim et al., 2017). Therefore, antifungal testing for such a combination drug is beneﬁcial, and its interpretation criteria should be established. Phospholipase enzyme plays an important role in host tissue invasion by disrupting the epithelial cell membrane. In general, C. albicans strains isolated from blood samples had higher enzyme activity than other anatomical sites (Ibrahim et al., 1995; Mohan Das and Ballal, 2008). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.01194/full#supplementary-material ETHICS STATEMENT This study was approved by the Siriraj Institutional Review Board, No. SI 091/2016. Informed consent was not required in this work. Both hemolysin and bioﬁlm formation are important virulence factors for C. albicans (Mayer et al., 2013). Hemolysin facilitates iron acquisition in disseminated candidiasis (Tsang et al., 2007), while bioﬁlm formation protects the yeast from a harsh environment including nutrient storage, metabolic cooperation, and acquisition of new genetic traits (Mayer et al., 2013). In our study, C. albicans highly expressed both hemolytic activity (100%) and bioﬁlm formation (82.6%). This suggests that both are important virulence factors for bloodstream infection. FUNDING The study was supported by the Siriraj Research Development Fund, grant number (IO) R016033021, Faculty of Medicine Siriraj Hospital, Mahidol University to PN, and Siriraj Graduate Scholarship to LP. CONCLUSION In conclusion, the molecular epidemiology among C. albicans blood strains in Thailand showed high genetic diversity. No signiﬁcant association existed between patient fatality rate and the MLST clades. Clade 17 strains as a predominant clade among isolates showed signiﬁcant association with hemolytic activity. This ﬁnding requires further strain sampling and pathogenetic study. AUTHOR CONTRIBUTIONS PN, PC, and KS designed the study. LP and SP performed the experiments and analyzed the data. LP, SP, and PN wrote the manuscript. All authors read and approved the manuscript. p Globally, C. albicans showed high susceptibility toward ﬂuconazole, voriconazole, and echinocandins. Azole resistance was generally region dependent and uncommonly found in C. albicans (Lyon et al., 2010; Tan et al., 2016). All C. albicans strains in this study showed high susceptibility to anidulafungin, micafungin, and caspofungin, similar to previous studies from Australia, the Asia-Paciﬁc, and Italy (Prigitano et al., 2016; Tan et al., 2016; Chapman et al., 2017). Only one (2.17%) C. albicans isolate was resistant to voriconazole and ﬂuconazole. This isolate was identiﬁed as DST2527, which resisted both voriconazole and ﬂuconazole. This ﬁnding contrasts with another study from Taiwan (Wang et al., 2015). Therefore, association of DST2527 with resistance is unlikely. Moreover, when categorizing strains based on ECVs, all C. albicans strains also had wild-type MICs for amphotericin B, similar to previous studies (Petlum et al., 2013; DISCUSSION For example, blood strains of C. albicans from Saudi Arabia (Fotedar and Al-Hedaithy, 2005), the European SENTRY program (Borst and Fluit, 2003), Brazil (Mattei et al., 2013), and Turkey (Atalay et al., 2015) showed 100, 71, 78, and 88% positive phospholipase activity, respectively. Similarly, all C. albicans in this study showed positive phospholipase activity. However, despite their similar function regarding host invasion, unlike phospholipase, proteinase activity was typically undetectable in blood strains, whereas 95% of the strains from other sources had proteinase activity (Kantarcioglu and Yucel, 2002). In this study, proteinase activity was undetected in all C. albicans strains. 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Microbiol. 43, 5601–5613. doi: 10.1128/jcm.43.11.5601-5613.2005 Yigit, N., Aktas, E., Dagistan, S., and Ayyildiz, A. (2011). Investigating bioﬁlm production, coagulase and hemolytic activity in Candida species isolated from denture stomatitis patients. Eurasian J. Med. 43, 27–32. doi: 10.5152/eajm. 2011.06 Tsai, M. H., Wang, S. H., Hsu, J. F., Lin, L. C., Chu, S. M., Huang, H. R., et al. (2015). Clinical and molecular characteristics of bloodstream infections caused by Candida albicans in children from 2003 to 2011. Clin. Microbiol. Infect. 21:1018.e1-8. doi: 10.1016/j.cmi.2015.06.024 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Tsang, C. S., Chu, F. C., Leung, W. K., Jin, L. J., Samaranayake, L. P., and Siu, S. C. (2007). Phospholipase, proteinase and haemolytic activities of Candida albicans isolated from oral cavities of patients with type 2 diabetes mellitus. J. Med. Microbiol. 56, 1393–1398. doi: 10.1099/jmm.0.47303-0 Copyright © 2019 Pham, Pharkjaksu, Chongtrakool, Suwannakarn and Ngamskulrungroj. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Microbiology \| www.frontiersin.org June 2019 \| Volume 10 \| Article 1194 REFERENCES 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. 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https://openalex.org/W2110917704	https://europepmc.org/articles/pmc3447949?pdf=render	English	null	The Kcnq1ot1 Long Non-Coding RNA Affects Chromatin Conformation and Expression of Kcnq1, but Does Not Regulate Its Imprinting in the Developing Heart	PLOS genetics	2,012	cc-by	10,649	The Kcnq1ot1 Long Non-Coding RNA Affects Chromatin Conformation and Expression of Kcnq1, but Does Not Regulate Its Imprinting in the Developing Heart Lisa Korostowski, Natalie Sedlak, Nora Engel* Lisa Korostowski, Natalie Sedlak, Nora Engel els Institute for Cancer Research/Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of Am nstitute for Cancer Research/Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America Abstract Funding: This work was supported by National Institutes of Health K22CA140361-01, R01GM093066. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: noraengel@temple.edu Received February 27, 2012; Accepted August 1, 2012; Published September 20, 2012 Copyright: 2012 Korostowski et al. This is an open-access article distributed under the terms of the Creative Commons Att unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health K22CA140361-01, R01GM093066. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by National Institutes of Health K22CA140361-01, R01GM093066. The funders had no role in s analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: noraengel@temple.edu September 2012 \| Volume 8 \| Issue 9 \| e1002956 Citation: Korostowski L, Sedlak N, Engel N (2012) The Kcnq1ot1 Long Non-Coding RNA Affects Chromatin Conform Regulate Its Imprinting in the Developing Heart. PLoS Genet 8(9): e1002956. doi:10.1371/journal.pgen.1002956 Editor: Anne C. Ferguson-Smith, University of Cambridge, United Kingdom ation: Korostowski L, Sedlak N, Engel N (2012) The Kcnq1ot1 Long Non-Coding RNA Affects Chromatin Conformation and Expression o gulate Its Imprinting in the Developing Heart. PLoS Genet 8(9): e1002956. doi:10.1371/journal.pgen.1002956 PLOS Genetics \| www.plosgenetics.org Abstract Although many of the questions raised by the discovery of imprinting have been answered, we have not yet accounted for tissue- or stage-specific imprinting. The Kcnq1 imprinted domain exhibits complex tissue-specific expression patterns co- existing with a domain-wide cis-acting control element. Transcription of the paternally expressed antisense non-coding RNA Kcnq1ot1 silences some neighboring genes in the embryo, while others are unaffected. Kcnq1 is imprinted in early cardiac development but becomes biallelic after midgestation. To explore this phenomenon and the role of Kcnq1ot1, we used allele-specific assays and chromosome conformational studies in wild-type mice and mice with a premature termination mutation for Kcnq1ot1. We show that Kcnq1 imprinting in early heart is established and maintained independently of Kcnq1ot1 expression, thus excluding a role for Kcnq1ot1 in repressing Kcnq1, even while silencing other genes in the domain. The exact timing of the mono- to biallelic transition is strain-dependent, with the CAST/EiJ allele becoming activated earlier and acquiring higher levels than the C57BL/6J allele. Unexpectedly, Kcnq1ot1 itself also switches to biallelic expression specifically in the heart, suggesting that tissue-specific loss of imprinting may be common during embryogenesis. The maternal Kcnq1ot1 transcript is shorter than the paternal ncRNA, and its activation depends on an alternative transcriptional start site that bypasses the maternally methylated promoter. Production of Kcnq1ot1 on the maternal chromosome does not silence Cdkn1c. We find that in later developmental stages, however, Kcnq1ot1 has a role in modulating Kcnq1 levels, since its absence leads to overexpression of Kcnq1, an event accompanied by an aberrant three-dimensional structure of the chromatin. Thus, our studies reveal regulatory mechanisms within the Kcnq1 imprinted domain that operate exclusively in the heart on Kcnq1, a gene crucial for heart development and function. We also uncover a novel mechanism by which an antisense non-coding RNA affects transcription through regulating chromatin flexibility and access to enhancers. Citation: Korostowski L, Sedlak N, Engel N (2012) The Kcnq1ot1 Long Non-Coding RNA Affects Chromatin Conformation and Expression of Kcnq1, but Does Not Regulate Its Imprinting in the Developing Heart. PLoS Genet 8(9): e1002956. doi:10.1371/journal.pgen.1002956 Editor: Anne C. Ferguson-Smith, University of Cambridge, United Kingdom Received February 27, 2012; Accepted August 1, 2012; Published September 20, 2012 Copyright: 2012 Korostowski 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. Author Summary Based on our previous studies, we hypothesized that paternal expression of Kcnq1ot1 represses Kcnq1 in cis, but this effect is countered when enhancer-driven activity of Kcnq1 becomes established during heart development. heart-specific enhancers [18]. Based on our previous studies, we hypothesized that paternal expression of Kcnq1ot1 represses Kcnq1 in cis, but this effect is countered when enhancer-driven activity of Kcnq1 becomes established during heart development. Defects in KCNQ1 are responsible for congenital long QT syndrome, a cardiac disorder that can result in serious cardiac arrhythmias [19]. There is a wide range of phenotypes, with some individuals remaining mostly asymptomatic and others presenting severe symptoms. Understanding the molecular mechanisms underlying the phenotypic variability of this disease will require fully elucidating the epigenetic profile of Kcnq1 expression during cardiac development. We investigated function of Kcnq1ot1 transcription in regulating Kcnq1 in the heart in wild-type mice and in a mutant mouse model in which the Kcnq1ot1 transcript is truncated. Our studies show that imprinted Kcnq1 expression in early heart development is not dependent on the Kcnq1ot1 transcript or on its transcriptional status. In fact, Kcnq1ot1 itself suffers a loss of imprinting during cardiac development, although it remains monoallelic in other tissues. We also show that the ncRNA does have a role in later embryogenesis for proper regulation of Kcnq1 levels in the embryonic heart by way of modulating the three- dimensional chromatin structure of the locus. q Our data clearly indicate that cardiac development is accom- panied by regulatory events in the Kcnq1 domain that are very distinct from its expression and imprinting status in other tissues. To determine if the loss of imprinting in the heart affected genes in the region other than Kcnq1 and Kcnq1ot1, we tested the allele- specific expression of Cdk1nc and Slc22a18 using either RT-PCR followed by allele specific restriction digest or sequencing analysis (Figure S3). Interestingly, both Cdkn1c and Slc22a18 maintained their maternal expression patterns throughout heart development. This confirms that transcription of the maternal allele of Kcnq1ot1 that occurs in the heart does not have repressive capability. As anticipated, and as previously reported [14], Cdkn1c and Slc22a18 maintain their imprinting expression in both P2 brain and liver samples (Figure S2A, S2B). The maternal Kcnq1ot1 ncRNA emerges from an alternative promoter and is shorter than its paternal counterpart The promoter of the Kcnq1ot1 gene lies immediately upstream of two CG-rich regions that harbor a maternal-specific gametic methylation mark (KvDMR). To determine if activation of the maternal Kcnq1ot1 allele is due to loss of methylation at the KvDMR, we performed methylation-specific PCR (data not shown) and bisulfite mutagenesis sequencing (Figure S4). Our results show that the maternal KvDMR methylation mark is not lost in neonatal heart. Author Summary Author Summary Imprinted genes express only one copy and the active allele depends on the parental origin. These genes are usually organized in groups and are subject to regulation over long distances. Misregulation in these domains can lead to developmental diseases and cancer. The paternally expressed Kcnq1ot1 non-coding RNA emerges from an intron of the Kcnq1 gene in antisense direction and silences several neighboring genes. The Kcnq1 gene is initially imprinted but becomes biallelic during develop- ment of the heart. To understand the mechanisms underlying this behavior, we performed expression and conformational studies of the Kcnq1 domain during this transition and found that the early stage imprinting of Kcnq1 is not dependent on transcription of Kcnq1ot1, thus revealing the existence of a distinct mechanism of silencing of the paternal Kcnq1 allele. Surprisingly, Kcnq1ot1 loses its imprinted expression specifically in the heart in the same time frame as Kcnq1. Absence of Kcnq1ot1 does lead to overexpression of Kcnq1 in later cardiac development, an event characterized by increased chromatin flexibility allowing the Kcnq1 promoter to establish ectopic regulatory contacts. Thus, we have uncovered novel regulatory mechanisms that will help our understanding of tissue specificity in imprinted domains and how they can go awry in disease. Previous reports have shown that the paternal Kcnq1ot1 ncRNA acts in cis by coating the chromosome [12,11]. We used dual DNA and RNA fluorescent in situ hybridization (FISH) to determine if the maternal Kcnq1ot1 transcript accumulates locally in the heart. RNA and DNA FISH was performed in primary cardiomyocytes that are biallelic for Kcnq1ot1 (Figure S2). Results show that the maternal transcript co-localizes with the DNA strand it is being transcribed from. This suggests that the reactivated maternal Kcnq1ot1 transcript does associate with the chromatin in cis. As shown in Figure 1C, levels of Kcnq1ot1 mRNA emerging from the maternal allele almost equal the paternal levels, suggesting that maternal expression is not merely due to ‘‘leaky’’ transcription. In fact, the transition to biallelic expression results in an increase in total Kcnq1ot1 expression when compared to tissues in which Kcnq1ot1 remains monoallelic (Figure S1B), Thus, the paternal activation of Kcnq1 does not interfere with Kcnq1ot1 transcription in cis [18] and, furthermore, initiation of Kcnq1ot1 expression from the maternal allele does not silence the already active maternal Kcnq1 allele. heart-specific enhancers [18]. Introduction cis [7,8]. In both the embryo and the placenta, the polycomb group complex is reported to play a major role in silencing. In the placental lineage, repressive histone modifications are necessary to maintain the imprinting status of the genes surrounding Kcnq1ot1 in the absence of differentially methylated regions [9]. In the embryo, only genes with stable DNA methylation are silenced by the ncRNA [10,11,12]. Although the role of Kcnq1ot1 in the establishment of allelic repression is still unclear, evidence has emerged suggesting that maintenance of silencing results from the act of transcription through the locus rather than as a function of the RNA molecule per se, at least in cultured embryonic and trophoblastic cells [13]. The genome is transcribed into a host of non-coding RNAs, some of which are designated as long intergenic (linc) RNAs. These have regulatory roles in gene activation and repression, and have been hypothesized to serve as guides for non-sequence-specific histone modifying enzymes [1]. One class of lincRNAs includes macro- RNAs, which can range in size up to hundreds of kilobases. Several macroRNAs have been studied intensively in the fields of genomic imprinting and X-inactivation [2,3,4,5]. The role of Xist in X- inactivation has been a long-standing paradigm for understanding how silencing of imprinted genes is achieved in domains that encode other macroRNAs. One prevalent model of imprinted gene silencing is that the non-coding (nc) RNA spreads along the chromatin, recruiting histone modifying repressive enzymes and rendering the region refractory to transcriptional activation. The silencing capacity of Kcnq1ot1 across the Kcnq1 domain is not universal. At least one gene, Cdkn1c, is repressed by a mechanism independent of Kcnq1ot1 in certain tissues [14]. On the other hand, some genes in the domain escape silencing, either ubiquitously or in a tissue-specific manner [15,16,17]. Kcnq1, one such exception, is expressed from the maternal allele during early embryogenesis and transitions to biallelic expression during fetal heart development. This transition coincides with conformational changes involving the interaction between the Kcnq1 promoter and The Kcnq1ot1 imprinted ncRNA is transcribed from the paternal allele, emerging from intron 11 of the Kcnq1 gene in antisense direction. In contrast to most long ncRNAs, Kcnq1ot1 is highly expressed and detectable in every tissue examined [3,6]. Kcnq1ot1 transcription has a role in repressing several neighboring genes in September 2012 \| Volume 8 \| Issue 9 \| e1002956 1 PLOS Genetics \| www.plosgenetics.org Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Activation of the paternal Kcnq1 allele could interfere with spreading of the antisense Kcnq1ot1 RNA, or it could impact its expression, thus limiting its role in silencing. To determine if the paternal Kcnq1ot1 allele is still expressed after paternal Kcnq1 activation, we carried out allele-specific RT-PCRs during normal heart development in progeny from reciprocal crosses between C57BL/6J and B6(CAST7) mice. B6(CAST7) are a consomic strain in which chromosome 7 is derived from Mus musculus castaneus [20]. Unexpectedly, we found that not only does the paternal Kcnq1ot1 remain active, but the maternal allele is now transcribed as well. This loss of imprinted expression for Kcnq1ot1 is independent of strain-specific effects, is specific to the heart and does not occur in other tissues (Figure 1B, Figure S1A). Kcnq1 is imprinted in the absence of Kcnq1ot1 in the early stage heart Our data show that in the context of the neonatal heart, Kcnq1ot1 does not silence Kcnq1. During early development, however, Kcnq1 exhibits monoallelic expression and it was assumed that Kcnq1ot1 was responsible for repressing Kcnq1 in cis (Figure S5A). To test this assumption, we examined a mutant mouse model in which the Kcnq1ot1 transcript is severely truncated due to the inclusion of a transcriptional termination site 1.5 kb down- stream of the transcriptional start site [8]. In this mouse, designated as K-term, transmission of the mutation from males results in loss of paternal silencing of genes in the Kcnq1 domain in the placenta and late embryonic stages. We examined whether the absence of Kcnq1ot1 leads to biallelic Kcnq1 cardiac expression from embryonic day 10.5 to postnatal day 2. Surprisingly, Kcnq1 did not lose imprinted expression and maintained the same profile in the K-term as in wild-type mice, i.e., expression was exclusively from the maternal allele in E10.5 hearts. Thereafter, expression became biallelic, as in wild-type hearts, although at a slightly earlier E12.5 dpc (Figure 3A). These results indicate that paternal-specific repression of Kcnq1 in early embryogenesis is independent of Kcnq1ot1 transcription and suggest that there are other mechanisms responsible for differential expression of the Kcnq1 gene. Figure 1. Cardiac expression profile of Kcnq1ot1. A) Imprinting pattern in the Kcnq1 domain in early development in the embryo. Arrows above the line represent maternal transcription and those below represent paternal transcription. B) Kcnq1ot1 imprinting as determined by RT-PCR followed by allele-specific restriction digest. The maternal Kcnq1ot1 becomes activated as development progresses in the heart. However, in brain and liver, Kcnq1ot1 remains monoallelic. M, maternal; P, paternal; N, non-digested; D, Digested; B, C57BL/6J; C, B6(CAST7). C) Quantitative analysis by qRT-PCR of Kcnq1ot1 expression throughout development. RNA levels were normalized to b-actin. doi:10.1371/journal.pgen.1002956.g001 Figure 1. Cardiac expression profile of Kcnq1ot1. A) Imprinting To determine if the maintenance of imprinting at Kcnq1 was specific to cardiac tissue, we tested Kcnq1 allelic expression in heads and bodies of the matched mutant embryos. In these, biallelic expression of Kcnq1 was seen in the absence of full-length Kcnq1ot1 (Figure 3A, left), indicating that maintenance of imprinted expression in K-term mutants may be restricted to the heart. These results also show that while the ncRNA is not responsible for imprinted expression of Kcnq1 in the heart, it does have a role in silencing Kcnq1 in tissues other than heart. Kcnq1 monoallelic expression is not due to methylation of its promoter To see if the maternal Kcnq1ot1 gene produces a full-length RNA, we scanned the region with allele-specific RT-PCR. As shown in Figure 2, the re-activated maternal transcript is truncated, producing an RNA of less than 44 kb long (Figure 2B and 2C, Figure S1B). To determine if early silencing of the paternal Kcnq1 allele is due to methylation of the promoter, bisulfite mutagenesis sequencing of the promoter was performed on E10.5 and P2 wild type hearts. Results show that the Kcnq1 promoter is not methylated in the early embryonic heart and thus, imprinted expression cannot be attributed to differential methylation (Figure S6). These results confirm our published 5-methylcytosine ChIP data in E7.5 whole embryos confirm that the CG-island at the Kcnq1 promoter is not methylated at any stage during development [18], as well as previous reports from others [22,23]. Kcnq1 is imprinted in the absence of Kcnq1ot1 in the early stage heart While imprinted expression of Kcnq1 in the early heart is independent of Kcnq1ot1, paternal silencing of both Cdkn1c and Slc22a18 does depend on Kcnq1ot1 transcription: upon paternal transmission of the K-term mutation, these genes lose their imprinting pattern and are biallelically expressed (Figure S3). analysis of an expressed single nucleotide polymorphism shows that although paternal transcripts are produced from both previously TSS and the newly identified TSS, the maternal transcripts are exclusively initiated at the cardiac-specific sites downstream of the CG-islands, thus bypassing the methylation mark. Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Figure 1. Cardiac expression profile of Kcnq1ot1. A) Imprinting pattern in the Kcnq1 domain in early development in the embryo. Arrows above the line represent maternal transcription and those below represent paternal transcription. B) Kcnq1ot1 imprinting as determined by RT-PCR followed by allele-specific restriction digest. The maternal Kcnq1ot1 becomes activated as development progresses in the heart. However, in brain and liver, Kcnq1ot1 remains monoallelic. M, maternal; P, paternal; N, non-digested; D, Digested; B, C57BL/6J; C, B6(CAST7). C) Quantitative analysis by qRT-PCR of Kcnq1ot1 expression throughout development. RNA levels were normalized to b-actin. doi:10.1371/journal.pgen.1002956.g001 (Figure S5B). Also, the C6B mono- to biallelic transition occurs two days later than in the reciprocal cross (Figure S5A), with the C57BL/6J paternal activation beginning at 16.5 dpc. In addition, the Kcnq1 expression level attained by the paternal copy is 75% of the maternal allele in the C6B cross, whereas maternal and paternal expression is equalized in the reciprocal cross (Figure S5C). These results show that there is a strong strain-specific genetic component on chromosome 7 that determines levels of Kcnq1 and affects the timing of paternal allele reactivation. The Kcnq1ot1 ncRNA loses imprinted expression in the heart The Kcnq1ot1 ncRNA loses imprinted expression in the heart The Kcnq1ot1 ncRNA loses imprinted expression in the heart The Kcnq1 domain exhibits complex expression and imprinting patterns, eliciting questions of how long-range regulatory mech- anisms coexist with locally limited ones (Figure 1A). The Kcnq1 gene switches from mono- to biallelic expression during mid- gestation in a tissue-specific manner. In the embryonic heart the transition occurs at approximately E14.5, coinciding with enhancers contacting the Kcnq1 promoter [18]. The timing for this is very consistent, with the switch occurring at the earliest at E13.5 and no later than E15.5. We proposed that tissue-specific enhancers compete with Kcnq1ot1 and create a chromatin environment that is antagonistic to the ncRNA. To determine if the maternal Kcnq1ot1 transcript is initiated at an alternative promoter, thus bypassing methylation on the maternal KvDMR, 59RACE was performed in neonatal heart (Figure 2A). We find that in the neonatal heart, transcripts are initiated at previously reported transcriptional start sites (TSS) [21] and at two additional downstream sites (Figure 2A). Sequence September 2012 \| Volume 8 \| Issue 9 \| e1002956 September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 2 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 September 2012 \| Volume 8 \| Issue 9 \| e1002956 Kcnq1 expression levels and loss of imprinting are subject to genetic background effects C) RT-PCRs on RNAs extracted from hearts of F1 hybrid progeny from a BxC cross, followed by allele-specific digestion, showing the maternal transcript absent after 33 kb. M, maternal; P, paternal; ND, non-digested; D, digested; B, C57BL/6J; C, B6(CAST7). doi:10.1371/journal.pgen.1002956.g002 Figure 2. Characterization of the maternal Kcnq1ot1 transcript. A) Schematic showing the regulatory sequences at the Kcnq1ot1 locus. Minimal promoter, enhancer and previously reported transcriptional start sites (, TSS) are from Fitzpatrick et al. ([21]. The silencing domain depicted was characterized by Mohammad et al. ([23]. Primers used for 59 RACE experiments in this report are designated A, B, C and nested primers, An, Bn and Cn. Transcriptional start sites (TSS) in heart and liver are depicted as bent arrows. A star indicates the relative location of a single nucleotide polymorphism (SNP) used to discriminate between parental transcripts. B) Schematic of the scan to determine the length of the Kcnq1ot1 maternal transcript, with primers amplifying fragments located at the indicated distances relative to the Kcnq1ot1 transcriptional start site. C) RT-PCRs on RNAs extracted from hearts of F1 hybrid progeny from a BxC cross, followed by allele-specific digestion, showing the maternal transcript absent after 33 kb. M, maternal; P, paternal; ND, non-digested; D, digested; B, C57BL/6J; C, B6(CAST7). doi:10.1371/journal.pgen.1002956.g002 Figure 2. Characterization of the maternal Kcnq1ot1 transcript. A) Schematic showing the regulatory sequences at the Kcnq1ot1 locus. Minimal promoter, enhancer and previously reported transcriptional start sites (, TSS) are from Fitzpatrick et al. ([21]. The silencing domain depicted was characterized by Mohammad et al. ([23]. Primers used for 59 RACE experiments in this report are designated A, B, C and nested primers, An, Bn and Cn. Transcriptional start sites (TSS) in heart and liver are depicted as bent arrows. A star indicates the relative location of a single nucleotide polymorphism (SNP) used to discriminate between parental transcripts. B) Schematic of the scan to determine the length of the Kcnq1ot1 maternal transcript, with primers amplifying fragments located at the indicated distances relative to the Kcnq1ot1 transcriptional start site. C) RT-PCRs on RNAs extracted from hearts of F1 hybrid progeny from a BxC cross, followed by allele-specific digestion, showing the maternal transcript absent after 33 kb. M, maternal; P, paternal; ND, non-digested; D, digested; B, C57BL/6J; C, B6(CAST7). doi:10.1371/journal.pgen.1002956.g002 Kcnq1 expression levels and loss of imprinting are subject to genetic background effects To determine the differences in genetic background effects on expression levels and the timing of the mono- to biallelic switch, we assayed relative mRNA abundance in B6C and C6B hearts during development (Figure S5). Early maternal expression of Kcnq1 is higher from the CAST/EiJ than from the C57BL/6J and continues to have a relatively higher expression level throughout development September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org 3 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Figure 2. Characterization of the maternal Kcnq1ot1 transcript. A) Schematic showing the regulatory sequences at the Kcnq1ot1 locus. Minimal promoter, enhancer and previously reported transcriptional start sites (, TSS) are from Fitzpatrick et al. ([21]. The silencing domain depicted was characterized by Mohammad et al. ([23]. Primers used for 59 RACE experiments in this report are designated A, B, C and nested primers, An, Bn and Cn. Transcriptional start sites (TSS) in heart and liver are depicted as bent arrows. A star indicates the relative location of a single nucleotide polymorphism (SNP) used to discriminate between parental transcripts. B) Schematic of the scan to determine the length of the Kcnq1ot1 maternal transcript, with primers amplifying fragments located at the indicated distances relative to the Kcnq1ot1 transcriptional start site. C) RT-PCRs on RNAs extracted from hearts of F1 hybrid progeny from a BxC cross, followed by allele-specific digestion, showing the maternal transcript absent after 33 kb. M, maternal; P, paternal; ND, non-digested; D, digested; B, C57BL/6J; C, B6(CAST7). doi:10.1371/journal.pgen.1002956.g002 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Figure 2. Characterization of the maternal Kcnq1ot1 transcript. A) Schematic showing the regulatory sequences at the Kcnq1ot1 locus. Minimal promoter, enhancer and previously reported transcriptional start sites (, TSS) are from Fitzpatrick et al. ([21]. The silencing domain depicted was characterized by Mohammad et al. ([23]. Primers used for 59 RACE experiments in this report are designated A, B, C and nested primers, An, Bn and Cn. Transcriptional start sites (TSS) in heart and liver are depicted as bent arrows. A star indicates the relative location of a single nucleotide polymorphism (SNP) used to discriminate between parental transcripts. B) Schematic of the scan to determine the length of the Kcnq1ot1 maternal transcript, with primers amplifying fragments located at the indicated distances relative to the Kcnq1ot1 transcriptional start site. Absence of Kcnq1ot1 leads to Kcnq1 overexpression in the heart To determine if the increase in mRNA levels of Kcnq1 in the mutant mice was due to increased transcription from one or both parental alleles, relative transcript abundance was determined in 14.5 dpc and neonatal hearts. Activation of the paternal Kcnq1 allele was progressive and levels reached 88% of the maternal allele RNA abundance (Figure 3C, Figure S5C). Thus, the overall increase in Kcnq1 expression follows the same dynamics as in the wild-type heart, i.e., the paternal allele becomes progressively activated and reaches levels similar to the maternal RNA. This activation coincides with tissue-specific enhancer-driven expres- sion in the wild-type mouse. Although Kcnq1ot1 is not responsible for Kcnq1 imprinted expression in the heart, we investigated if it had any repressive effect on Kcnq1 in later stages of heart development. To test this, we determined Kcnq1 mRNA abundance by qRT-PCR was performed on E10.5, E16.5 and P2 hearts in wild-type and K-term mice. Kcnq1 transcript levels were similar between wt and K-term hearts at E10.5. However, by E16.5, Kcnq1 abundance was significantly increased in K-term compared to wild type mice, indicating that Kcnq1ot1 transcription plays a role in regulating Kcnq1 levels (Figure 3B, Figure S5B). September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 4 nq1ot1 modulates three dimensional chromatin Chromosome Conformation Capture (3C) assays were per- gure 3. Kcnq1 expression in the heart during development when the truncated Kcnq1ot1 (K-term mutation) is inherited paternally. RT-PCR followed by allele-specific digests in E10.5 heads and bodies and throughout the development of the heart in F1 hybrid progeny of CAST7)6K-term mice. M, maternal; P, paternal; N, non-digested; D, digested; B, C57BL/6J; KT, K-term. B) qRT-PCR analysis of Kcnq1 expression in d-type and K-term mice. Transcripts were normalized to b-actin. A significant difference in expression was seen when comparing wild-type and K- m hearts at E16.5 and P2 Hearts. These differences had a p-value less than 0.05. C) Parental origin of Kcnq1 expression throughout cardiac velopment. The RT-PCR and allele specific bands were quantified and the ratio of paternal to maternal transcript was determined. :10.1371/journal.pgen.1002956.g003 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Figure 3. Kcnq1 expression in the heart during development when the truncated Kcnq1ot1 (K-term mutation) is inherited paternally. Absence of Kcnq1ot1 leads to Kcnq1 overexpression in the heart A) RT-PCR followed by allele-specific digests in E10.5 heads and bodies and throughout the development of the heart in F1 hybrid progeny of B6(CAST7)6K-term mice. M, maternal; P, paternal; N, non-digested; D, digested; B, C57BL/6J; KT, K-term. B) qRT-PCR analysis of Kcnq1 expression in wild-type and K-term mice. Transcripts were normalized to b-actin. A significant difference in expression was seen when comparing wild-type and K- term hearts at E16.5 and P2 Hearts. These differences had a p-value less than 0.05. C) Parental origin of Kcnq1 expression throughout cardiac development. The RT-PCR and allele specific bands were quantified and the ratio of paternal to maternal transcript was determined. doi:10.1371/journal.pgen.1002956.g003 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 To determine if the ectopic contact sites for the Kcnq1 promoter in K-term mice have the hallmarks of cis-acting regulatory sequences, we conducted chromatin immunoprecipitation (ChIP) assays (Figure 5A, 5B). Enhancers are typically occupied by p300 and have specific epigenetic marks, notably H3K4Me1 and H3K27Ac. In addition, some enhancers are highly conserved. Using ChIP analysis, the Kcnq1 region was scanned for p300, H3K27Ac and H3K4me1 occupancy in wild-type hearts, focusing on the sequences that exhibited interactions in the 3C assays. Our data reveal a genetic background effect on the level of expression of Kcnq1, with the CAST/EiJ allele exhibiting higher abundance than the C57BL/6J allele at all stages in the developing heart. In addition to being expressed less abundantly, a C57BL/6J paternal allele is reactivated at a later time point than a CAST/EiJ one. Whether these two features are related needs to be determined, but our own analyses and publicly available genomic data lead us to hypothesize that strain-specific cis-regulatory polymorphisms may explain both phenomena. For example, sequence differences in transcription factor binding or affinity for enhancers could determine higher levels of Kcnq1 expression from the CAST/EiJ allele, and greater accessibility to reactivation when inherited paternally. Several of the ectopic contact sites showed epigenetic hallmarks of regulatory sequences (Figure 5B). Specifically, regions amplified with primer sets 13 and 17 (+39 kb and +78 kb relative to TSS, respectively) have p300 occupancy, with H3K4Me1 and H3K27Ac modifications. Although we did not observe these features with primers sets 23 and 25, H3K4Me1 cardiac-specific peaks are reported by the ENCODE consortium (annotated on UCSC), as are the primer 13 and 17 signals. Only regions 13, 21 and 23 are highly conserved (www.dcode.org), and these only between mouse, rat, rhesus and human. Interestingly, the +189 kb peak lies within the Kcnq1ot1 promoter region. In fact, the Kcnq1 promoter does not make contacts beyond a CTCF binding site upstream (located in the Trpm5 gene) and two CTCF binding sites downstream in the Kcnq1ot1 promoter, suggesting that these may serve as boundaries to the movement of the Kcnq1 promoter, at least in the absence of Kcnq1ot1. In exploring the cardiac regulation of the Kcnq1 gene, our findings delineate two phases, independently of strain-specific differences: one, in early development, when Kcnq1 exhibits imprinted expression independently of Kcnq1ot1; and two, in the neonate, when Kcnq1 is biallelic and its expression levels are modulated by Kcnq1ot1. Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 enhancers acting on Kcnq1 also exert influence on the Kcnq1ot1 promoter, activating the previously silent maternal allele. for ectopic promoter contacts that increase the overall levels of Kcnq1. The 3C assays are targeted to regulatory elements and there is a paucity of polymorphisms in these regions. We were able to determine allele-specificity of contacts with one primer set (primer 27) by analyzing substrate prepared from heart from progeny of crosses between B6(CAST7) and K-term mice (Figure S7). PCR followed by allele-specific restriction digest showed ectopic contact between the Kcnq1 promoter and the Kcnq1ot1 promoter region only on the paternal chromosome, i.e., the one carrying the K-term mutation. The maternal methylation mark encompassing the Kcnq1ot1 promoter is not lost during the activation of transcription. This is in line with abundant data showing that gametic methylation is very stable [24], and suggests that the repressive effect of DNA methylation can be overcome. In fact, our data show that maternal transcription of Kcnq1ot1 ncRNA is initiated at an alternative promoter region downstream of the methylation mark. Several examples of alternative promoter usage bypassing DNA methyl- ation have been reported [25], and we propose that this mechanism may be commonly deployed to fine-tune expression of imprinted genes for tissue-specific needs. Analysis of brains from K-term mice showed a distinct pattern, with two associations that were not present in wild-type brains. These results suggest that the biological consequences of aberrant Kcnq1 promoter contacts are tissue-specific and reinforce that the Kcnq1 gene has multiple levels of regulation in the heart (Figure 4C). Interestingly, the Kcnq1ot1 molecule produced from the mater- nal chromosome is half the length of the paternal transcript. This could be because expression of maternal Kcnq1 from early stages blocks or competes with the Kcnq1ot1 in some way and impedes completion of the transcript. Another possibility is that an alternative transcriptional termination signal is used. These two mechanisms are not mutually exclusive. Surprisingly, the reacti- vated ncRNA does not repress Cdkn1c, even though it accumulates locally in a manner similar to the paternal Kcnq1ot1. It remains to be determined if this is because of the diminished length of the RNA or if cardiac cells do not provide repressive co-factors that are necessary for the spread of silencing. Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Our analysis of the K-term mutant mouse shows that Kcnq1 expression is imprinted in early cardiac development even in the absence of the ncRNA. This result excludes a role for Kcnq1ot1 in establishing or maintaining repression. Thus, there is an indepen- dent mechanism that silences the paternal Kcnq1 allele during early development. This mechanism is cardiac-specific, because absence of Kcnq1ot1 does release Kcnq1 from repression in tissues other than the heart. Although a secondary methylation mark at Cdkn1c is reportedly dependent on expression of Kcnq1ot1, the Kcnq1 CG- rich promoter is never methylated. Thus, monoallelic expression of Kcnq1 is likely due to chromatin conformation or trans-acting factors. Perhaps paternal expression of Kcnq1ot1 opens the chromatin and makes a tissue-specific silencer available to factors that repress Kcnq1. These factors would be present in early development and would disappear upon full maturation of the heart (Figure 6). Another possibility is that an inhibitory factor (IF) for Kcnq1 has a cognate bind site within a differentially methylated region. If the factor is methylation-sensitive, it would only be able to bind the unmethylated paternal allele, thus rendering the paternal Kcnq1 allele inactive. In fact, a silencing domain (SD) has been delimited downstream of the Kcnq1ot1 promoter, overlapping Kcnq1ot1 modulates three-dimensional chromatin conformation Chromosome Conformation Capture (3C) assays were per- formed on P2 hearts of K-term mutant mice, with the anchor primer located at the Kcnq1 promoter (Figure 4A). In contrast to the tight interactions seen in the wild-type heart, the 3C assays showed that the Kcnq1 promoter interacts with many additional sites throughout the region in the K-term mutants (Figure 4B). These results implicate the transcription of the Kcnq1ot1 RNA in regulating the specificity of promoter interactions. Thus, although the Kcnq1ot1 RNA does not silence Kcnq1, its absence is permissive We have previously shown that the Kcnq1 promoter interacts with an upstream region in a cardiac-specific fashion [18]. Moreover, this interaction is established during the transition from mono- to biallelic expression of Kcnq1. Because the loss of the ncRNA results in a large increase in Kcnq1 transcript in the heart, we investigated whether the three-dimensional conformation of the locus was altered in the K-term mice. September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 5 PLOS Genetics \| www.plosgenetics.org September 2012 \| Volume 8 \| Issue 9 \| e1002956 Discussion The findings presented in this report underscore the value of tissue- and stage-specific studies of imprinted domains and have yielded several surprising insights. Our data and studies of other imprinted genes confirm that loss of imprinting in specific tissues is a normal developmental event. We find that strain-specific effects influence expression levels and timing of imprinting loss. We also find two co-existing mechanisms responsible for imprinting within the Kcnq1 domain in the embryo. Importantly, by mapping the higher order structure of a specific region, we reveal a novel mechanism by which an antisense non-coding RNA can regulate transcription. Kcnq1ot1 is ubiquitously expressed and it had been assumed that it was imprinted in every tissue. In this study, we find that Kcnq1ot1 is biallelically expressed in the heart, with the transition from mono- to biallelic occurring in parallel with the Kcnq1 switch. This loss of imprinting is not seen in other tissues at that stage, indicating that there are mechanisms that shift the control of the region to meet tissue-specific needs. Perhaps the strong cardiac September 2012 \| Volume 8 \| Issue 9 \| e1002956 6 Figure 4. Chromosome conformation capture (3C) in the K-term mouse. A) Schematic of the Kcnq1 domain. The blue arrowhead i the anchor primer at the Kcnq1 promoter and vertical lines represent the regions investigated. The purple boxes represent Kcnq1 exons, the d box represents the Kcnq1ot1 gene. Comparison of the chromatin interaction profile in wild-type and K-term hearts (B), wild-type and K-term b and K-term hearts and brains (D). doi:10.1371/journal.pgen.1002956.g004 Kcnq1ot1 Does Not Regulate Imprinting o Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Figure 4. Chromosome conformation capture (3C) in the K-term mouse. A) Schematic of the Kcnq1 domain. The blue arrowhead indicates the anchor primer at the Kcnq1 promoter and vertical lines represent the regions investigated. The purple boxes represent Kcnq1 exons, the dark blue box represents the Kcnq1ot1 gene. Comparison of the chromatin interaction profile in wild-type and K-term hearts (B), wild-type and K-term brains (C) and K-term hearts and brains (D). doi:10.1371/journal.pgen.1002956.g004 one of the differentially methylated CG-islands (Figure 2A). Interestingly, our bisulfite sequencing results for the paternal KvDMR show a trend towards acquisition and spreading of methylation that could be explained by loss of the inhibitory factor binding at the SD (Figure S3). methylation-sensitive. PLOS Genetics \| www.plosgenetics.org September 2012 \| Volume 8 \| Issue 9 \| e1002956 Discussion CTCF could negatively impact expression of Kcnq1 in early embryonic stages by repressing it directly or blocking it from access to enhancers required at that stage (Figure 6, inhibitory factor (IF) would be CTCF). Enhancer activity has been shown for a sequence immediately upstream of the Kcnq1ot1 promoter, a position that would be blocked by CTCF [21]. Early paternal repression of Kcnq1 may also be due to the presence of CTCF on the paternal KvDMR [21]. CTCF binding is September 2012 \| Volume 8 \| Issue 9 \| e1002956 September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org PLOS Genetics \| www.plosgenetics.org 7 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Figure 5. Chromatin immunoprecipitation (ChIP) of selected regions of the Kcnq1 gene. A) Schematic of the Kcnq1 domain. The blue arrowhead indicates the anchor primer at the Kcnq1 promoter and vertical lines represents the primer regions investigated. The purple boxes represent Kcnq1 exons, the dark blue box represents the Kcnq1ot1 gene. B) ChIP analysis in wild-type heart for p300, H3K4Me1 and H3K27Ac at the interaction peaks observed in the 3C assays. The asterisks indicate frequent interactions in the K-term heart as determined by 3C. doi:10.1371/journal.pgen.1002956.g005 Figure 5. Chromatin immunoprecipitation (ChIP) of selected regions of the Kcnq1 gene. A) Schematic of the Kcnq1 domain. The blue arrowhead indicates the anchor primer at the Kcnq1 promoter and vertical lines represents the primer regions investigated. The purple boxes represent Kcnq1 exons, the dark blue box represents the Kcnq1ot1 gene. B) ChIP analysis in wild-type heart for p300, H3K4Me1 and H3K27Ac at the interaction peaks observed in the 3C assays. The asterisks indicate frequent interactions in the K-term heart as determined by 3C. doi:10.1371/journal.pgen.1002956.g005 cooperative and competitive dialogues between enhancers, silenc- ers and insulators [31,32] and that understanding complex genetic loci will require identifying all the regulatory sequences and determining their linear order, tissue-specificity and three-dimen- sional structure. Comparison of Kcnq1 abundance and domain conformation between wild-type and mutant mice reveals what may be the key role for Kcnq1ot1 in later cardiac development. Our results show that absence of the ncRNA leads to much increased Kcnq1 levels, accompanied by a wider range of promoter contacts with sequences throughout the region. Although several of the regions contacted exhibit the marks of regulatory sequences, the Kcnq1 promoter is only associated with them if Kcnq1ot1 is prematurely terminated. Mouse strains and crosses All wild-type tissues used in the experiments were either C57BL/6J or F1 progeny of reciprocal crosses between C57BL/ 6J and B6(CAST7) (a consomic strain harboring a chromosome 7 derived from Mus musculus castaneus on a C57BL/6J background) (BxC or CxB for short). For the K-term mouse samples, mutant males were crossed with B6(CAST7) females and tissues of F1 progeny were obtained (CxK-term). To show the transition in allelic expression for Kcnq1 and Kcnq1ot1, 2–3 embryos were analyzed per litter and 3–5 litters were analyzed at each stage of development. Tissue-specificity of Kcnq1ot1 repression has been previously reported, specifically for the neighboring Cdkn1c gene [14]. In fact, Cdkn1c imprinting is maintained in the kidney, liver and lung even with a truncated Kcnq1ot1. Several studies have also shown lineage- specific imprinting regulation in the placenta [11,29]. Indeed, these observations collectively underscore the need to identify cis- and trans-acting factors and the dynamics of their interactions to test hypotheses relative to the tissue-specific effects of Kcnq1ot1. Ethics statement This study was carried out on mice in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Temple University Animal Care and Use Committee (Protocol 3294). Discussion The ectopic interactions reflect a greater flexibility of the chromatin fiber in the absence of Kcnq1ot1 transcription and may contribute to the increased Kcnq1 levels. It is imperative to further elucidate the molecular basis of the tissue-specificity of this effect. Subnuclear localization of the Kcnq1 domain in the presence or absence of the Kcnq1ot1 RNA will be an interesting avenue to pursue, to determine whether the ncRNA can act to tether the region to specific nuclear compartments [1,26,27,28]. We also need to explore whether these mechanisms are more widely used by other ncRNA, especially those that are antisense to coding genes. PLOS Genetics \| www.plosgenetics.org September 2012 \| Volume 8 \| Issue 9 \| e1002956 RNA purification Overall, our results are in accordance with the idea that the Kcnq1ot1 ncRNA itself is not responsible for directly silencing neighboring genes [13]. We posit that distinct cis-acting elements are made available for regulatory contacts depending on whether the act of transcription occurs or not, as previously suggested [30]. These associations have an additional layer of regulation that imposes the tissue-specificity of the patterns. Tissues for total RNA extraction were collected at appropriate days of gestation from F1 progeny. Polymorphisms between strains were used to distinguish between parental alleles. RNA was extracted using either TRIzol Reagent (Invitrogen #15596-018) or the Roche High Pure RNA Isolation Kit (#11828665001), following the manufacturer’s protocol. All RNA samples were subjected to DNAse treatment using Turbo DNA-free (Ambion #AM1907). Three to five biological samples were collected for each tissue. In conclusion, our results paint picture of the regulation at the Kcnq1 domain that is much more complex than previously assumed. Imprinting centers, which regulate extended domains of genes, are limited and counteracted by transcriptional mechanisms that may have evolved in response to the emergence of imprinting. Our studies on the Kcnq1 domain underscore that the expression pattern of a domain is ultimately determined by Following the manufacturer’s instructions, complementary DNA synthesis was performed on total RNA using SuperScript II Reverse Transcriptase (Invitrogen #18064-014). A Reverse Transcriptase negative control was used to ensure there was no DNA contamination. September 2012 \| Volume 8 \| Issue 9 \| e1002956 September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 8 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 Figure 6. Model for regulation of Kcnq1 in the embryonic heart. WT, wild-type; K-term, mutant mouse, in which transcription of Kcnq1ot1 is terminated prematurely; IF, methylation sensitive inhibitory factor. Maternal (m) events are shown above and paternal (p) events below the chromosome; filled circles, methylated DNA, empty circles, unmethylated DNA. Curved arrows represent interactions, bent arrows depict transcription. Ovals represent enhancers, which are inactive (light gray) at 10.5 dpc and active (black) at 14.5–16.5 dpc. doi:10.1371/journal.pgen.1002956.g006 Figure 6. Model for regulation of Kcnq1 in the embryonic heart. WT, wild-type; K-term, mutant mouse, in which transcription of Kcnq1ot1 is terminated prematurely; IF, methylation sensitive inhibitory factor. Maternal (m) events are shown above and paternal (p) events below the chromosome; filled circles, methylated DNA, empty circles, unmethylated DNA. Curved arrows represent interactions, bent arrows depict transcription. RNA purification Ovals represent enhancers, which are inactive (light gray) at 10.5 dpc and active (black) at 14.5–16.5 dpc. doi:10.1371/journal.pgen.1002956.g006 Real-time qRT–PCR and control templates. Following the PCR, restriction digests were performed with NlaIII, StuI, and Taqa1 (New England Biolabs) respectively. PCR and digestion products were run on 7% polyacrylamide gels and quantified using the Kodak Gel Logic 2000 imaging system. The ratio of paternal to maternal band intensities was calculated for Kcnq1. The Slc22a18 product was run on a 2% agarose gel and extracted using the E.Z.N.A Gel Extraction Kit (Omega Bio-tek, Inc.) The purified PCR product was then sent for sequencing analysis by Europhins MWG Operon. Sequences were aligned with Geneious Pro 4.6.5. The following primers were used to amplify the transcripts: Transcript levels of Kcnq1, Kcnq1ot1 and b-actin were analyzed on the ABI Step One Plus system. Reactions were conducted using the SYBR Green PCR Master Mix (ABI #4309155) in a final reaction volume of 20 ml. The PCR was performed under the following conditions: an initial denaturing step for 10 minutes at 95uC, an amplification step for 45 cycles of 95uC for 20 seconds, 55uC degrees for 30 seconds and 72uC for 30 seconds, and a final elongation step at 72uC for 2 minutes. The Kcnq1 transcript was detected using the following primers: 59-CAAAGACCGTG GCAGTAAC-39 and 59-CCTTCATTGCTGGCTACAAC-39. Kcnq1ot1 was detected using 59-GGTCTGAGGTAGGGATC AGG-39 and 59-GGCACACGGTATGAGAAAAGATTG-39. The Kcnq1 and Kcnq1ot1 levels were normalized to b-actin, using the primers 59-TGTTACCAACTGGGACGACA-39 and 59- CCATCACAATGCCTGTGGTA-39. Each qRT-PCR reaction was performed in triplicate with three biological replicates together with a negative control. The DDCT values of the Kcnq1 transcript were normalized to the b-actin transcript. The standard deviation for each ratio was determined and the error bars represent one standard deviation from the average ratio. A t-test and p-value was obtained for all comparisons. g p p y p Kcnq1 59-CATCGGTGCCCGTCTGAACAGG-39 and 59-TT GCTGGGTAGGAAGAGCTCAG-39 with NlaIII Kcnq1 59-CATCGGTGCCCGTCTGAACAGG-39 and 59-TT GCTGGGTAGGAAGAGCTCAG-39 with NlaIII Kcnq1ot1 59-GGTCTGAGGTAGGGATCAGG-39 and 59- GG CACACGGTATGAGAAAAGATTG-39 with StuI Cdkn1c 59-CGGACGATGGAAGAACTCTGG-39 and 59-TA CACCTTGGGACCAGCGTACTCC-39 with Taqa1 Slc22a18 59-GTGCTGCCTGTATTCCTGGT-39 and 59-CA Kcnq1 59-CATCGGTGCCCGTCTGAACAGG-39 and 59-TT GCTGGGTAGGAAGAGCTCAG-39 with NlaIII Kcnq1ot1 59-GGTCTGAGGTAGGGATCAGG-39 and 59- GG CACACGGTATGAGAAAAGATTG-39 with StuI Cdkn1c 59-CGGACGATGGAAGAACTCTGG-39 and 59-TA CACCTTGGGACCAGCGTACTCC-39 with Taqa1 Slc22a18 59-GTGCTGCCTGTATTCCTGGT-39 and 59-CA GTCCCACAACAGCAAAGA-39 Kcnq1ot1 59-GGTCTGAGGTAGGGATCAGG-39 and 59- GG CACACGGTATGAGAAAAGATTG-39 with StuI Cdkn1c 59-CGGACGATGGAAGAACTCTGG-39 and 59-TA CACCTTGGGACCAGCGTACTCC-39 with Taqa1 Slc22a18 59-GTGCTGCCTGTATTCCTGGT-39 and 59-CA GTCCCACAACAGCAAAGA-39 Kcnq1ot1 gene scan The following primers and associated restriction enzymes were used to scan the Kcnq1ot1 maternal transcript in P2 hearts from reciprocal crosses of C57BL/6J and B6(CAST7) mice: ChIP assays y Primary tissues were collected at neonatal stage P2, chopped into 1 mm pieces and cross-linked in 1% formaldehyde for 10 minutes. The formaldehyde was quenched with 0.125 M glycine for 5 minutes. The cells were spun down and the cell pellet was washed twice in PBS before re-suspension in lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40). Nuclei were extracted from the cells by incubating on ice for 10 minutes for brain tissue and 30 minutes for heart tissue, followed by 15 strokes in a dounce homogenizer. Nuclei were washed twice with PBS and resuspended in digestion buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2 and 4% NP-40). 107 nuclei were sonicated and 8 ug of chromatin was used for each IP, with 10% saved as the input. Protein A beads were primed with their respective antibody in IP buffer (20 mM Tris pH 8, 2 mM EDTA, 1% Triton X-100 and 150 mM NaCl) for 2 hours at 4uC. Chromatin was pre-cleared with IgG in IP buffer at 4uC for 1 hour. Chromatin and antibody/bead complexes were incubated overnight and washed 3 times with buffer #1 (20 mM Tris pH 8, 2 mM EDTA, 0.1% SDS and 150 mM NaCl), twice with buffer #2 (20 mM Tris pH 7.5, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 500 mM NaCl), once with buffer #3 (10 mM Tris pH 8, 1 mM EDTA, 0.25 M LiCl and 1% NP-40) and twice with TE. The chromatin was eluted by incubating chromatin/antibody/bead complex at 65uC for 15 minutes in a buffer containing 25 mM Tris pH 7.5, 10 mM EDTA and 0.5% SDS. A final concentration of 0.5% SDS and 1.5 ug/ul of Proteinase K was added to all samples including the input. The samples were incubated for 1 hour at 42uC and de- crosslinked overnight at 65uC in a shaking water bath, followed by phenol:chloroform extraction and ethanol precipitation. Pellets were re-suspended in 10 mM Tris. p 60 kb 59-GATCAGCATGGGTTATTGGA-39 and 59 –ATT AAGGGACCACAGCAAGG-39 with HpaI 60 kb 59-GATCAGCATGGGTTATTGGA-39 and 59 –ATT AAGGGACCACAGCAAGG-39 with HpaI C: 5 -CTGCTCTTCCTTACTCTAAGCACTGT Cn: 59-TAAGCCTTTGTTTGCCTCCTCTGGCCTAGAA GGCC Relative positions of the primers are shown in Figure 2A. Bands of interest were recovered after gel electrophoresis, cloned (TOPO-TA Cloning Kit, Life Technologies #K4500-01) and transformed into competent E.coli cells. Plasmid DNA was prepared from bacteria (Spin Mini Prep Kit (QIAGEN #30020)and sequenced (Eurofins MWG Operon). Sequences were aligned with Geneious Pro 4.6.5 [34]. The ChIP substrates were amplified using the ABI step-one plus machine and Sybr green reagents using the same conditions as the qRT-PCR assays. The data was analyzed using the percent input method. A full list of primers can be found in Table S2. Bisulfite mutagenesis sequencing 1 ug of DNA was mutagenized using the EZ DNA Methylation- Gold Kit (Zymo Research #5005) following the manufacturer’s protocol. Amplification of the Kcnq1ot1 CpG island was performed Allele-specific RT–PCR and quantification Allele-specific RT–PCR and quantification The Kcnq1, Kcnq1ot1, Cdkn1c, and Slc22a18 transcripts were amplified using Ruby Taq Master Mix (Affymetrix - #71191) in a 15 ml reaction. PCR reactions were performed with experimental 2 kb Kcnq1ot1 59-GGTCTGAGGTAGGGATCAGG-39 and 59- GGCACACGGTATGAGAAAAGATTG-39 with StuI September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 9 59 RACE assays 59 RACE assays to determine the Kcnq1ot1 start site were conducted using the 59 RACE system (Invitrogen), with RNA from three independent samples of neonatal hearts and livers. Primers for cDNA synthesis and nested PCR amplification (combined with a universal adapter primer) were as follows: An: 59-GCAACCACTGCGGCCTCCACCCCAAGTTCC CAATC CAATC B: 59-ATGACGAAACAAGATAAGACCTCAC B: 59-ATGACGAAACAAGATAAGACCTCAC Bn: 59-GGACAAACACTGAGGAGGATCGCGTTGAGCA AAGC C: 59-CTGCTCTTCCTTACTCTAAGCACTGT Cn: 59-TAAGCCTTTGTTTGCCTCCTCTGGCCTAGAA GGCC hromosome conformation capture (3C) assays 3C was performed as described previously [18]. Digestion efficiency at each restriction site was determined by comparing amplification with primers spanning the restriction site to amplification with primers immediately downstream on the digested template. No restriction bias was observed across the region assayed by 3C [18]. as previously described [33]. Primers for the Kcnq1 promoter were: KBS-F: 59-GGTTGGTGTATTGTAAGTGT KBS-R: 59- CTAACAACAATAACTACCC KBS-R: 59- CTAACAACAATAACTACCC PCR products were cloned into the pCR2.1-TOPO vector using a TOPO-TA Cloning Kit (Life Technologies #K4500-01). The bacterial colonies were cultured and plasmids purified using a Spin Mini Prep Kit (QIAGEN #30020) and plasmids were sent to Eurofins MWG Operon for sequencing. Sequences were analyzed with Geneious Pro 4.6.5. Data were obtained from three independent biological samples, with at least 10 clones sequenced from each. PCR efficiency of each combination of primers was assessed with a control template prepared from three BACs encompassing the Kcnq1ot1 region analyzed, as described previously. All PCR reactions were performed with RubyTaqTM (USB) as follows: 67u 200, 72u 200 for 15 cycles, with a decrease in the annealing temperature of 2u every 3 cycles, followed by 25 cycles of 55u 200, 72u 200 and a final extension step of 72u 19300. The linear range for each primer pair was determined by serial dilution. PCR reactions were performed with experimental and control templates in parallel and PCR products were run on 7% polyacrylamide gels and quantified using the Kodak Gel Logic 2000 imaging system. Crosslinking frequencies were calculated from duplicate PCR analyses of three independent 3C preparations. Interaction frequencies for Kcnq1 and Kcnq1ot1 were normalized to a control interaction to allow comparisons between different tissues and cell types. A full list of primers can be found in Table S1. Acknowledgments We thank Christine Soto and May Truong-Cao for technical support, Andrew Fedoriw for help with the RNA-FISH protocol, Temple University School of Medicine Confocal Core Facility for use of their microscope, Bettina Buffaro for aid in obtaining the images, and Keith Latham and Carmen Sapienza for comments on the manuscript. We also thank Deborah Mancini-Dinardo and Shirley Tilghman for sharing the K-term mutant mouse. We thank Christine Soto and May Truong-Cao for technical support, Andrew Fedoriw for help with the RNA-FISH protocol, Temple University School of Medicine Confocal Core Facility for use of their microscope, Bettina Buffaro for aid in obtaining the images, and Keith Latham and Carmen Sapienza for comments on the manuscript. We also thank Deborah Mancini-Dinardo and Shirley Tilghman for sharing the K-term mutant mouse. Figure S3 Allele-specific expression of imprinted genes in the Kcnq1 Domain. A) BxC, CxB and CxK-term Cdkn1c imprinting as determined by RT-PCR followed by allele-specific restriction digest in P2 Heart, Liver and Brain. M, maternal; P, paternal; ND, non-digested; D, Digested; B, C57BL/6J; C, B6(CAST7); KT, K- term. B) Sequencing analysis of Slc22a18 to determine allelic expression in P2 Heart, Liver and Brain. Asterisks indicates location of polymorphism. (TIF) Supporting Information Figure S6 Methylation analysis of the Kcnq1 promoter. A) Schematic of the Kcnq1 promoter analyzed by bisulfite mutagenesis sequencing. B) Representative methylation results for the Kcnq1 promoter. Filled in circles represent methylated CpGs, open circles represent non-methylated CpGs, numbers represent strands with the same pattern. Figure S1 Allele-specific analyses of Kcnq1ot1 in F1 hybrid mice from crosses between B6(CAST7) (C) and C57BL/6J (B) mice (CxB). A) Kcnq1ot1 imprinting pattern as determined by RT-PCR followed by allele-specific restriction digest. The primers are located 2 kb downstream of the canonical transcriptional start site. M, maternal; P, paternal; N, non-digested; D, Digested; B, C57BL/6J; C, B6(CAST7). B) Scanning of Kcnq1ot1 RNA for progeny from CxB crosses by RT-PCRs followed by allele-specific digestion. Primers are depicted in Figure 2. Results show absence of the maternal transcript 45 kb downstream of the transcriptional start site. M, maternal; P, paternal; N, non-digested; D, digested; B, C57BL/6J; C, B6(CAST7). C) Quantitative analysis by qRT- PCR of Kcnq1ot1 expression throughout development. RNA levels were normalized to b-actin. (TIF) Figure S7 Allele-Specific 3C at +189 kb to TSS. A 3C PCR was performed using a CxKT Heart substrate with the anchor at the Kcnq1 promoter and varying primer placed 189 kb downstream. The sample was then digested to determine allele of origin. B, BAC positive control; +L, 3C product; D, Digested 3C product; -L, 3C negative control; KT, K-term allele; P, Paternal. (TIF) Table S1 3C Primers. Primers used for the 3C scan on wild type and K-term samples. The primers are listed 59 to 39 across the domain and were used in conjunction with the anchor primer to determine if a 3C PCR product was present. (DOCX) Figure S2 RNA and DNA fluorescent in situ hybridization (FISH) against Kcnq1ot1. RNA (red, top left) and DNA (green, top right) FISH in primary cardiomyocytes with probes designed against Kcnq1ot1. The nucleus is stained blue with DAPI (bottom left). A merge of all three (bottom right) shows two signals for RNA (maternal and paternal transcripts) and that they completely overlap with the DNA signal within the nucleus. Approximately 100 nuclei were analyzed for RNA-DNA FISH expression and 74% of the nuclei analyzed were positive for two RNA signals. (TIF) Table S2 ChIP Primers. Left and right primers used for the ChIP ABI analysis. The primers are listed 59 to 39 across the domain and correlate to the 3C region scanned. (DOCX) Author Contributions Conceived and designed the experiments: LK NE. Performed the experiments: LK NS NE. Analyzed the data: LK NE. Wrote the paper: LK NE. Conceived and designed the experiments: LK NE. Performed the experiments: LK NS NE. Analyzed the data: LK NE. Wrote the paper: LK NE. Figure S4 KvDMR Methylation. A) Schematic of the CG-islands analyzed by bisulfite sequencing. B) Representative methylation 2. Sleutels F, Zwart R, Barlow DP (2002) The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415: 810–813. 1. Lee JT (2009) Lessons from X-chromosome inactivation: long ncRNA as guides and tethers to the epigenome. Genes Dev 23: 1831–1842. Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 results for the KvDMR on the maternal and paternal strands of E10.5 and P2 heart and P2 brain. Filled in circles represent methylated CpGs, open circles represent non-methylated CpGs. (TIF) Primary cardiomyocyte collection and culture y y y Collection of cardiomyoctes was performed as described (Sreejit, et. al., 2008) with the following modifications. The hearts were incubated in Hanks Balanced Salt Solution (HBSS) for 10 minutes on ice and the cells were cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine, penicillin (100 u/mL) and streptomycin (100 mg/mL). After the fibroblasts settled, the cardiomyocytes were collected and plated directly onto 4 well #1.5 sterile chambered glass coverslips (Thermo Fisher Scientific). PLOS Genetics \| www.plosgenetics.org September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 10 RNA and DNA FISH Cells were fixed and permeabilized using Life Technology’s FIX AND PERM kit following the manufacturer’s protocol. RNA FISH was performed as previously described (Golding, et. al., 2011). The probes for hybridization were made using Life Technology’s BioPrime DNA Labeling kit with fosmids WI1- 2505B3 and WI1-2753P18 (CHORI) following the manufacturer’s protocol. Cy3-dCTP (GE) was used for RNA detection and FITC- dUTP (Roche) was used for DNA detection. After the washes for the RNA probe hybridization, DNA FISH was performed incubating the cells with 0.01 N HCl with 0.1 mg/ml (0.01%) pepsin for 3 minutes at room temperature. The cells were rinsed with PBS and dehydrated with a series of ethanol washes. The DNA was denatured for 30 minutes at 85uC with 70% formamide in 26SSC. The cells were rinsed with 26SSC and the probe was hybridized overnight. The following day, excess probe was washed with SSC, mounted and stained with Vectashield augmented with DAPI (Vector Laboratories). Cells were imaged on a Leica Sp5 Confocal Microscope with Z-stacking. Figure S5 Allele-specific analyses of Kcnq1 in F1 hybrid mice from crosses between B6(CAST7) (C) and C57BL/6J (B) mice (CxB). A) Kcnq1 imprinting pattern as determined by RT-PCR followed by allele-specific restriction digest. M, maternal; P, paternal; N, non-digested; D, Digested; B, C57BL/6J; C, B6(CAST7). While the BxC cross shows a mono- to biallelic transition at 14.5 dpc, the CxB cross shows full biallelic expression at post neonatal day. B) qRT-PCR analysis of Kcnq1 expression in BxC and CxB hearts throughout development. Transcripts were normalized to b-actin and compared against BxC E10.5 heart using the DDCT method. C) Parental origin of Kcnq1 expression throughout cardiac development. The RT-PCR and allele specific bands were quantified and the ratio of paternal to maternal transcript was determined. Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 3. Smilinich NJ, Day CD, Fitzpatrick GV, Caldwell GM, Lossie AC, et al. (1999) A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome. Proc Natl Acad Sci U S A 96: 8064–8069. 19. Bokil NJ, Baisden JM, Radford DJ, Summers KM (2010) Molecular genetics of long QT syndrome. Mol Genet Metab 101: 1–8. 20. Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE, et al. (2003) Disruption of imprinted gene methylation and expression in cloned preimplan- tation stage mouse embryos. Biol Reprod 69: 902–914. 4. Borsoni G, Tonlorenzi R, Simmler MC, Dandolo L, Arnaud D, et al. (1991) Characterization of a murine gene expressed from the inactive X chromosome. Nature 351: 325–328. g y p 21. Fitzpatrick GV, Pugacheva EM, Shin JY, Abdullaev Z, Yang Y, et al. (2007) Allele-specific binding of CTCF to the multipartite imprinting control region KvDMR1. Mol Cell Biol 27: 2636–2647. 5. Lee JT, Lu N (1999) Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell 99: 47–57. 22. Yatsuki H, Joh K, Higashimoto K, Soejima H, Arai Y, et al. (2002) Domain regulation of imprinting cluster in Kip2/Lit1 subdomain on mouse chromosome 7F4/F5: large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative imprinting control region. Genome Res 12: 1860–1870. 6. Lee MP, Hu R, Johnson LA, Feinberg AP (1997) Human KVLQT1 gene shows tissue-specific imprinting and encompasses Beckwith-Wiedemann syndrome chromosomal rearrangements. Nature Genet 15: 181–185. g y y putative imprinting control region. Genome Res 12: 1860–1870. 7. Fitzpatrick GV, Soloway PD, Higgins MJ (2002) Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1. Nat Genet 32: 426–431. 23. Mohammad F, Mondal T, Guseva N, Pandey GK, Kanduri C (2010) Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1. Development 137: 2493–2499. 8. Mancini-Dinardo D, Steele SJ, Levorse JM, Ingram RS, Tilghman SM (2006) Elongation of the Kcnq1ot1 transcript is required for genomic imprinting of neighboring genes. Genes Dev 20: 1268–1282. 24. Woodfine K, Huddleston JE, Murrell A (2011) Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue. Epigenetics Chromatin 4: 1–13. g g g 9. Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, et al. Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 (2004) Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation. Nat Genet 36: 1291–1295. 25. Chotalia M, Smallwood SA, Ruf N, Dawson C, Lucifero D, et al. (2009) Transcription is required for establishment of germline methylation marks at imprinted genes. Genes Dev 23: 105–117. independent of DNA methylation. Nat Genet 36: 1291–1295. 10. Umlauf D, Goto Y, Cao R, Cerqueira F, Wagschal A, et al. (2004) Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes. Nat Genet 36: 1296–1300. p g 26. Mondal T, Kanduri C (2012) Noncoding RNA scaffolds in pluripotency. Circ Res 110: 1162–1165. 27. Laurent GS, Savva YA, Kapranov P (2012) Dark matter RNA: an intelligent scaffold for the dynamic regulation of the nuclear information landscape. Front Genet 3: 1–11. 11. Pandey RR, Mondal T, Mohammad F, Enroth S, Redrup L, et al. (2008) Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol Cell 32: 232–246. 28. Wang KC, Chang HY (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell 43: 904–914. 12. Terranova R, Yokobayashi S, Stadler MB, Otte AP, van Lohuizen M, et al. (2008) Polycomb Group Proteins Ezh2 and Rnf2 Direct Genomic Contraction and Imprinted Repression in Early Mouse Embryos. Dev Cell 15: 668–679. 29. Redrup L, Branco MR, Perdeaux ER, Krueger C, Lewis A, et al. (2009) The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing. Development 136: 525–530. 13. Golding MC, Magri LS, Zhang L, Lalone SA, Higgins MJ, et al. Depletion of Kcnq1ot1 non-coding RNA does not affect imprinting maintenance in stem cells. Development 138: 3667–3678. 30. Pauler FM, Koerner MV, Barlow DP (2007) Silencing by imprinted noncoding RNAs: is transcription the answer? Trends Genet 23: 284–292. p 14. Shin JY, Fitzpatrick GV, Higgins MJ (2008) Two distinct mechanisms of silencing by the KvDMR1 imprinting control region. Embo J 27: 168–178. 31. Bonifer C (2000) Developmental regulation of eukaryotic gene loci: which cis- regulatory information is required? Trends Genet 16: 310–315. 15. Gould TD, Pfeifer K (1998) Imprinting of mouse Kvlqt1 is developmentally regulated. Hum Mol Genet 7: 483–487. 32. Engel N, Bartolomei MS (2003) Mechanisms of insulator function in gene regulation and genomic imprinting. Int Rev Cytol 232: 89–127. g 16. Latos PA, Barlow DP (2009) Regulation of imprinted expression by macro non- coding RNAs. References 2. Sleutels F, Zwart R, Barlow DP (2002) The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415: 810–813. 1. Lee JT (2009) Lessons from X-chromosome inactivation: long ncRNA as guides and tethers to the epigenome. Genes Dev 23: 1831–1842. September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 11 Kcnq1ot1 Does Not Regulate Imprinting of Kcnq1 RNA Biol 6: 100–106. 33. Rivera RM, Stein P, Weaver JR, Mager J, Schultz RM, et al. (2008) Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum Mol Genet 17: 1–14. 17. Paulsen M, Davies KR, Bowden LM, Villar AJ, Franck O, et al. (1998) Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5. Hum Mol Genet 7: 1149–1159. 34. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2011) Geneious v5.4. http://www.geneious.com 18. Korostowski L, Raval A, Breuer G, Engel N (2011) Enhancer-driven chromatin interactions during development promote escape from silencing by a long non- coding RNA. Epigenetics Chromatin 4: 21–32. September 2012 \| Volume 8 \| Issue 9 \| e1002956 PLOS Genetics \| www.plosgenetics.org 12
https://openalex.org/W2123601953	https://www.scielo.br/j/acb/a/gM6XNrCW6s9DSQTYqdNJnYt/?lang=pt&format=pdf	Portuguese	null	Efeito do tamoxifeno no perfil das proteínas plasmáticas em condição de diabetes mellitus tipo 1	Acta Cirúrgica Brasileira	2,005	cc-by	6,604	1 Trabalho realizado pelo Programa de Pós-Graduação em Ciências Farmacêuticas da Universidade Federal do Rio Grande do Norte, Natal – RN, Brasil. 2 Aluna de Mestrado do Programa de Pós-Graduação em Ciências Farmacêuticas, Centro de Ciências da Saúde, UFRN. 3 Farmacêutico Bioquímico graduado pela UFRN. 4 Acadêmico de Farmácia da UFRN, bolsista do programa de Iniciação Cientifica PIBIC/ CNPq. 5 Mestre em Bioquímica – Departamento de Bioquímica da UFRN, Farmacêutico da Oncoclínica São Marcos, Natal – RN. 6 Professora, Doutora, do Departamento de Patologia da UFRN. 7 Professora, Doutora, Coordenadora do Programa de Pós-Graduação em Ciências Farmacêuticas da UFRN. 8 Professora, Doutora, Vice-coordenadora do Programa de Pós-Graduação em Ciências Farmacêuticas da UFRN Efeito do tamoxifeno no perfil das proteínas plasmáticas em condição de diabetes mellitus tipo 1 1 Teresa Cristina P. Silva2, Saul B. Mota3, Maria Margareth C. Almeida2, Elaine Cristina S. Ferreira2, Marcela A. G. Ururahy4, João Felipe Bezerra4, Ney M. L. Pereira5, Ana M. O. Ramos6, Maria das Graças Almeida7, Adriana A. Rezende 8 Silva TCP, Mota SB, Almeida MMC, Ferreira ECS, Ururahy MAG, Bezerra JF, Pereira NML, Ramos AMO, Almeida MG, Rezende AA. Efeito do tamoxifeno no perfil das proteínas plasmáticas em condição de diabetes mellitus tipo 1. Acta Cir Bras [serial on line] Available from: URL: htt://www.scielo.br/acb. RESUMO – Objetivo: Considerando-se que importantes avanços científicos têm sido obtidos através de estudos com Diabetes mellitus experimental, e que a ação do tamoxifeno em humanos permanece obscura, o presente trabalho objetiva acompanhar as modificações promovidas pelo diabetes e tamoxifeno no perfil eletroforético das proteínas plasmáticas. Métodos: Foram utilizados 27 ratos fêmeas Wistar (180-220g peso corporal), divididos randomicamente em 5 grupos: C1 (n=3, receberam veículo), C2 (n=3, sem tratamento), T (n=5, tratados com tamoxifeno, 0,3mg/kg/dia), D (n=8, diabéticos experimentais por estreptozotocina, 45mg/Kg) e DT (n=8, diabéticos tratados com tamoxifeno). A eletroforese foi realizada em acetato de celulose, pH 8,6-8,8, cuba TECNOW, e as fitas foram coradas em Ponceau S. As proteínas totais foram determinadas pelo método do Biureto (Kit Labtest). Os proteinogramas foram obtidos em densitômetro BioSystems BTS-235. Resultados: Albumina diminuiu progressivamente nos grupos T, D e DT; a fração a 1 aumentou nos grupos T e DT; a fração a 2 aumentou nos grupos T e D, havendo efeito aditivo no grupo DT; a fração b aumentou nos grupos T e D; a fração g aumentou nos grupos T, D e DT. Conclusão: Os resultados indicam uma resposta de fase aguda, com efeito aditivo do tamoxifeno e diabetes, sugerindo uma provável lesão hepática. DESCRITORES: Tamoxifeno. Proteínas plasmáticas. Diabetes mellitus. Eletroforese de proteínas. com aumento do risco de câncer endometrial, trombose, embolia pulmonar e esteatose hepática 2,5,6. Introdução Primeiro, a proteína C-reativa (PCR) e a 1- antiquimotripsina; depois, dentro das primeiras 12 horas, a a 1 glicoproteína ácida (AAG); em seguida, a a 1-antitripsina (AAT), haptoglobina, C4 e fibrinogênio; e, finalmente, C3 e ceruloplasmina. As proteínas reagentes de fase aguda são consideradas indicadores mais fiéis da resposta sistêmica fren- te aos processos inflamatórios e infecciosos, quando comparadas a outras variáveis, tais como febre, aumento da taxa de hemossedimentação e/ou presença de leucocitose associada a neutrofilia. Embora a resposta de fase aguda não seja específica, pode ser usada na prática clínica como parte do diagnóstico, tanto na avaliação da intensidade da atividade inflamatória como na discriminação de doenças inflamatórias das não-inflamatórias. Além disso, são úteis na monitorização da resolução tecidual de traumas ou inflamação e também na avaliação da resposta orgânica ao tratamento. Dessa forma, a detecção de um processo inflamatório agudo necessita de medida sensível e rápida de proteínas de fase agu- da 9,10,11,12,13,14,15. Na maior parte das condições patológicas que envolvem as proteínas plasmáticas, as alterações abrangem não apenas O Diabetes mellitus tipo 1 é uma doença inflamatória autoimune associada com alterações na permeabilidade vascular, levan- do a muitas complicações incluindo nefropatia, retinopatia, hipertensão, e neuropatia. A disfunção endotelial tem sido consistentemente demonstrada em modelos animais de dia- betes, bem como em pacientes com diabetes tipo 1 e tipo 2, sendo sugerido que o processo inflamatório está envolvido na patogênese da disfunção endotelial e da aterosclerose. Estes dados são confirmados pela observação de níveis ele- vados de proteína C reativa, um marcador de inflamação sistêmica, em pacientes com intolerância a glicose. Além dis- so, correlações entre proteína C reativa e marcadores da disfunção endotelial tem sido relatadas tanto em pacientes diabéticos tipo 1 como em pacientes diabéticos tipo 2. Dentre os fatores que podem contribuir para o estado inflamatório vascular no diabetes estão o estresse oxidativo, a hiperglicemia, os produtos de glicação avançados (AGEs) e a oxidação do LDL (lipoproteína de baixa densidade). Tanto a inflamação intra-arterial como o estímulo extravascular pode induzir ativação da resposta de fase aguda, levando a um aumento de parâmetros inflamatórios como a proteína C reativa 16. Introdução A inflamação aguda é geralmente acompanhada por uma res- posta sistêmica, conhecida como resposta de fase aguda, que é caracterizada por uma rápida alteração nos níveis das várias proteínas plasmáticas. Há liberação de amplo espectro de mediadores por parte dos macrófagos teciduais e monócitos sanguíneos, dos quais as citocinas, principalmente o fator de necrose tumoral (TNF) e as interleucinas IL-1 e IL-6, são as principais mediadoras da síntese das proteínas de fase aguda séricas. Durante a resposta inflamatória, a ligação destas citocinas aos seus respectivos receptores nos hepatócitos induz a produção do fator de transcrição específico do fígado NF-IL6, que promove a transcrição dos genes que codificam diversas proteínas de fase aguda. Coincidentemente, os ní- veis do fator de transcrição C/EBP, que é expresso constitutivamente nos hepatócitos e que promove a transcri- ção da albumina e transferrina, diminuem e os níveis de albumina e transferrina, conseqüentemente diminuem. Desta maneira o aumento da síntese de proteínas reagentes de fase aguda positivas é acompanhado de uma diminuição na sínte- se de pré-albumina, albumina e transferrina (proteínas reagentes negativos da fase aguda), de modo que só ocorre um leve aumento do total de proteínas do plasma. O aumento da concentração das proteínas de fase aguda séricas, desig- nadas como reagentes de fase aguda (RFA), ocorre de forma proporcional à intensidade da agressão e da destruição tecidual. Supõe-se que todas tomem parte no complexo pro- cesso defensivo da inflamação, para tanto, apresentam ação antiproteásica e antioxidante, podendo agir, como ativadores do sistema complemento. Os níveis plasmáticos das proteí- nas individuais da fase aguda sobem com velocidades diferentes. Primeiro, a proteína C-reativa (PCR) e a 1- antiquimotripsina; depois, dentro das primeiras 12 horas, a a 1 glicoproteína ácida (AAG); em seguida, a a 1-antitripsina (AAT), haptoglobina, C4 e fibrinogênio; e, finalmente, C3 e ceruloplasmina. As proteínas reagentes de fase aguda são consideradas indicadores mais fiéis da resposta sistêmica fren- te aos processos inflamatórios e infecciosos, quando comparadas a outras variáveis, tais como febre, aumento da taxa de hemossedimentação e/ou presença de leucocitose associada a neutrofilia. Embora a resposta de fase aguda não seja específica, pode ser usada na prática clínica como parte do diagnóstico, tanto na avaliação da intensidade da atividade inflamatória como na discriminação de doenças inflamatórias das não-inflamatórias. Introdução O tamoxifeno (TAM) é o agente antiestrogênico não esteroidal comumente utilizado no tratamento do câncer de mama e mais recentemente seu uso tem sido aprovado na quimioprevenção em mulheres com elevado risco de desen- volvimento de câncer de mama. Atua bloqueando a ação do estrogênio, que por sua vez, estimula a divisão celular na mama 1,2,3. O TAM pode produzir efeito estrogênico total ou parcial, bem como efeito antagonista, dependendo do teci- do alvo e da espécie estudada. Existem dois receptores estrogênicos (RE), a e b, onde as diferenças entre estes subtipos de receptores em relação à afinidade de ligação e distribuição nos tecidos poderiam contribuir para explicar a ação seletiva dos agonistas ou antagonistas do RE em dife- rentes órgãos e sistemas. Este fármaco apresenta ação agonista em determinados tecidos, como o endometrial (au- mentando o risco de câncer de endométrio), o ósseo (prevenindo a osteoporose pré e pós-menopausa) e o hepá- tico (metabolismo lipoprotéico), e apresenta ação antagonista na mama 1,4,5. O tratamento com tamoxifeno está associado As proteínas são polímeros de aminoácidos unidos por liga- ções covalentes denominadas de ligações peptídicas. Os diferentes grupamentos R encontrados nos aminoácidos in- fluenciam na estrutura, funcionalidade e nas propriedades das proteínas individuais, que podem ser usadas para a sua separação, identificação e dosagem 7. Dentre as técnicas de separação de proteínas encontra-se a eletroforese, que se refere à migração de solutos ou partículas carregadas sob a influência de um campo elétrico. A velocidade de migração eletroforética depende de muitos fatores, que incluem carga elétrica global da molécula, tamanho e forma da molécula, intensidade do campo elétrico, propriedades do meio de sus- tentação e temperatura de operação. Em um proteinograma obtido do soro normal, costumam ser observadas duas frações, sendo uma de albumina e outra composta pelas globulinas que são divididas em a 1, a 2, b e g globulinas 8. O processo infamatório agudo decorrente de infecções, da- nos teciduais ou provenientes de outros estímulos, induz 86 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 uma série complexa de reações que tem como finalidade inibir a continuidade do dano tecidual, isolando e destruindo o organismo agressor e ativando o processo de reparação ne- cessário para o retorno do organismo às funções normais. Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 87 Introdução Além disso, são úteis na monitorização da resolução tecidual de traumas ou inflamação e também na avaliação da resposta orgânica ao tratamento Dessa forma a plo dos componentes protéicos, dando lugar a quadros mais característicos dos processos fisiopatológicos. A interpreta- ção racional, com base no conhecimento do significado fisiológico e fisiopatológico de cada componente, e das as- sociações entre as alterações dos diversos componentes, pode trazer indicações úteis ao diagnóstico 8. uma série complexa de reações que tem como finalidade inibir a continuidade do dano tecidual, isolando e destruindo o organismo agressor e ativando o processo de reparação ne- cessário para o retorno do organismo às funções normais. A inflamação aguda é geralmente acompanhada por uma res- posta sistêmica, conhecida como resposta de fase aguda, que é caracterizada por uma rápida alteração nos níveis das várias proteínas plasmáticas. Há liberação de amplo espectro de mediadores por parte dos macrófagos teciduais e monócitos sanguíneos, dos quais as citocinas, principalmente o fator de necrose tumoral (TNF) e as interleucinas IL-1 e IL-6, são as principais mediadoras da síntese das proteínas de fase aguda séricas. Durante a resposta inflamatória, a ligação destas citocinas aos seus respectivos receptores nos hepatócitos induz a produção do fator de transcrição específico do fígado NF-IL6, que promove a transcrição dos genes que codificam diversas proteínas de fase aguda. Coincidentemente, os ní- veis do fator de transcrição C/EBP, que é expresso constitutivamente nos hepatócitos e que promove a transcri- ção da albumina e transferrina, diminuem e os níveis de albumina e transferrina, conseqüentemente diminuem. Desta maneira o aumento da síntese de proteínas reagentes de fase aguda positivas é acompanhado de uma diminuição na sínte- se de pré-albumina, albumina e transferrina (proteínas reagentes negativos da fase aguda), de modo que só ocorre um leve aumento do total de proteínas do plasma. O aumento da concentração das proteínas de fase aguda séricas, desig- nadas como reagentes de fase aguda (RFA), ocorre de forma proporcional à intensidade da agressão e da destruição tecidual. Supõe-se que todas tomem parte no complexo pro- cesso defensivo da inflamação, para tanto, apresentam ação antiproteásica e antioxidante, podendo agir, como ativadores do sistema complemento. Os níveis plasmáticos das proteí- nas individuais da fase aguda sobem com velocidades diferentes. Determinação da concentração de glicose Determinação da concentração de glicose Indução do diabetes mellitus Após jejum alimentar não hídrico, ad libitum, por cerca de 12 horas, ratos Wistar fêmeas de aproximadamente 180-220g re- ceberam, por via intraperitoneal, estreptozotocina (Sigma) dissolvida em tampão citrato de sódio 0,01M, pH 4,5, na pro- porção de 45 mg/kg de peso corporal 19. Eletroforese em acetato de celulose A eletroforese das proteínas plasmáticas foi realizada utili- zando-se fitas de acetato de celulose Midifilm (5,7 x 14 cm – Biomidi), e como amostra o soro dos animais experimentais. Foi utilizada uma cuba eletroforética TECNOW e a migração das proteínas séricas foi realizada em tampão Tris-Veronal (Biomidi) pH 8,6 – 8,8. As fitas de acetato de celulose foram previamente incubadas no mesmo tampão por dez minutos para em seguida serem posicionadas na cuba e receberem as amostras. A aplicação ocorreu no pólo negativo da cuba uti- lizando cerca de 3 a 5 ml de soro. Após migração de 50 minutos as fitas foram coradas em Ponceau S 5g/L, por aproximada- mente 8 minutos, e descoradas em solução aquosa de ácido acético a 5%. A seguir, foram fixadas em metanol PA por 1 minuto e transparentizadas em solução de 85% metanol, 14% ácido acético PA, 1% glicerina por 2 minutos. Finalmente fo- ram secas em estufa à 60º C, durante 5 minutos, e a leitura do resultado e quantificação das frações protéicas foi realizada por densitometria, usando o densitômetro BioSystems BTS 235. O aparelho mede a superfície de cada pico e deduz a percentagem. Utilizando a concentração das proteínas totais, previamente determinada, o aparelho deduz a quantidade de cada fração em g/dL. Durante o período de 30 dias, foi feita pesagem semanal dos animais e observação do quadro de poliúria, polidipsia e polifagia para os animais diabéticos experimentais. Foi reali- zada a avaliação das fases do ciclo estral dos animais de acordo com DUARTE et al 19. Após o período de 30 dias, os animais foram sacrificados por deslocamento cervical. Todos os procedimentos em relação aos animais foram exe- cutados de acordo com o Guia padrão para Cuidados e Uso de animais de laboratórios 20. Indução do diabetes mellitus Preparo das amostras de sangue Preparo das amostras de sangue As amostras de sangue destinadas à avaliação foram obtidas dos animais submetidos a jejum alimentar não hídrico, ad libitum, por cerca de 8-10 horas, sendo estas analisadas no período de 30 dias. Os animais foram sacrificados por deslo- camento cervical e submetidos à coleta de cerca de 6mL de sangue. Considerando que importantes avanços científicos têm sido obtidos através de estudos realizados em animais com Dia- betes mellitus experimental e que a ação do tamoxifeno em humanos ainda permanece obscura, o presente trabalho tem por objetivo acompanhar as modificações promovidas pelo DM, bem como pelo TAM, no perfil eletroforético das prote- ínas plasmáticas. O sangue coletado, em um tubo sem anticoagulante, foi centrifugado utilizando uma centrífuga de bancada não refri- gerada Sigma 2-3 à 1.500 min -1, durante 10 minutos. O soro obtido foi utilizado para a dosagem de glicose, proteínas to- tais e posterior eletroforese de proteínas. Animais A determinação da concentração de glicose foi realizada uti- lizando-se o método da enzima glicose oxidase peroxidase (kit LABTEST), em um espectrofotômetro RA 50 (Bayer Diag- nósticos, Irlanda, 1998). Foram utilizados 27 ratos fêmeas da linhagem Wistar, de peso entre 180g - 220g, fornecidos pelo Biotério do Centro de Ciên- cias da Saúde da Universidade Federal do Rio Grande do Norte, mantidos em gaiolas com no máximo três animais, com água e ração comercial purina ad libitum, e com ciclo claro- escuro 12/12h. Determinação da concentração de proteínas totais A determinação da concentração das proteínas totais foi rea- lizada utilizando o método do Biureto, onde as ligações peptídicas das proteínas (-HN-CO-) reagem com íons cúpricos em meio alcalino (Reagente do Biureto) formando um comple- xo de coloração violeta, cuja absorbância medida em 545 nm (espectrofotômetro RA 50 - Bayer Diagnósticos, Irlanda, 1998) é diretamente proporcional à concentração de proteínas na amostra. Os animais foram divididos em cinco grupos: Grupo (C1): n=3 animais recebendo apenas veículo (sorbitol + tween 20 + carboximetilcelulose - CMC); Grupo (C2): n=3 animais sem qualquer tipo de tratamento; Grupo (T): n=5 animais com tra- tamento exclusivo com tamoxifeno; Grupo (D) n=8 animais diabéticos experimentais por estreptozotocina; e Grupo (DT): n=8 animais diabéticos experimentais em tratamento com tamoxifeno. Eletroforese em acetato de celulose Introdução GOMES et al, estudaram um grupo de pacientes com diabetes mellitus tipo 1 sem complicações clínicas macro e microvasculares para determinar se os níveis de proteínas de fase aguda (alfa 1 glicoproteína ácida, proteína C reativa e fibrinogênio) estavam alterados e se esses níveis de proteí- nas de fase aguda estavam relacionados com o controle glicêmico. De acordo com os resultados obtidos, as proteí- nas de fase aguda, um conhecido marcador de inflamação, apresentaram-se aumentadas em pacientes com diabetes mellitus tipo 1 quando comparados aos controles, indepen- dentemente do controle glicêmico 17. A variedade de proteínas séricas encontradas em ratos é diferente daquelas encontradas em humanos. Algumas pro- teínas séricas estão presentes em concentrações muito mais baixa em ratos do que em humanos, como por exemplo, a haptoglobina em condições basais. Outras proteínas estão presentes em concentrações bem mais elevadas em ratos, como é o caso da proteína C reativa. Algumas proteínas são espécie especifica para ratos, como é o caso da expressão da a1 macroglobulina em condições basais e da expressão da proteína serina inibidora 3 durante um processo inflama- tório 18. Recentes investigações sobre proteínas séricas têm demons- trado que as diferenças qualitativas e quantitativas entre as espécies de ratos (Sprague-Dawley, Lewis e Wistar Kyoto) são mínimas, pelo menos em condições basais. Do mesmo Na maior parte das condições patológicas que envolvem as proteínas plasmáticas, as alterações abrangem não apenas uma determinada proteína, mas um grupo razoavelmente am- Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 87 sorbitol 10% contendo carboximetilcelulose (CMC) 0,5% e 10 gotas de Tween 20. modo outros estudos mostraram que a idade pouco influen- ciou no padrão das proteínas séricas dos ratos estudados, porém GIANAZZA et al 18 encontrou diversas diferenças no perfil protéico entre machos e fêmeas: a hemopexina e a pro- teína C reativa são aproximadamente 50% e 25%, respectivamente mais altas em fêmeas, enquanto que a a1- antitripsina é aproximadamente 30% mais alta em machos 18. 88 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 Tratamento com tamoxifeno Os animais foram tratados diariamente com tamoxifeno, por via oral, em uma proporção de 0,3mg/kg/dia, por um período de 30 dias. O tamoxifeno foi preparado em solução aquosa de 88 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 Caracterização do estado diabético Análise estatística Os dados obtidos foram submetidos à análise de variância (ANOVA) para saber se existia diferença significativa entre os grupos estudados para cada fração do proteinograma, e em seguida o teste de Tukey foi aplicado para detectar quais as médias apresentavam diferença significativa. O nível de rejeição da hipótese de nulidade foi fixado em 5% (p £ 0,05). Foi utilizado o programa STATISTICA for Windows versão 5.1 e o programa Excel 2000 MicrosoftÒ para a montagem dos bancos de dados e gráficos. A caracterização do estado diabético experimental dos animais utilizados foi realizada através do acompanhamento da glicemia (Figura 1) e da evolução do peso corporal (Figura 2) de cada animal, por um período de 30 dias. Os animais diabéticos apresen- taram-se hiperglicêmicos 24 horas após a administração de estreptozotocina, com valores médios de glicemia sempre superi- ores a 250 mg/dL. Em relação ao ganho de peso corporal os animais controles atingiram um peso médio de 210g enquanto que os diabéti- cos experimentais o peso médio final foi de 180 - 190g. Avaliação do ciclo estral A avaliação geral dos proteinogramas revelou que todos os ani- mais apresentaram perfis eletroforéticos dentro do padrão geralmente obtido para humanos, onde as proteínas séricas são separadas em 2 frações distintas, sendo uma de albumina e outra composta pelas globulinas, entre elas a 1, a 2, b e g globulinas. A Tabela 1 mostra os respectivos valores da média e desvio pa- drão das frações protéicas obtidas para cada grupo estudado, no período de 30 dias, e a Figura 3 mostra a representação gráfica do comportamento das frações protéicas para cada grupo estu- dado, a partir dos quais será possível avaliar as possíveis alterações observadas em função do tratamento realizado. As fases do ciclo estral dos animais controles apresentaram- se regulares durante todo o período de estudo, entretanto para os grupos T, D e DT observou-se que as fases do ciclo estral apresentaram-se irregulares ou mesmo estacionadas durante horas ou mesmo dias na mesma fase. Considerando que a irregularidade do ciclo estral não interferiu no perfil eletroforético das proteínas plasmáticas, todos os animais foram utilizados (resultados não mostrados). Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 89 FIGURA 1 – Glicemia dos animais controle com veículo (C1), con- trole sem veículo (C2), tratados com tamoxifeno (T), diabéticos (D) e diabéticos tratados com tamoxifeno (DT), duran- te o período de 30 dias. Os resultados são ex- pressos como média ± desvio padrão. * (p < 0,05) vs C1, C2, T FIGURA 2 – Peso cor- poral dos animais controle com veículo (C1), controle sem veí- culo (C2), tratados com tamoxifeno (T), diabéti- cos (D) e diabéticos tratados com tamoxifeno (DT), durante o período de 30 dias. Os resulta- dos são expressos como média ± desvio padrão. * (p < 0,05) vs D, DT FIGURA 1 – Glicemia dos animais controle com veículo (C1), con- trole sem veículo (C2), tratados com tamoxifeno (T), diabéticos (D) e diabéticos tratados com tamoxifeno (DT), duran- te o período de 30 dias. Os resultados são ex- pressos como média ± desvio padrão. * (p < 0,05) vs C1, C2, T FIGURA 2 – Peso cor- poral dos animais controle com veículo (C1), controle sem veí- culo (C2), tratados com tamoxifeno (T), diabéti- cos (D) e diabéticos tratados com tamoxifeno (DT), durante o período de 30 dias. Os resulta- dos são expressos como média ± desvio padrão. FIGURA 1 – Glicemia dos animais controle com veículo (C1), con- trole sem veículo (C2), tratados com tamoxifeno (T), diabéticos (D) e diabéticos tratados com tamoxifeno (DT), duran- te o período de 30 dias. Os resultados são ex- pressos como média ± desvio padrão. Avaliação do ciclo estral * (p < 0,05) vs D, DT Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 89 Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 89 TABELA 1 – Valores relativos e absolutos das frações protéicas obti- das a partir do proteinograma dos ani- mais controle com veículo (C1), controle sem veículo (C2), trata- dos com tamoxifeno (T), diabéticos (D) e diabéti- cos tratados com tamoxifeno (DT) Os valores são expressos como média ± Desvio Padrão. (p < 0,05) vs C1, C2. ♦(p< 0,05) vs C1, C2, T, D. + (p< 0,05) vs C1, C2, T. FIGURA 3 – Evolução das frações protéicas dos animais controle com veículo (C1), con- trole sem veículo (C2), tratados com tamoxifeno (T), diabéticos (D) e diabéticos tratados com tamoxifeno (DT), duran- te o período de 30 dias TABELA 1 – Valores relativos e absolutos das frações protéicas obti- das a partir do proteinograma dos ani- mais controle com veículo (C1), controle sem veículo (C2), trata- dos com tamoxifeno (T), diabéticos (D) e diabéti- cos tratados com tamoxifeno (DT) Os valores são expressos como média ± Desvio Padrão. (p < 0,05) vs C1, C2. ♦(p< 0,05) vs C1, C2, T, D. + (p< 0,05) vs C1, C2, T. FIGURA 3 – Evolução das frações protéicas dos animais controle com veículo (C1), con- trole sem veículo (C2), tratados com tamoxifeno (T), diabéticos (D) e diabéticos tratados com tamoxifeno (DT), duran- te o período de 30 dias (p< 0,05) vs C1, C2, T, D. + (p< 0,05) vs C1, C2, T. FIGURA 3 – Evolução das frações protéicas dos animais controle com veículo (C1), con- trole sem veículo (C2), tratados com tamoxifeno (T), diabéticos (D) e diabéticos tratados com tamoxifeno (DT), duran- te o período de 30 dias + (p< 0,05) vs C1, C2, T. (p< 0,05) vs C1, C2, T. 90 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 Discussão Como pode ser observado na Figura 3, o grupo T apresentou um aumento e diminuição significativa da fração a 1 e albumina, respectivamente, caracterizando um processo de fase aguda, seguido de um leve aumento das g globulinas indicando o início de um processo de defesa. Para o grupo D houve uma diminuição progressiva e signifi- cativa da fração albumina quando comparada ao grupo concentração de albumina livre e aumentando a concentra- ção de frutosamina 7,8. A diminuição da albumina não poderia estar associada a uma provável microalbuminúria, geralmen- te causada pela nefropatia diabética, visto que o período de 30 dias seria insuficiente para desencadear uma lesão renal. Além disso, a concentração sérica de creatinina manteve-se dentro dos valores de referência durante o período de 30 dias o que também descarta a possibilidade de uma possível lesão renal (resultados não mostrados). As frações a 1 e a 2 permane- ceram praticamente inalteradas em relação ao grupo controle, mas a análise da fração b revelou um aumento significativo quando comparado ao grupo controle e até mesmo em rela- ção ao grupo tratado com tamoxifeno, o que poderia ser resultante do aumento do colesterol e conseqüentemente das b-lipoproteínas em função do Diabetes mellitus 7. A fração g apresentou um aumento significativo quando comparado ao grupo controle e ao grupo tratado com TAM, provavelmente devido a um possível processo inflamatório associado ao DM, visto que nesta fração se encontram as imunoglobulinas e a proteína C reativa. A avaliação geral dos resultados para o grupo D, como pode ser observado na Figura 3, revela um quadro mais característico de um processo de defesa uma vez que as frações b e g apresentaram aumentos mais representa- tivos seguido de diminuição de albumina. Quando a condição do diabetes foi associada ao tratamento com TAM, a redução da fração albumina foi ainda mais signi- ficativa quando comparada aos demais grupos. Esta redução está relacionada a um efeito da associação entre DM e o TAM e suas respectivas alterações hepáticas. A redução da fração albumina poderia ser avaliada como um processo inici- al da esteatose hepática provocada pela ação do tamoxifeno junto com o DM, ou mesmo pelo comprometimento da sínte- se protéica, uma vez que a biotransformação do TAM ocorre em nível hepático e seus metabólitos causam lesão do DNA dos hepatócitos 6,21. Discussão animais considerados diabéticos mantiveram o nível glicêmico superior ao dos controles. A administração do veículo em um dos grupos controles (C1) foi realizada para verificar se a constituição do veículo poderia contribuir de maneira significativa no estado de hiperglicemia gerado pela condição do diabetes. Os animais do grupo C1 apresenta- ram valores de glicemia aproximados à dos animais do grupo C2, mostrando que a constituição do veículo utilizado para administração do TAM não alterou os níveis de glicemia dos mesmos. O Diabetes mellitus tipo 1 é uma doença inflamatória autoimune que acomete milhares de pessoas em todo mun- do comprometendo a produtividade, qualidade de vida e sobrevida das mesmas, e que está associada a alterações nos níveis das proteínas plasmáticas 16,17. Os níveis das pro- teínas plasmáticas também são alterados pelo tratamento com tamoxifeno, uma vez que o mesmo induz uma série de alterações hepáticas que pode levar ao comprometimento da síntese protéica, visto que a maioria das proteínas plasmáticas são sintetizadas pelo fígado, com exceção das imunoglobulinas 6,21. Como pode ser observado na Tabela 1 e na Figura 3, os resul- tados obtidos para o grupo C1 (animais que receberam veículo) não apresentaram alterações significativas na con- centração relativa e absoluta das frações protéicas quando comparados aos do C2 (animais que não receberam veículo), portanto a utilização do veículo não foi responsável pelas alterações observadas. Além disso, os valores de ambos os grupos ficaram dentro dos valores de referência geralmente indicados para cada fração do proteinograma, possibilitando a utilização do grupo controle com veículo (C1) como parâmetro de comparação para os demais grupos. O modelo utilizado para estudar o efeito do diabetes e do tamoxifeno nos níveis das proteínas plasmáticas foram ra- tos diabéticos experimentais por estreptozotocina. A redução ou ausência de ganho de peso corporal dos animais diabé- ticos associada à evolução glicêmica e ao quadro visível de poliúria, polifagia e polidipsia não deixou dúvidas acerca do estado diabético dos animais utilizados, durante o período de 30 dias. Observou-se ainda, que ao longo de 30 dias os 90 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 Em relação ao grupo T foi possível observar alterações nas concentrações das frações albumina e a 1, a 2, b e g globulinas quando comparado ao grupo controle (Tabela 1). Discussão Em relação à albumina, classificada como uma proteína de fase aguda negativa, houve uma redução significativa em relação ao gru- po controle. Para a fração a 1, constituída pelas proteínas a 1 antitripsina (principal constituinte), mucoproteínas e glicoproteína a 1 ácida, houve um aumento não significativo quando comparado ao grupo controle. Este aumento pode ser justificado pelo fato das proteínas a 1 antitripsina e a 1 glicoproteína ácida serem reagentes de fase aguda positiva podendo apresentarem-se elevadas devido a um possível processo inflamatório causado pelo TAM. Por outro lado, este aumento também poderia estar relacionado ao efeito do hiperestrogenismo sobre a a 1antitripsina, já que o TAM pode produzir efeitos agonistas ao do estrógeno em locais que possuem receptor de estrogênio, entre eles o fígado 7,8. Já a fração a 2, constituída pelas proteínas haptoglobulina, a 2 macroglobulina e ceruloplasmina, apresentou um aumento não significativo quando comparado ao grupo controle. Deve ser observado que os valores obtidos para a fração a2 em relação ao grupo controle (3,0% ± 0,41) foram inferiores aos valores de referência para humanos (5,7 – 11,5%), diferente- mente das demais frações cujos valores foram sempre próximos aos indicados para humanos. Isto pode ser justificado, pois segundo GIANAZZA et al 18, a haptoglobina está presente em ratos em concentrações inferiores a dos humanos. O au- mento desta fração geralmente está associado a situações em que grandes quantidades de proteínas plasmáticas de baixo peso molecular, como a albumina, apresentam-se reduzidas, uma vez que para manter a pressão oncótica apenas molécu- las de alto peso molecular como a a2-macroglobulina seria capaz de compensar esta função 7. Em relação à fração b, onde estão presentes a transferrina, os componentes C3 e C4 do sistema complemento, fibrinogênio, b-lipoproteína, entre outras, também pode ser observado um aumento não signifi- cativo quando comparado ao grupo controle. Este aumento poderá estar relacionado à elevação da transferrina, uma vez que segundo CHRISTENSON e SILVERMAN 7 a transferrina se encontra elevada na terapia com estrogênio. Consideran- do que o TAM pode produzir efeito estrogênico total ou parcial, este pode provocar uma elevação da transferrina e conseqüentemente da fração b. A análise da fração g mostrou um aumento não significativo quando comparado ao grupo controle. Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 91 Discussão Estes resultados são reforçados pelo aumento da ordem de 15 vezes da atividade das enzimas alanina aminotransferase (ALT) e aspartato aminotransferase (AST) no soro dos animais quando da associação do DM e TAM (resultados não mostrados) as quais são reconhecidas como marcadores hepáticos específicos. Estes resultados são semelhantes aos relatados na literatura, que demonstraram esteatose hepática em pacientes bem como em animais quan- do submetidos ao tratamento com TAM 6,21. A fração a1 apresentou um aumento não significativo quando compara- do ao grupo controle; este aumento poderia estar possivelmente relacionado a um processo de fase aguda e/ ou a um possível efeito específico do TAM sobre a a 1 antitripsina, uma vez que esta proteína representa cerca de 90% da constituição protéica da fração a 1. Considerando que resultados semelhantes foram obtidos apenas para o grupo tratado com TAM, mas não para o grupo D, o aumento da a 1 globulina no grupo DT foi causado principalmente pelo TAM. O aumento da fração a2 foi bastante significativo chegando a representar a somatória dos valores obtidos em condição de DM e do tratamento com TAM. Este aumento poderia ser explicado devido à progressiva diminuição da concentração Para o grupo D houve uma diminuição progressiva e signifi- cativa da fração albumina quando comparada ao grupo controle, podendo esta estar associada a um possível pro- cesso inflamatório, visto que a albumina é um reagente de fase aguda negativa 16. Além disso, esta redução poderá está associada às glicações não-enzimáticas causadas pelo esta- do de hiperglicemia do Diabetes mellitus, diminuindo assim a Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 91 6. Cai Q, Bensen M, Greene R, Kirchner J. Tamoxifen-induced transient multifocal hepatic fatty infiltration. Am J Gastroenterol 2000;95:277- 9. da albumina, fazendo com que a fração a2, principalmente a a2 macroglobulina, se encontre mais elevada de modo a manter a pressão oncótica. Na região b não houve diferença signifi- cativa com relação ao grupo D, mantendo-se um aumento significativo em relação aos demais grupos, provavelmente devido ao efeito do Diabetes mellitus sobre as lipoproteínas. Em relação à fração g foi observado um aumento significativo quando comparado ao grupo controle, provavelmente devi- do a um processo inflamatório, visto que nesta fração se encontram as imunoglobulinas, que sofrem um aumento policlonal na maioria dos processos inflamatórios. Conclusão A avaliação do proteinograma dos animais diabéticos trata- dos com tamoxifeno mostrou que a utilização do TAM por um período de 30 dias em condição de DM provocou alterações nos níveis das proteínas plasmáticas caracterizando uma res- posta de fase aguda, que poderia estar relacionada a uma possível lesão hepática. Porém como o período de estudo foi curto, não foi possível observar um efeito muito agressivo, que poderá ser observado na continuidade do estudo em períodos mais prolongados. Entretanto estes resultados são representativos no que diz respeito a possível ação deletéria do TAM em nível hepático, além de confirmar os possíveis efeitos aditivos que o TAM poderia causar em pacientes dia- béticos do tipo 1. 12. Bayne C, Gerwick L. The acute phase response and innate immunity of fish. Devel Comp Immunol 2001; 25: 725-43. 13. Wu X, Thomas SJ, Rendón DN, Sanford AP, Wolf SE. Insulin decreases hepatic acute phase protein levels in severely burned children. Surgery 2004; 135: 196-202. 14. Corrêa CR, Burini RC. Proteínas plasmáticas reativas de fase aguda. J Bras Patol 2000; 36: 26-34. 15. Goldsby RA, Kindt TJ, Osborne BA. Kuby – Imunologia. 4ed. Rio de Janeiro: Revinter; 2002. Discussão Além dis- so, o aumento da proteína C reativa (PCR), que é a primeira proteína de fase aguda positiva a se elevar na vigência de um processo inflamatório poderá também estar contribuindo já que a ação do TAM leva à liberação de substâncias autógenas tóxicas por tecidos danificados e a PCR teria a função de remover essas substâncias tóxicas do sangue 7,8. 7. Christenson RH, Silverman LM. Aminoácidos e proteínas: In: Burtis CA, Ashwood ER. Fundamentos de química clínica. 4ed. Rio de Janeiro: Guanabara Koogan; 1996. p. 234-74. 8. Naoum PC. Eletroforese: técnicas e diagnósticos. 2ed. São Paulo: Santos Editora; 1999. 9. Corrêa CR, Angeleli AYO, Camargo NR, Barbosa L, Burini RC. Comparação entre a relação PCR/albumina e o índice prog- nóstico inflamatório nutricional (IPIN). J Bras Patol Med Lab 2002; 38: 183-90. 10. Pichet G, Bresolin PL, Pareira O, Jaworski MC, Santos CM, Pinto AP. Mucoproteína versus alfa-1-glicoproteína ácida: o que quantificar? J Bras Patol Med Lab 2002; 38: 87-91. 11. Kogika M, Pereira DA, Elias F, Notomi MK, Delayte EH, Kawahara R, Hagiwara MK. Determinação sérica de haptoglobina, ceruloplasmina e á-glicoproteína ácida em cães com gastroenterite hemorrágica. Ciência Rural 2003; 33: 513- 7. Referências 16. Tan K. Dyslipidaemia, inflammation and endothelial dysfunction in diabetes mellitus. Int Congr Ser 2004; 1262: 511-4. 1. Gebrim LH, Lima GR. Quimioprevenção primária e secundá- ria do carcinoma de mama. In: Prado FC, Ramos J, Valle JR. Atualização Terapêutica 2003. 21ed. São Paulo: Artes Médi- cas; 2003. 17. Gomes MB, Piccirillo LJ, Nogueira VG, Matos HJ. Acute- phase proteins among patients with type 1 diabetes. Diabetes Metab 2003; 29: 405-11. 2. Sharma M, Shubert DE, Sharma M, Lewis J, McGarrigle BP, Bofinger DP, Olson JR. Biotransformation of tamoxifen in a human endometrial explant culture model. Chem Biol Interact 2003; 146:237-49. 18. Gianazza E, Eberini I, Villa P, Fratelli M, Pinna C, Wait R, Gemeiner M, Miller I. Monitoring effects of drug treatment in rat models of disease by serum proteins analysis. J Chomatogr B 2002; 771: 107-30. 3. Bonanni B, Johansson H, Gandini S, Guerrieri-Gonzaga A, Sandri MT, Mariette F, Lien EA, Decensi A. Effect of tamoxifen at low doses on ultrasensitive C-reactive protein in healthy women. J Thromb Haemost 2003;1:2149-52. 19. Duarte VMG, Ramos AMO, Rezende LA, Macedo UBO, Neto-Brandão J, Almeida MG, Rezende AA. Osteopenia: a bone disorder associated with diabetes mellitus. J Bone Miner Metab 2005; 23: 58-68. 4. Pelletier G. Localization of androgen and estrogen receptors in rat and primate tissues. Histol Histopathol 2000;15:1261-70. 20. Guide for the care and use of laboratory animals, US National Conucil, 1996. 5. Lewis LS, Brown K, Carthew P, Lim C, Martín EA, Styles J, White I. Chemoprevention of breast cancer by tamoxifen: risk and opportunities. Crit Rev Toxicol 2000;30:571-94. 21. White IH. The tamoxifen dilemma. Carcinogenesis 1999; 20: 1153-60. 92 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 Silva TCP, Mota SB, Almeida MMC, Ferreira ECS, Ururahy MAG, Bezerra JF, Pereira NML, Ramos AMO, Almeida MG, Rezende AA. Effect of tamoxifen on plasma proteins in diabetes mellitus type 1. Acta Cir Bras [serial on line] Available from: URL: htt:// www.scielo.br/acb. Silva TCP, Mota SB, Almeida MMC, Ferreira ECS, Ururahy MAG, Bezerra JF, Pereira NML, Ramos AMO, Almeida MG, Rezende AA. Effect of tamoxifen on plasma proteins in diabetes mellitus type 1. Acta Cir Bras [serial on line] Available from: URL: htt:// www.scielo.br/acb. Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 93 Referências ABSTRACT – Purpose: Considering that important scientific advances have been obtained through studies based on experimental Diabetes mellitus, and that tamoxifen action in humans remains unknown, the aim of the present work is to follow the modifications promoted by diabetes and tamoxifen in the electrophoretic profile of plasmatic proteins. Methods: It was used 27 Wistar female rats (180-250 body weight), randomicaly divided into five groups: C1 (n=3, received vehicle), C2 (n=3, no treatment), T (n=5, treated with tamoxifen, 0.3mg/Kg/day), D (n=8, experimental diabetes by estreptozotocin, 45mg/Kg and DT (n=8, diabetic treated with tamoxifen). The electrophoresis was accomplished in cellulose acetate. pH 8.6–8.8, TECNOW chamber, and the strains were stained by Ponceau S. The total proteins were determined by the Biuret method (Labtest). Proteinograms were obtained in densitometer BioSystems BTS-235. Results: Albumin decreased progressively in the groups T, D and DT; á1 fraction increased in groups T and DT; á2 fraction increased in groups T and D, including a synergic effect in group DT; â fraction increased in groups T and D; ã fraction increased in groups T, D and DT. Conclusions: The results indicate an acute phase resposta, with synergic effect of tamoxifen and diabetes, suggesting a probable hepatic lesion. KEYWORDS: Tamoxifen. Plasmatic proteins. Diabetes mellitus. Electrophoresis of proteins. Endereço para correspondência: Adriana Augusto de Rezende Rua Pastor Gabino Brelaz, 1401, Bloco A, apto 101, Capim Macio, CEP: 59082-010 Telefones: (84) 207 9305 (84) 215 4377 / 215 4345 e-mail: adrirezende@yahoo.com Conflito de interesses: nenhum Fonte de financiamento: nenhuma Endereço para correspondência: Adriana Augusto de Rezende Rua Pastor Gabino Brelaz, 1401, Bloco A, apto 101, Capim Macio, CEP: 59082-010 Telefones: (84) 207 9305 (84) 215 4377 / 215 4345 e-mail: adrirezende@yahoo.com Conflito de interesses: nenhum Fonte de financiamento: nenhuma
https://openalex.org/W2111430155	http://www.ajnr.org/content/ajnr/33/4/643.full.pdf	English	null	The Impact of Thromboemboli Histology on the Performance of a Mechanical Thrombectomy Device	American journal of neuroradiology	2,011	cc-by	5,531	of October 23, 2024. This information is current as Thrombectomy Device the Performance of a Mechanical The Impact of Thromboemboli Histology on Vinuela, J.W. Sayre, Y. Murayama and F. Vinuela I. Yuki, I. Kan, H.V. Vinters, R.H. Kim, A. Golshan, F.A. http://www.ajnr.org/content/33/4/643 https://doi.org/10.3174/ajnr.A2842 doi: 2012, 33 (4) 643-648 AJNR Am J Neuroradiol Thrombectomy Device the Performance of a Mechanical The Impact of Thromboemboli Histology on Vinuela, J.W. Sayre, Y. Murayama and F. Vinuela I. Yuki, I. Kan, H.V. Vinters, R.H. Kim, A. Golshan, F.A. http://www.ajnr.org/content/33/4/643 https://doi.org/10.3174/ajnr.A2842 doi: 2012, 33 (4) 643-648 AJNR Am J Neuroradiol of October 23, 2024. This information is current as Please address correspondence to Ichiro Yuki, MD, Division of Interventional Neuroradi- ology UCLA Medical Center and David Geffen School of Medicine at UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095-1721; e-mail: iyuki@mednet.ucla.edu Received March 30, 2011; accepted after revision June 23. From the Division of Interventional Neuroradiology (I.Y., I.K., R.H.K., A.G., F.A.V., F.V.), Department of Radiological Sciences, and Section of Neuropathology and Departments of Pathology and Laboratory Medicine and Neurology (H.V.V.), David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California; Department of Biostatistics (J.W.S.), UCLA School of Public Health, Los Angeles, Los Angeles, California; and Department of the Neurosurgery (Y.M.), Jikei University of Medicine, Tokyo, Japan. Harry V. Vinters is supported, in part by, the Daljit S. and Elaine Sarkaria Chair in Diagnostic Medicine and Specialized Programs of Translational Research in Acute Stroke, grant 1P50NS044378. Pl dd d t I hi Y ki MD Di i i f I t ti l N di AJNR Am J Neuroradiol 33:643–48 Apr 2012 www.ajnr.org 643 http://dx.doi.org/10.3174/ajnr.A2842 ORIGINAL RESEARCH I. Yuki I. Kan H.V. Vinters R.H. Kim A. Golshan F.A. Vinuela J.W. Sayre Y. Murayama F. Vinuela I. Yuki I. Kan H.V. Vinters R.H. Kim A. Golshan F.A. Vinuela J.W. Sayre Y. Murayama F. Vinuela BACKGROUND AND PURPOSE: Recently we published a novel method of thrombus preparation for use in a swine model for evaluation of thrombectomy designs. The clot (fibrin rich clot) is characterized by its similarity in histologic characteristics to the thromboemboli recovered from stroke patients. The purpose of this latest study was to evaluate if the performance of a mechanical thrombectomy device was affected by the histologic characteristics of thromboembolus. Erythrocyte rich clot, which was created using exogenous thrombin, and the novel experimental clot with abundance of fibrin/cellular component were used for comparison. The Merci clot retriever was used for the evaluation and the angiographic outcomes were analyzed. MATERIALS AND METHODS: Two histologically different types of experimental clot, a conventionally used thrombin-induced clot (erythrocyte-rich clot) and a novel experimental clot that is similar in histologic characteristics to the thromboemboli recovered from patients with stroke (fibrin-rich clot), were prepared. Eight extracranial arteries in swine were occluded with erythrocyte-rich clot (group A), and 8 were occluded with fibrin-rich clot (group B), and MT by using the Merci clot retriever device was performed. Angiographic results in each group were evaluated. RESULTS: A total of 48 attempts at MT were made. The average number of attempts to achieve TIMI grade II or III recanalization was 2.75 times in group A and 4.5 times in group B (P .001), respectively. The mean time to achieve recanalization was 15.5 minutes in group A and 81.5 minutes in group B (P .01). Every vessel in group A showed recanalization (100%), whereas only 3 of 8 samples (37.5%) achieved recanalization in group B. CONCLUSIONS: In this model, arteries occluded by fibrin-rich clot demonstrated a significantly lower recanalization rate, lower final TIMI score, and a longer mean recanalization time than did arteries occluded by erythrocyte-rich clot. The angiographic outcome of MT by using the Merci clot retriever system was influenced by the histologic characteristics of the occluding thromboembolus. Received March 30, 2011; accepted after revision June 23. From the Division of Interventional Neuroradiology (I.Y., I.K., R.H.K., A.G., F.A.V., F.V.), Department of Radiological Sciences, and Section of Neuropathology and Departments of Pathology and Laboratory Medicine and Neurology (H.V.V.), David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California; Department of Biostatistics (J.W.S.), UCLA School of Public Health, Los Angeles, Los Angeles, California; and Department of the Neurosurgery (Y.M.), Jikei University of Medicine, Tokyo, Japan. Harry V. Vinters is supported, in part by, the Daljit S. and Elaine Sarkaria Chair in Diagnostic Medicine and Specialized Programs of Translational Research in Acute Stroke, grant 1P50NS044378. Please address correspondence to Ichiro Yuki, MD, Division of Interventional Neuroradi- ology UCLA Medical Center and David Geffen School of Medicine at UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095-1721; e-mail: iyuki@mednet.ucla.edu http://dx.doi.org/10.3174/ajnr.A2842 Thrombus Preparation Several samples of each clot type (fibrin- and erythrocyte-rich) were randomly selected and microscopically examined to evaluate their histologic characteristics (Fig 1B). The erythrocyte-rich samples were homogeneous in composition (composed almost exclusively of red blood cells) (Fig 1B, left). The fibrin-rich samples were complex in nature with well-defined fibrin-, cellular-, and erythrocyte-rich lay- ers, respectively (Fig 1B, right). Preparation of Erythrocyte-Rich Clot. A conventional experi- mental clot (thrombin-induced clot) was prepared. The detailed preparation process is described elsewhere.10,11 Briefly, 10 mL of swine whole blood was mixed with 25 IU of bovine thrombin (Dade Behring, Newark, Delaware). One-half a gram of barium sulfate was mixed with the blood for 10 seconds, and the mixture was kept in a silicone tube for 60 minutes at room temperature. After the incuba- tion period, the created clot was removed from the silicone tube and cut to a length of 2 cm (Fig 1A). Occlusion of Extracranial Arteries in Swine with Different Types of Experimental Clot. With the swine under general anesthe- sia, maintained with isoflurane (2%–5%), an 8F femoral sheath was placed in its right femoral artery. A high-resolution angiographic sys- tem (Allura Xper FD 10; Philips Healthcare, Best, the Netherlands) was used for selective digital subtraction angiography of each target artery. An 8F balloon-guiding catheter (Concentric Medical) was ad- vanced through the femoral sheath and placed at the origin of the target vessel. A control angiogram was obtained. The silicone tube containing a prepared clot was then connected to the guiding cathe- ter, and approximately 5 mL of saline was injected to push the clot Preparation of Fibrin-Rich Clot. The detailed preparation pro- cess of fibrin-rich clot is described elsewhere.9 Briefly, a syringe con- taining 1 g of barium sulfate powder was used to aspirate 30 mL of whole blood from an 8F sheath placed in the right femoral artery. The syringe was then gently rotated to allow mixing of the whole blood and barium sulfate powder to obtain radiopacity. To minimize the anticoagulant effect of barium, which is due to absorption of vitamin Kdependent coagulation factor 4,12 we paid special attention to rotational motion during mixing. Subsequently, the contents of the Comparison between the erythrocyte-rich clot group and the fibrin-rich clot group Erythrocyte-Rich Clot (Group A) Fibrin-Rich Clot (Group B) Level of Significance Total No. of treated vessels 8 8 N/A Total No. INTERVENTIONAL ORIGINAL RESEARCH INTERVENTIONAL ORIGINAL RESEARCH ABBREVIATIONS: IMAX internal maxillary artery; LA lingual artery; MT mechanical throm- bectomy; SCA superficial cervical artery; TIMI thrombolysis in myocardial infarction; UCLA University of California, Los Angeles ORIGINAL RESEARCH M know how the performance of an MT device is influenced by thromboemboli of differing histopathology. M T is an emerging treatment option for acute stroke.1-7 However, frequently 2 angiographically identical oc- cluded vessels in patients with acute stroke will respond differ- ently to MT for reasons that are not angiographically evident. One hypothesis is that the performance of the MT device is influenced by the histologic characteristics of thromboem- bolus (eg, fibrin/cellular component), but current image tech- nology cannot differentiate thromboemboli of varying histo- logic characteristics. Given that the goal of MT is to optimize target-vessel recanalization and procedure time, it is crucial to A recent article reported that contrary to traditional teach- ing, approximately 75% of thromboemboli recovered from patients with stroke by MT showed complex histologic char- acteristics (a mixture of fibrin, erythrocytes, and nucleated cells).8 Moreover, the article showed that red clots composed solely of erythrocytes were uncommon. We recently reported a new method for creating clot (fi- brin-rich clot) with histologic characteristics similar to those of thromboemboli recovered from patients with stroke.9 This fibrin-rich clot is composed of histologically distinct layers: a fibrin-predominant layer, a cellular-predominant layer, and an erythrocyte-predominant layer. In contrast, the clot that is conventionally used in experimental thrombectomy models is homogeneous and predominantly composed of erythrocytes (erythrocyte-rich clot) with a meshwork of interspersed fibrin. The purpose of this study was to determine whether the effi- cacy of MT is influenced by thromboemboli having differing compositions. After embolization of the target vessels with clot of varying histology (fibrin- and erythrocyte-rich), we tested the performance of the Merci clot retriever device (Con- centric Medical, Mountain View, California) on each clot type. AJNR Am J Neuroradiol 33:643–48 Apr 2012 www.ajnr.org 643 AJNR Am J Neuroradiol 33:643–48 Apr 2012 www.ajnr.org 643 643 Fig 1. A, Macroscopic view of 2 different experimental clots. A novel experimental clot (fibrin-rich clot) (left) is prepared by manually excising the fibrin-rich layer of swine clot, which is created by natural sedimentation. Fibrin-rich clot shows more solid and elastic mechanical properties than conventional thrombin-induced experimental clot (erythrocyte-rich clot) (right). Animals All animal experiments were conducted in accordance with policies established by the UCLA chancellor’s animal research committee and the National Institutes of Health guidelines. A total of 16 extracranial arteries in 5 Yorkshire swine (male and female animals; age range, 3–4 months; weight range, 30–40 kg) were used in this study. INTERVENTIONAL ORIGINAL RESEARCH F indicates fibrin component; Arrows, the cellular component; E, the erythrocyte component. Materials and Methods syringe were incubated at room temperature, allowing separation of the whole blood components through precipitation, with plasma on top, fibrin/platelets/white blood cells in the middle, and red blood cells in the bottom. After plain sedimentation (2 hours), the solid component (fibrin-rich and erythrocyte-rich layers) was removed from the syringe. Next, the fibrin-rich layer was manually resected by using a number 11 scalpel, and pieces measuring 5 mm in diameter and 20 mm in length were prepared for injection (fibrin-rich clot) (Fig 1A). INTERVENTIONAL ORIGINAL RESEARCH B, Microscopic view of the experimental clots versus thromboembolus recovered from a patient with acute stoke (H&E, original magnification 40). A microscopic view of an erythrocyte-rich clot (left) demonstrates a thrombus composed predominantly of erythrocytes with interspersed fibrin and barium sulfate deposits. A thromboembolus recovered from a patient with stroke (center) shows distinct layers of fibrin, nucleated cells, and erythrocytes. A fibrin-rich clot (right) produces a layered pattern of fibrin, nucleated cells, and erythrocytes similar to that seen in the clot recovered from the patient with stroke (center). F indicates fibrin component; Arrows, the cellular component; E, the erythrocyte component. Fig 1. A, Macroscopic view of 2 different experimental clots. A novel experimental clot (fibrin-rich clot) (left) is prepared by manually excising the fibrin-rich layer of swine clot, which is created by natural sedimentation. Fibrin-rich clot shows more solid and elastic mechanical properties than conventional thrombin-induced experimental clot (erythrocyte-rich clot) (right). B, Microscopic view of the experimental clots versus thromboembolus recovered from a patient with acute stoke (H&E, original magnification 40). A microscopic view of an erythrocyte-rich clot (left) demonstrates a thrombus composed predominantly of erythrocytes with interspersed fibrin and barium sulfate deposits. A thromboembolus recovered from a patient with stroke (center) shows distinct layers of fibrin, nucleated cells, and erythrocytes. A fibrin-rich clot (right) produces a layered pattern of fibrin, nucleated cells, and erythrocytes similar to that seen in the clot recovered from the patient with stroke (center). F indicates fibrin component; Arrows, the cellular component; E, the erythrocyte component. Fig 1. A, Macroscopic view of 2 different experimental clots. A novel experimental clot (fibrin-rich clot) (left) is prepared by manually excising the fibrin-rich layer of swine clot, which is created by natural sedimentation. Fibrin-rich clot shows more solid and elastic mechanical properties than conventional thrombin-induced experimental clot (erythrocyte-rich clot) (right). B, Microscopic view of the experimental clots versus thromboembolus recovered from a patient with acute stoke (H&E, original magnification 40). A microscopic view of an erythrocyte-rich clot (left) demonstrates a thrombus composed predominantly of erythrocytes with interspersed fibrin and barium sulfate deposits. A thromboembolus recovered from a patient with stroke (center) shows distinct layers of fibrin, nucleated cells, and erythrocytes. A fibrin-rich clot (right) produces a layered pattern of fibrin, nucleated cells, and erythrocytes similar to that seen in the clot recovered from the patient with stroke (center). Thrombus Preparation of attempts (times) 14 34 P .001 Average No. of attempts per vessel (times) 2.75 4.5 P .001 Mean procedural time (min) 15.5 81.5 P .01 Final TIMI grade 0 0 4 2 0 1 N/A 3 5 2 4 3 1 Note:—N/A indicates not applicable. 644 Yuki AJNR 33 Apr 2012 www.ajnr.org Comparison between the erythrocyte-rich clot group and the fibrin-rich clot group Fig 2. Bar graphs showing the number of attempts required to achieve the angiographic end point in each treated vessel. Every target vessel in group A achieved recanalization of TIMI grade 2 or greater, whereas only 3 of 8 vessels achieved recanalization in group B. None of the treated vessels that underwent 5 attempts of retrieval demonstrated significant recanalization (1 vessel with TIMI 1 and 4 vessels with TIMI 0). Fig 2. Bar graphs showing the number of attempts required to achieve the angiographic end point in each treated vessel. Every target vessel in group A achieved recanalization of TIMI grade 2 or greater, whereas only 3 of 8 vessels achieved recanalization in group B. None of the treated vessels that underwent 5 attempts of retrieval demonstrated significant recanalization (1 vessel with TIMI 1 and 4 vessels with TIMI 0). A postretrieval angiogram of the target artery was obtained and evaluated by using the TIMI scale. “Recanalization” was defined as TIMI 2 or greater in this study. The retrieval attempt could be re- peated up to 5 times per target artery. If no recanalization was achieved after 5 attempts, the retrieval was rated as a failure. We collected the following data: 1) the number of attempts required to achieve recanalization, 2) distal migration or fragmentation of thrombus, 3) final angiographic result in the TIMI (0–3) scale, 4) the time (minutes) required to achieve the final result, and 5) vessel dis- section or perforation. The presence of distal migration of thrombo- embolus and thrombus fragmentation was evaluated by comparing the preprocedural and postrecanalization angiograms. If the treated artery remained completely occluded in the postprocedural angio- gram, the assessment was not performed. into the guiding catheter. Next, another 5 mL of saline was injected, under fluoroscopy, to inject the clot from the guiding catheter into the target artery. Target vessels included the LA, IMAX, and SCA. Vessels with complete occlusion were considered successfully embolized and used in the study. Statistical Data Analysis Statistical Data Analysis Each vessel tested in the study was considered to be an independent data point, and statistical analysis comparing the 2 groups (groups A and B) was performed. The Wilcoxon signed rank test (an unpaired nonparametric test) was used to compare the 2 groups in the follow- ing areas: 1) average number of attempts required to achieve the final angiographic result, 2) time (minutes) to achieve the final result, and 3) final TIMI grade. Thrombus Preparation Vessels without complete occlusion after clot injec- tion were excluded, and intervention was not performed. The target vessels occluded with conventional thrombin-induced (erythrocyte- rich) clot were categorized as group A, and the vessels occluded with fibrin-rich clot were categorized as group B. MT by Using Merci Clot Retriever System The use of extracranial vessels in swine to assess the efficacy of MT has been well established elsewhere.10,11,13 After the injection of experi- mental clot, the guiding catheter was removed for 5 minutes to restore arterial flow and allow embedding of the thrombus. The Merci clot retriever system was used for MT. The thromboembolus was crossed with a Transcend 14 guidewire (Boston Scientific, Natick, Massachu- setts) loaded in an 18-L microcatheter (Concentric Medical), and the distal segment of the target vessel was then catheterized. A superse- lective angiogram was obtained via the microcatheter to confirm po- sitioning of the catheter distal to the occlusion site. Depending on the size of the vessel, either a V2.5 or 3.0 (firm) thrombectomy device was selected. Next, a Merci clot retriever was deployed distal to the clot. The balloon catheter tip was inflated with 0.8 mL of 50% contrast solution. If the tip of the catheter was nearly-occlusive/occlusive at the origin of the vessel, the balloon was not inflated. Then, an attempt was made to pull the thrombus into the guiding catheter while gently aspirating. When the retriever system was immediately outside the guiding catheter, 30 mL of blood was vigorously aspirated while re- moving the Merci device. The balloon was then deflated, and both the Merci device and aspirate were examined for evidence of thrombus. Results The average number of retrieval attempts to achieve the angiographic end point was 2.75 times in group A and 4.5 times in group B (P .001). In group A, 3 vessels demonstrated TIMI grade 3 recanalization with 1 retrieval at- tempt, 2 vessels showed TIMI grade 3 recanalization after 2 attempts, 2 vessels achieved TIMI grade 2 recanalization after 2 attempts, and 1 vessel showed TIMI grade 2 recanalization after 3 attempts. In group B, 1 vessel achieved TIMI 3 recana- lization after 2 attempts, 1 vessel achieved TIMI 2 recanaliza- tion after 3 attempts, and 1 vessel achieved TIMI grade 2 re- canalization after 4 attempts. The remaining 5 target vessels in catheterization through the occlusion site was successfully achieved. Fourteen retrieval attempts were made to achieve the final angiographic results in the target arteries in group A, whereas 34 attempts were made in group B (P .001). Every target vessel (100%) in group A achieved recanalization of TIMI grade 2 or greater, whereas only 3 of 8 vessels (37.5%) achieved recanalization of TIMI grade 2 or greater in group B. Figures 2 and 3 show the typical angiographic findings from each group. The average number of retrieval attempts to achieve the angiographic end point was 2.75 times in group A and 4.5 times in group B (P .001). In group A, 3 vessels demonstrated TIMI grade 3 recanalization with 1 retrieval at- tempt, 2 vessels showed TIMI grade 3 recanalization after 2 attempts, 2 vessels achieved TIMI grade 2 recanalization after 2 attempts, and 1 vessel showed TIMI grade 2 recanalization after 3 attempts. In group B, 1 vessel achieved TIMI 3 recana- lization after 2 attempts, 1 vessel achieved TIMI 2 recanaliza- tion after 3 attempts, and 1 vessel achieved TIMI grade 2 re- canalization after 4 attempts. The remaining 5 target vessels in group B did not demonstrate significant vessel recanalization (1 vessel with TIMI 1 after 5 attempts and 4 vessels with TIMI 0 after 5 attempts) (Fig 2). The average time required to achieve the final angiographic result was 15.5 minutes in group A and 81.5 minutes in group B (P .01). Figures 3 and 4 show the typical angiographic findings from each group. All of the 8 target vessels in group A demonstrated recanalization (TIMI 2 or greater), and 7 of 8 demonstrated distal migration of the fragmented clot. Results A total of 16 vessels (6 LAs, 8 SCAs, and 2 IMAXs) were oc- cluded, and 48 retrieval attempts were made. A total of 8 ves- sels were successfully occluded with erythrocyte-rich clot (group A), and 8 vessels were occluded with fibrin-rich clot (group B). The Table summarizes the data and statistical sig- nificance between the 2 groups. In every target vessel, micro- AJNR Am J Neuroradiol 33:643–48 Apr 2012 www.ajnr.org 645 AJNR Am J Neuroradiol 33:643–48 Apr 2012 www.ajnr.org 645 645 Fig 3 an e dural poste Comp tion devic and c gram sidua seen Fig 3. Angiographic findings of a target vessel occluded wit an erythrocyte-rich clot and treated with MT. A, Preproce dural angiogram of a right superficial cervical artery (antero posterior view) shows the branching pattern of the artery. B Complete occlusion of the vessel was confirmed after injec tion of an erythrocyte-rich clot. C, A Merci clot retrieve device (V2.5 firm) was deployed distal to the occlusion site and clot retrieval was performed. D, A postprocedural angio gram shows recanalization of the treated artery. Small re sidual thrombi distal to the bifurcation of the artery are als seen (arrow). Fig 3. Angiographic findings of a target vessel occluded with an erythrocyte-rich clot and treated with MT. A, Preproce- dural angiogram of a right superficial cervical artery (antero- posterior view) shows the branching pattern of the artery. B, Complete occlusion of the vessel was confirmed after injec- tion of an erythrocyte-rich clot. C, A Merci clot retriever device (V2.5 firm) was deployed distal to the occlusion site, and clot retrieval was performed. D, A postprocedural angio- gram shows recanalization of the treated artery. Small re- sidual thrombi distal to the bifurcation of the artery are also seen (arrow). catheterization through the occlusion site was successfully achieved. Fourteen retrieval attempts were made to achieve the final angiographic results in the target arteries in group A, whereas 34 attempts were made in group B (P .001). Every target vessel (100%) in group A achieved recanalization of TIMI grade 2 or greater, whereas only 3 of 8 vessels (37.5%) achieved recanalization of TIMI grade 2 or greater in group B. Figures 2 and 3 show the typical angiographic findings from each group. Results In group B, only 3 of the 8 target vessels demonstrated recanali- zation, and 2 of 3 demonstrated distal migration of the frag- mented clot. No vessel dissection or perforation was observed in the postprocedural angiograms. We also observed the following trends: Erythrocyte-rich clots (group A) were more easily mobilized and demonstrated more fragmentation in comparison with fibrin-rich clots (group B). Complete stretching of the device at the site of occlusion was more frequently seen in group B. In group A, once the clot was engaged, it was typically successfully recov- Yuki AJNR 33 Apr 2012 www.ajnr.org 646 ered, whereas in group B recovery was typically unsuccessful after clot capture. Discussion achieve recanalization (TIMI grade 2 or 3) compared with vessels occluded by erythrocyte-rich clot (4.5 times versus 2.8 times respectively, P .001). Consequently, the mean time to the angiographic end point in the fibrin-rich group was longer Fig 4. Angiographic findings of a target artery occluded with a fibrin-rich clot and treated with MT. A, A preprocedural angiogram of a left superficial cervical artery shows its branching pattern. B, Complete occlusion of the vessel was confirmed after injecting a fibrin-rich clot. C, A Merci clot retriever device (V2.5 firm) was deployed distal to the clot, and clot retrieval was performed. D, After 5 attempts at clot retrieval, the postprocedural angiogram shows that the treated vessel remains occluded. Fig 4. Angiographic findings of a target artery occluded with a fibrin-rich clot and treated with MT. A, A preprocedural angiogram of a left superficial cervical artery shows its branching pattern. B, Complete occlusion of the vessel was confirmed after injecting a fibrin-rich clot. C, A Merci clot retriever device (V2.5 firm) was deployed distal to the clot, and clot retrieval was performed. D, After 5 attempts at clot retrieval, the postprocedural angiogram shows that the treated vessel remains occluded. ered, whereas in group B recovery was typically unsuccessful after clot capture. ered, whereas in group B recovery was typically unsuccessful after clot capture. achieve recanalization (TIMI grade 2 or 3) compared with vessels occluded by erythrocyte-rich clot (4.5 times versus 2.8 times respectively, P .001). Consequently, the mean time to the angiographic end point in the fibrin-rich group was longer than that in the erythrocyte-rich group (81.5 minutes versus 15.5 minutes, P .01). Results Furthermore, every vessel occluded with erythrocyte-rich clot showed recanalization defined as a final TIMI grade of 2 or 3 (100%), whereas only 3 of 8 (37.5%) vessels occluded with fibrin-rich clot achieved recanalization. Fig 4. Angiographic findings of a target artery occluded with a fibrin-rich clot and treated with MT. A, A preprocedural angiogram of a left superficial cervical artery shows its branching pattern. B, Complete occlusion of the vessel was confirmed after injecting a fibrin-rich clot. C, A Merci clot retriever device (V2.5 firm) was deployed distal to the clot, and clot retrieval was performed. D, After 5 attempts at clot retrieval, the postprocedural angiogram shows that the treated vessel remains occluded. Histologic Characteristics of Thromboemboli Recovered from Patients with Stroke Marder et al8 performed histologic analysis on 25 thrombo- emboli from 25 separate patients with acute stroke, recovered with MT. The study showed that a majority (75%) of the re- covered thromboemboli were fibrin-rich and that so called “red clots” composed uniquely of erythrocytes were uncom- mon. In this study, we created 2 histologically distinct types of clot: 1 with fibrin/cellular/erythrocyte components, which mimic clot recovered from patients with acute stroke, and 1 composed predominantly of erythrocytes. These results suggest that vessels occluded with fibrin-rich clot are relatively more resistant to MT than those occluded with erythrocyte-rich clot. There are a few interpretations as to why recovering fibrin-rich clot is more challenging: 1) One is that the fibrin and cellular components of fibrin-rich clot make it stiff/hard. The stiff nature of the fibrin-rich clot may reduce the probability of successful engagement by the MT device, in turn decreasing the chances of successful clot cap- ture. The stiff/noncompliant nature of fibrin-rich clot may also explain why complete stretching of the device at the oc- clusion site is more frequently observed in group B. 2) It is also AJNR Am J Neuroradiol 33:643–48 Apr 2012 www.ajnr.org 64 Other Potential Factors That Influence the Performance of MT Devices 6. Nogueira RG, Schwamm LH, Hirsch JA. Endovascular approaches to acute stroke. Part 1. Drugs, devices, and data. AJNR Am J Neuroradiol 2009;30: 649–61 6. Nogueira RG, Schwamm LH, Hirsch JA. Endovascular approaches to acute stroke. Part 1. Drugs, devices, and data. AJNR Am J Neuroradiol 2009;30: 649–61 There are other factors that potentially impact the perfor- mance of the MT devices. Yin et al15 described 5 postmortem cases of patients with acute stroke who underwent MT with unsuccessful recanalization. Four of 5 patients showed prom- inent atheromata, often with significant luminal stenosis in the treated lesion. Although limited by a small sample size, the study by Yin et al presents another potential explanation for resistance to MT (baseline vessel stenosis). 7. Nogueira RG, Yoo AJ, Buonanno FS, et al. Endovascular approaches to acute stroke. Part 2. A comprehensive review of studies and trials. AJNR Am J Neu- roradiol 2009;30:859–75 7. Nogueira RG, Yoo AJ, Buonanno FS, et al. Endovascular approaches to acute stroke. Part 2. A comprehensive review of studies and trials. AJNR Am J Neu- roradiol 2009;30:859–75 8. Marder VJ, Chute DJ, Starkman S, et al. Analysis of thrombi retrieved from cerebral arteries of patients with acute ischemic stroke. Stroke 2006;37: 2086–93 9. Kan I, Yuki I, Murayama Y, et al. A novel method of thrombus preparation for use in a swine model for evaluation of thrombectomy devices. AJNR Am J Neuroradiol 2010;31:1741–43. Epub 2010 Jan 21 10. GrallaJ,SchrothG,RemondaL,etal.Adedicatedanimalmodelformechanical thrombectomy in acute stroke. AJNR Am J Neuroradiol 2006;27:1357–61 Thrombectomy Device Performance Can Be Influenced by the Histologic Characteristics of Thromboemboli In our experience, target vessels occluded by fibrin-rich thromboemboli required more attempts at thrombectomy to 647 AJNR Am J Neuroradiol 33:643–48 Apr 2012 www.ajnr.org possible that erythrocyte-rich clot fragments easily make piecemeal clot retrieval feasible, whereas fibrin-rich clot is less likely to fragment, preventing piecemeal thrombectomy, due to its more rigid nature. This partially explains why emboliza- tion in distal branches is frequently seen in the erythrocyte- rich clot group. 3) Last, on more than 1 occasion, a fibrin-rich clot was successfully captured by the MT device, but the cap- tured clot would not pass through the guiding catheter tip. The explanation, while speculative on our part, may be secondary to the mechanical properties of fibrin-rich clot (ie, the non- compliant nature of the fibrin-rich-clot and its tendency to disengage from the MT device at the tip of the guiding catheter). clot. This would make information from an animal model (ie, MT device performance as a function of clot histology) clini- cally applicable and could potentially play a role in procedure planning. References 1. Gobin YP, Starkman S, Duckwiler GR, et al. MERCI 1: a phase 1 study of Me- chanical Embolus Removal in Cerebral Ischemia. Stroke 2004;35:2848–54 2. Smith WS, Sung G, Saver J, et al. Mechanical thrombectomy for acute ischemic stroke: final results of the Multi MERCI trial. Stroke 2008;39:1205–12 3. Bose A, Henkes H, Alfke K, et al. The Penumbra System: a mechanical device for the treatment of acute stroke due to thromboembolism. AJNR Am J Neu- roradiol 2008;29:1409–13 4. Castano C, Dorado L, Guerrero C, et al. Mechanical thrombectomy with the Solitaire AB device in large artery occlusions of the anterior circulation: a pilot study. Stroke 41:1836–40 5. Josephson SA, Saver JL, Smith WS. Comparison of mechanical embolectomy and intraarterial thrombolysis in acute ischemic stroke within the MCA: MERCI and Multi MERCI compared to PROACT II. Neurocrit Care 2009;10: 43–49 Limitations of the Study The main limitation was the small sample size of our study. The use of only 1 MT device, the Merci clot retriever, which was necessary given our limited sample size, was another lim- itation. Finally the use of only 1 clot size (2.0 0.5 cm) was also a limitation. In an animal experiment, Gralla et al14 re- ported that occlusion length is highly associated with the suc- cess rate in MT. Additional investigation with larger numbers of subject animals is necessary to further test many of the hy- potheses we have set forth. Disclosures: Harry V. Vinters—UNRELATED: Stock/Stock Options: have significant share holdings in Minnesota Mining and Manufacturing Company, Glaxo-Smith-Kline, Teva Pharma, and Pfizer. I received dividends from all these companies. Fernando A. Vinuela— RELATED: Provision of Writing Assistance, Medicines, Equipment, or Administrative Sup- port: Volunteer. The variable radiopacity of the fibrin-rich clot was a tech- nical limitation in this study. To minimize the anticoagulant effect of barium (explained by absorption of vitamin Kde- pendent coagulation factors),12 we carefully controlled the mixture of barium and whole blood. Given the limited mix- ture time of barium and whole blood, there was heterogeneity in the radiopacity of the fibrin-rich clot samples. Therefore, developing a technique that consistently produces fibrin-rich clot with homogeneous radiopacity is requisite. Conclusions To evaluate whether the efficacy of mechanical thrombectomy is influenced by the histologic characteristics of thromboem- boli, swine vessels occluded by 2 different types of experimen- tal clot were treated with a MT device, and the angiographic outcomes were analyzed. Target vessels occluded by fibrin- rich clot demonstrated significantly lower recanalization rates, longer mean recanalization time, and lower final TIMI scores compared with the vessels occluded by erythrocyte-rich clot. In our study, the performance of the Merci clot retriever sys- tem was influenced by the histologic characteristics of the thromboemboli. Disclosures: Harry V. Vinters—UNRELATED: Stock/Stock Options: have significant share holdings in Minnesota Mining and Manufacturing Company, Glaxo-Smith-Kline, Teva Pharma, and Pfizer. I received dividends from all these companies. Fernando A. Vinuela— RELATED: Provision of Writing Assistance, Medicines, Equipment, or Administrative Sup- port: Volunteer. Future Applications of a Stroke Model by Using Fibrin-Rich Clot 11. Gralla J, Schroth G, Remonda L, et al. Mechanical thrombectomy for acute ischemic stroke: thrombus-device interaction, efficiency, and complications in vivo. Stroke 2006;37:3019–24 It is critical to evaluate the devices under challenging and re- producible conditions, which is best done by using an animal model, to make improvements on existing and experimental MT devices. Given that the mechanical and histologic features of a thromboembolus are potentially among the key variables in predicting MT device performance, the use of a model with conventional thrombin-induced as well as fibrin-rich clot may contribute to the further refinement of MT devices. 12. Zucker MB, Owen J. Non-decalcified barium sulfate-adsorbed plasma: a po- tentially useful reagent for studying blood clotting, platelets or complement. Thromb Haemost 1982;47:182–84 13. Brekenfeld C, Schroth G, El-Koussy M, et al. Mechanical thromboembolec- tomy for acute ischemic stroke: comparison of the catch thrombectomy de- vice and the Merci retriever in vivo. Stroke 2008;39:1213–19 14. Gralla J, Burkhardt M, Schroth G, et al. Occlusion length is a crucial determi- nant of efficiency and complication rate in thrombectomy for acute ischemic stroke. AJNR Am J Neuroradiol 2008;29:247–52 15. Yin NS, Benavides S, Starkman S, et al. Autopsy findings after intracranial thrombectomy for acute ischemic stroke: a clinicopathologic study of 5 pa- tients. Stroke 2010;41:938–47 Furthermore, future improvements in diagnostic imaging technology may allow histologic characterization of in situ Yuki AJNR 33 Apr 2012 www.ajnr.org 648
https://openalex.org/W4367314968	https://www.frontiersin.org/articles/10.3389/fnagi.2023.1139789/pdf	English	null	Identification and validation of biomarkers based on cellular senescence in mild cognitive impairment	Frontiers in aging neuroscience	2,023	cc-by	10,279	OPEN ACCESS OPEN ACCESS EDITED BY Rong Chen, University of Maryland, United States REVIEWED BY Mauro Montalbano, University of Texas Medical Branch at Galveston, United States Ferit Tuzer, Drexel University, United States CORRESPONDENCE Haiyun Wang why819@126.com †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Neurocognitive Aging and Behavior, a section of the journal Frontiers in Aging Neuroscience RECEIVED 07 January 2023 ACCEPTED 30 March 2023 PUBLISHED 28 April 2023 CITATION Ma S, Xia T, Wang X and Wang H (2023) Identification and validation of biomarkers based on cellular senescence in mild cognitive impairment. Front. Aging Neurosci. 15:1139789. d i 10 3389/f i 2023 1139789 Songmei Ma 1,2†, Tong Xia 1†, Xinyi Wang 1,3,4,5 and Haiyun Wang 1,3,4,5 1 Department of Anesthesiology, The Third Central Clinical College of Tianjin Medical University, Tianjin, China, 2 Department of Anesthesiology, The First People’s Hospital of Shangqiu, Shangqiu, Henan, China, 3 Tianjin Key Laboratory of Extracorporeal Life Support for Critical Diseases, Tianjin, China, 4 Artificial Cell Engineering Technology Research Center, Tianjin, China, 5 Tianjin Institute of Hepatobiliary Disease, Tianjin, China Background: Mild cognitive impairment (MCI), a syndrome defined as decline of cognitive function greater than expected for an individual’s age and education level, occurs in up to 22.7% of elderly patients in United States, causing the heavy psychological and economic burdens to families and society. Cellular senescence (CS) is a stress response that accompanies permanent cell-cycle arrest, which has been reported to be a fundamental pathological mechanism of many age-related diseases. This study aims to explore biomarkers and potential therapeutic targets in MCI based on CS. Ma S, Xia T, Wang X and Wang H (2023) Identification and validation of biomarkers based on cellular senescence in mild cognitive impairment. Methods: The mRNA expression profiles of peripheral blood samples from patients in MCI and non-MCI group were download from gene expression omnibus (GEO) database (GSE63060 for training and GSE18309 for external validation), CS-related genes were obtained from CellAge database. Weighted gene co-expression network analysis (WGCNA) was conducted to discover the key relationships behind the co-expression modules. The differentially expressed CS-related genes would be obtained through overlapping among the above datasets. Then, pathway and GO enrichment analyses were performed to further elucidate the mechanism of MCI. The protein–protein interaction network was used to extract hub genes and the logistic regression was performed to distinguish the MCI patients from controls. TYPE Original Research PUBLISHED 28 April 2023 DOI 10.3389/fnagi.2023.1139789 TYPE Original Research PUBLISHED 28 April 2023 DOI 10.3389/fnagi.2023.1139789 1. Introduction Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease, and other types of dementia are more likely to occur as people age. Cell senescence was first discovered in vitro after large numbers of cultured fibroblasts were examined (Hayflick, 1965), and there is increasing evidence suggesting that cellular senescence plays an important role in age-related pathologies (Campisi and Robert, 2014; Muñoz-Espín and Serrano, 2014). Given the consideration of the important role cellular senescence plays in cognitive decline, we downloaded and analyzed the gene expression dataset about MCI (GSE63060) from GEO and senescence-related genes from the CellAge Database, hub genes associated with cellular senescence on MCI were identified in this study, 14 pairs of clinical samples were collected, patients with or without MCI were matched on the basis of age and education. Then multi-dimensional interaction networks were conducted to help researchers further understand the mechanism of MCI and develop novel treatments to alleviate the symptoms of MCI or slow its progression. Mild cognitive impairment (MCI), an intermediate stage between normal cognitive aging and dementia, is defined as poor cognitive performance on neurocognitive tests without significant impairment of instrumental activities of daily living (ADL). Etiologies associated with MCI mainly include degenerative and vascular processes, psychiatric causes, and some comorbidities like hypertension, diabetes mellitus and hyperlipidemia. The latest US Census data show that the adjusted prevalence of all-cause MCI was 22.7% (Rajan et al., 2021). With the follow-up duration lasting over 5 years, the annual rate of progression to dementia has been estimated to 5–15% (Dunne et al., 2021). So far, there is a roadblock to the early diagnosis and effective treatments due to the lack of consistency with screening for MCI and unclarified molecular mechanism. With the increasing aging population, the prevalence of MCI among the elderly warrants attention to explore the mechanism and identify the markers linked to the diagnosis of MCI. Such markers may offer researchers a point of intervention to minimize the future burden of dementia. Cellular senescence (CS) is a stress response that can be induced by DNA injury, telomere dysfunction, organelle stress, among others, which is characterized by an essentially irreversible proliferative arrest, abnormal metabolism of mitochondria as well as lysosomes. Senescent cells have been recognized as a fundamental driving factor of some senile chronic diseases, cancer and other age-related diseases (Saez- Atienzar and Masliah, 2020; Wissler Gerdes et al., 2020; Cortesi et al., 2021). 2.1. Data source All the microarray data after normalization were analyzed by R software. Microarray expression data for MCI patients and controls were downloaded from the Gene Expression Omnibus (GEO) database. The sample source of both GSE63060 and GSE18309 is from human blood. The GSE63060 dataset that included 99 controls and 80 MCI samples was used as training set, while GSE18309 dataset which included 3 controls and 3 MCI samples was used as external validation sets. 279 CS-related genes were acquired from Cell Age database.1 OPEN ACCESS The hub gene-drug network, hub gene-miRNA network as well as transcription factor-gene regulatory network were used to analyze potential therapeutic targets for MCI. Front. Aging Neurosci. 15:1139789. doi: 10.3389/fnagi.2023.1139789 COPYRIGHT © 2023 Ma, Xia, Wang and Wang. 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. Results: Eight CS-related genes were identified as key gene signatures in MCI group, which were mainly enriched in the regulation of response to DNA damage stimulus, Sin3 complex and transcription corepressor activity. The receiver operating characteristic curves of logistic regression diagnostic model were constructed and presented great diagnostic value in both training and validation set. Conclusion: Eight CS-related hub genes – SMARCA4, GAPDH, SMARCB1, RUNX1, SRC, TRIM28, TXN, and PRPF19 – serve as candidate biomarkers for MCI and display the excellent diagnostic value. Furthermore, we also provide a theoretical basis for targeted therapy against MCI through the above hub genes. 01 Frontiers in Aging Neuroscience frontiersin.org Ma et al. Ma et al. 10.3389/fnagi.2023.1139789 10.3389/fnagi.2023.1139789 mild cognitive impairment, cellular senescence, diagnostic model, biomarker, elderly patients 1 https://genomics.senescence.info/cells/ 1. Introduction Senescent cells display three major characteristics – loss of proliferation or regeneration capacity, alteration of metabolic functions and resistance to apoptosis, and secretion of an array of pathogenically active molecules, termed senescence-associated secretary phenotype (SASP) (Khosla et al., 2020), a suite of pro-inflammatory cytokines which could not only improve tissue homeostasis through promoting myofibroblast differentiation as well as wound healing in young people (Baker et al., 2016), they generate a proinflammatory environment and recruit immune cells to accelerate degenerative changes (Muñoz-Espín and Serrano, 2014). When a lot of immune cells were brought to the brain, it would further induce neuroinflammation and exert an impact to cognitive performance (Gaikwad et al., 2021; Guerrero et al., 2021; Sikora et al., 2021). Besides, mitochondrial dysfunction can be often found in senescent cells, such changes would make energy metabolism in these senescent cells prone to be disordered and consequently generate more free radicals damaging senescent cells themselves or the cells around (Balaban et al., 2005). It is reported that age-related accumulation of senescent cells may dysregulate neural stem cells directly causing the decline of brain function in hippocampus, which may be a potential mechanism in MCI (Fatt et al., 2022). Moreover, the removal of these cells could significantly reduce the infiltration of peripheral immune cells into the brain parenchyma and alleviate aging-related inflammatory response and cognitive function (Zhang et al., 2022). 2.3. Weighted gene co-expression network analysis A weighted co-expression network was constructed for the expression profile data of the GSE63060 dataset with the help of the WGCNA package of R software (Langfelder and Horvath, 2008). Firstly, samples were clustered to assess the presence of any outliers. Then, the automatic network construction function was used to get the co-expression network. An ideal soft threshold (β) was selected and verified with the help of the “pick Soft Threshold” function. The matrix data were then transformed into an adjacency matrix, followed by clustering, to identify modules based on the topological overlap. After completing the calculation of module eigengene (ME) and merging similar modules in the clustering tree according to ME, a hierarchical clustering dendrogram was drawn. Modules were combined with phenotypic data to measure the significance of genes as well as clinical information, and analyze the correlation between modules and clinical features. In this way we could find out which modules are most relevant to MCI. After overlapping DEGs, module genes and CS-related genes, we obtained the final DEGs associated with CS-related genes (hereinafter referred to as CS-DEGs). To effectively differentiate the MCI from controls, the logistic regression diagnostic model was constructed using the glm function in R language (Friedman et al., 2010). The gene expression was seen as continuous variable and sample type as binary classification variable. The stepwise regression method was used to screen the variables with p < 0.05, and the final adjusted model was established based on the screened variables. The diagnostic and predictive performance to MCI of the logistic regression model would be evaluated by receiver operating characteristic (ROC) analysis. 2.4. Functional annotation and pathway enrichment analysis Functional enrichment analysis of CS-DEGs was performed in three domains of Gene Ontology (GO) database using the R package “clusterProfiler” (The Gene Ontology Consortium, 2017), including biological process (BP), cellular component (CC), and molecular function (MF). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database contains datasets of pathways involving biological functions, diseases, chemicals, and drugs (Kanehisa and Goto, 2000). The enrichment analysis was carried out by “cluster Profiler” package to determine the biological functions of the genes and relevant pathways. p < 0.05 was considered statistically significant. “Omic Circos” package was used to analyze and display the location of genes on chromosomes. 2.8. Construction of gene-drug interaction network and regulatory network of hub genes In order to explore the potential therapeutic drugs for MCI, drugs targeting proteins encoded by key genes were identified through the DGIdb database.3 Then we used NetworkAnalyst database4 and starBase databases5 to predict the transcription factors (TFs) and miRNAs related to hub genes. The association between hub genes and their TFs or miRNAs were integrated into a regulatory network using Cytoscape software. 2.7. Correlation and functional similarity analysis of hub genes and gene set enrichment analyses analysis The “ggpubr” package was used to perform Spearman correlation analysis on hub genes. Moreover, the functional similarity among proteins was evaluated using the geometric mean of semantic similarities in CCs and MFs through the GOSemSim package (Yu et al., 2010). Functional similarity, the geometric mean of their semantic similarities in GO-MF and GO-CC terms, is designed for measuring the strength of the relationship between each protein and its partners by considering function and location of proteins. Moreover, GSEA of hub genes were performed using the “GSEABase” packages, KEGG gene sets were used as a reference, with p < 0.05 being a statistically significant difference. 2.2. Acquirement of differentially expressed genes Differential expression genes (DEGs) were analyzed between the controls and MCI patients using the “limma” package in R software, the cutoff values were adj.P.Val < 0.05. The heatmap cluster and the volcano plot about DEGs were created using the “pheatmap” and “ggplots” packages via R software. 1 https://genomics.senescence.info/cells/ Frontiers in Aging Neuroscience 02 frontiersin.org Ma et al. 10.3389/fnagi.2023.1139789 3 https://dgidb.genome.wustl.edu/ 4 https://www.networkanalyst.ca/ 5 https://starbase.sysu.edu.cn/index.php 3 https://dgidb.genome.wustl.edu/ 4 https://www.networkanalyst.ca/ 3.2. Go and KEGG enrichment analysis of CS-DEGs We overlapped 2,394 module genes and DEGs to obtain 1865 MCI-related DEGs (Figure 3A). Furthermore, after intersecting the CS-related genes (CGRs) with MCI-related DEGs, we found total 33 CS-DEGs (Figure 3B). Localization analysis revealed that these 33 CS-DEGs were mainly located in 14 pairs of chromosomes (Figure 3C). In GO analysis, the above 33 CS-DEGs were classified into biological process (BP), cellular component (CC), and molecular function (MF). Among BP classes, CS-DEGs were mainly enriched in the response to DNA damage stimulus and reactive oxygen species. In the CC category, the genes expressed were mostly enriched in transcription corepressor activity, transcription coregulator activity and core promoter sequence-specific DNA binding. The pathways enriched by GO-MF were principally associated with Sin3 complex, npBAF complex and XY body (Figure 4A). GO enrichment analysis showed that CS-DEGs were mainly enriched in transcription corepressor activity, transcription coregulator activity and response to DNA damage stimulus (Figure 4B). Next, we performed KEGG pathway enrichment analysis to further understand gene biological functions of CS-DEGs and it did not reveal significantly enriched pathways. TABLE 1 Primer sequences for QRT-PCR. TABLE 1 Primer sequences for QRT-PCR. Gene Primer sequence (5′ → 3′) SMARCA4 Forward CAAAGACAAGCACATCCTCGCC Reverse GCCACATAGTGCGTGTTGAGCA SMARCB1 Forward GGCATCAGAAGACCTACGCCTT Reverse CTCCATCTCAGCGTCTGTCAGA RUNX1 Forward CCACCTACCACAGAGCCATCAA Reverse TTCACTGAGCCGCTCGGAAAAG SRC Forward CTGCTTTGGCGAGGTGTGGATG Reverse CCACAGCATACAACTGCACCAG TRIM28 Forward CAAGATTGTGGCAGAGCGTCCT Reverse CATAGCCTTCCTGCACCTCCAT TXN Forward GTAGTTGACTTCTCAGCCACGTG Reverse CTGACAGTCATCCACATCTACTTC PRPF19 Forward TGGGCTTTCTCTGACATCCAGAC Reverse CCTGTTCCAAAGATGAGTCCGTC β-actin Forward CACCATTGGCAATGAGCGGTTC Reverse AGGTCTTTGCGGATGTCCACGT 3.1. Identification of DEGs and co-expression network construction A total of 5,017 DEGs, including 2,605 up-regulated genes and 2,412 down-regulated genes, were obtained when comparing MCI cases to controls (Figure 1A, adj.P.val < 0.05). The heatmap showed the most significant 10 up-regulated and down-regulated genes (Figure 1B). 2.10. RNA extraction and quantitative real-time-PCR The co-expression network was constructed by WGCNA to further identify genes strongly associated with MCI, followed by clustering of samples and 8 outliers need to be culled (Figure 2A). A soft threshold of β = 10 was selected for consistency with the scale-free network. 19 modules were identified based on average hierarchical clustering and dynamic tree clipping (Figures 2B,C). The correlations of the above 19 modules with MCI and controls were presented via heat maps, with two key modules including green and brown contained a total of 2,394 genes demonstrating the highest correlation with MCI (cor = −0.5, p < 0.001 in green module; cor = 0.49, p < 0.001 in brown module; Figure 2D). The blood samples were collected before surgery. The peripheral blood mononuclear cells (PBMCs) were extracted using Lymphocyte Separation Medium (Human) (P8610, Solarbio, Beijing, China), washed with 1 × PBS (FZ1258, Solarbio, Beijing, China) 3 times and then total RNA from PBMCs was extracted by the TRIzol reagent (15596026, Thermo Fisher Scientific Inc., Waltham, MA, United States). The Nano Drop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) was utilized to measure the concentration and purity of the extracted RNA, with the A260/ A280 between 1.8 and 2.0. The cDNA synthesis was conducted using the reverse transcription kit (RR036A, Takara, Japan). Using β-actin as a reference, we performed QRT-PCR with the SYBR Premix Ex Taq II (RR820A, Takara, Japan) on an ABI 7500 instrument (Applied Biosystems, Foster City, CA, United States), with 3 duplicates each well. Primer sequences (Sangon Biotech, Shanghai, China) for reference and candidate genes are shown in Table 1. The 2−∆∆Ct method was applied to calculate the relative expression level of mRNA. 3. Results with healthy controls according to age and gender. All subjects underwent a structured interview and a battery of neuropsychological assessments, such as MMSE, MoCA and Clinical Dementia Rating (CDR). MCI was identified as follows: MMSE < 27 (17–27 for illiterate individuals, 20–27 for participants with elementary school education, and 24–27 for those with middle school education and above; Mitchell, 2009), MoCA scores in the range of 15–24 (Memoria et al., 2013), and CDR equal to 0.5. 2.9. Sample collection To further explore the interaction among the obtained CS-DEGs, we used the Search Tool for the Retrieval of Interacting Genes (STRING)2 to construct a PPI network (Szklarczyk et al., 2015). Then, Cytoscape plugin-Analyze Network was used to screen the significant modules with the degree >3 in the PPI network, and hub genes from CS-DEGs were obtained. The “ggpubr” package was used to perform Spearman correlation analysis on these hub genes. This study was approved by the Medical Ethics Committee of the Third Central Clinical College of Tianjin Medical University (approval number: IRB2022-011-02), which complied with the Declaration of Helsinki. All subjects signed informed consent. Patients with the age of more than 65 years and undergoing lumbar decompression and fusion were enrolled. Total of 14 MCI patients and 14 controls were enrolled in the study, and subjects undergoing MCI were matched Frontiers in Aging Neuroscience Frontiers in Aging Neuroscience frontiersin.org 03 Ma et al. 10.3389/fnagi.2023.1139789 Frontiers in Aging Neuroscience 3.3. PPI network construction and module analysis To further analyze the interaction of 33 CS-DEGs, Analysis of PPI network was performed using STRING and visualized by Cytoscape under default parameters (Figure 5A). By using Analyze Network plugin, the 8 genes – SMARCA4, GAPDH, SMARCB1, RUNX1, SRC, TRIM28, TXN, and PRPF19 – were selected as potential hub genes (Figures 5B,C). The expressions of all genes except TXN were Frontiers in Aging Neuroscience 04 frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. A B FIGURE 1 Identification of differentially expressed genes (DEGs) between MCI and Control group. (A) Volcano plot of DEGs expression profile. (B) heatmap of the top 10 up-regulated and down-regulated DEGs, respectively. A C D B FIGURE 2 A B FIGURE 1 Identification of differentially expressed genes (DEGs) between MCI and Control group. (A) Volcano plot of DEGs expression profile. (B) heatmap of the top 10 up-regulated and down-regulated DEGs, respectively. B A A A B FIGURE 1 Identification of differentially expressed genes (DEGs) between MCI and Control group. (A) Volcano plot of DEGs expression profile. (B) heatmap of the top 10 up-regulated and down-regulated DEGs, respectively. A C D B FIGURE 2 Identification of significant modules and genes of GSE63060 by WGCNA. (A) Clustering dendrogram of samples. (B) Network topology analysis with different soft thresholds. The scale-free R2 was 0.90 and the soft threshold was 10. (C) A cluster dendrogram of module-specific colors showed 19 co-expressed gene modules between MCI and controls. (D) Heatmap of correlation between disease groupings and gene modules. A B B A A C D C D FIGURE 2 Identification of significant modules and genes of GSE63060 by WGCNA. (A) Clustering dendrogram of samples. (B) Network topology analysis with different soft thresholds. The scale-free R2 was 0.90 and the soft threshold was 10. (C) A cluster dendrogram of module-specific colors showed 19 co-expressed gene modules between MCI and controls. (D) Heatmap of correlation between disease groupings and gene modules. 05 Frontiers in Aging Neuroscience frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. A B C FIGURE 3 The intersection of MCI-related modules and cellular senescence-related genes as well as their locations on chromosomes. (A) There were 5,017 DEGs in the GSE63060 dataset and 2,394 genes in the blue module of the GSE63060 dataset, and 1865 genes were obtained as key DEGs by overlapping the two datasets. 3.4. The logistic regression model for the diagnosis of MCI By performing GSEA analysis of each hub gene, we found that all hub genes were associated with “KEGG_RIBOSOME” and “KEGG_ PARKINSONS_DISEASE.” The top five important pathways associated with PRPF19 and RUNX1 were the same. PRPF19, RUNX1, SMARCA4, SMARCB1, SRC, TRIM28, and TXN were related to “KEGG_OXIDATIVE PHOSPHORYLATION.” PRPF19, RUNX1, SMARCA4, SMARCB1, TRIM28, and TXN were associated with “KEGG_ALZHEIMERS_DISEASE” (Figures 8A–H). These indicated that MCI shares common features with AD and Parkinson’s disease. In order to further elucidate the clinical predictive value of the 8 hub genes, we applied the logistic regression model. Taken together, the logistic regression model established based on the hub genes could effectively identify individuals with or without MCI. The results of ROC curve in training set (AUC = 0.816) and validation set (AUC = 0.889) show the great predictive power of the model and suggest that these eight genes may be potential treatment targets for patients with MCI (Figures 6A,B). 3.3. PPI network construction and module analysis (B) There were 1865 key DEGs and 279 cellular senescence-related genes (CRGs) from Cell Age database, and 33 genes were obtained as CS-DEGs by overlapping the two datasets. (C) Chromosome locations of the 33 CS-DEGs. A C B B FIGURE 3 The intersection of MCI-related modules and cellular senescence-related genes as well as their locations on chromosomes. (A) There were 5,017 DEGs in the GSE63060 dataset and 2,394 genes in the blue module of the GSE63060 dataset, and 1865 genes were obtained as key DEGs by overlapping the two datasets. (B) There were 1865 key DEGs and 279 cellular senescence-related genes (CRGs) from Cell Age database, and 33 genes were obtained as CS-DEGs by overlapping the two datasets. (C) Chromosome locations of the 33 CS-DEGs. upregulated in MCI and all displayed difference between groups (Figure 5D). upregulated in MCI and all displayed difference between groups (Figure 5D). correlation (cor = 0.68), followed by the correlation between SMARCB1 and PRPF19 (cor = 0.62; Figure 7A). Moreover, we ranked hub genes based on the average functional similarity relationships among proteins within the interactome of 8 hub genes. SMARCA4, SMARCB1 and TRIM28 were the three top-ranked proteins potentially playing key roles in MCI (Figure 7B). (Figure 9). we entered the 8 hub genes (SMARCA4, GAPDH, SMARCB1, RUNX1, SRC, TRIM28, TXN, and PRPF19) in starBase and NetworkAnalys database as search terms, and the searched results about miRNAs-genes and TFs-genes network were constructed by Cytoscape software. Among them, miRNAs targeting at least three hub genes were selected to construct the network. Finally, the network composed of 8 hub genes, 385 miRNAs and 39 TFs were established (Figure 10). 3.6. Construction of hub gene-drug interaction network and regulatory network of hub genes The correlation results showed that TXN had the negative correlation with other hub genes. Among them, TXN had the strong negative correlation with TRIM28 (cor = −0.74), the next was PRPF19 (cor = −0.69). PRPF19 and TRIM28 had the strongest positive In order to explore the interaction between 8 hub genes and potential treatment of MCI, drugs associated with the key genes were identified by DGIdb database. The interested genes Frontiers in Aging Neuroscience 06 frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. A B FIGURE 4 Gene ontology enrichment analysis. (A) Enrichment bar graph. X-axis indicates the number of CS-DEGs. Y-axis represents GO terms. All GO terms are grouped into three ontologies: biological process (BP), cellular component (CC) and molecular function (MF). The color of bars represents the adj.P.Val. (B) Chord plot shows that the links were straight forward to show the relations between genes and some key GO terms. B A B A FIGURE 4 Gene ontology enrichment analysis. (A) Enrichment bar graph. X-axis indicates the number of CS-DEGs. Y-axis represents GO terms. All GO terms are grouped into three ontologies: biological process (BP), cellular component (CC) and molecular function (MF). The color of bars represents the adj.P.Val. (B) Chord plot shows that the links were straight forward to show the relations between genes and some key GO terms. SMARCB1 shows no statistical difference between MCI group and non-MCI group. SMARCB1 shows no statistical difference between MCI group and non-MCI group. (SMARCA4, GAPDH, SMARCB1, RUNX1, SRC, TRIM28, TXN, and PRPF19) are used as search terms and determined the extant drugs targeting these genes. Plenty of drugs could affect the expression of these eight hub genes except for PRPF19 and TRIM28, and a total of 124 drug-gene interaction pairs are in this network. Among them, SRC, RUNX1 and GAPDH were the top 3 hub genes targeted by 56, 35, 15 drugs, respectively (Figure 9). 4. Conclusion This work has shown that the mRNA levels of SMARCA4, GAPDH, SMARCB1, RUNX1, SRC, TRIM28, TXN and PRPF19 in PBMCs are effective diagnostic biomarkers in MCI. Multifactorial interaction networks related to CRGs have been successfully constructed and these results would largely contribute to the understanding of the pathogenesis of MCI and provide the foundation to explore novel therapeutic targets for MCI. 5. Discussion Then we recruited patients for clinical study to validate the above findings. After analyzing the basic demographic data for the enrolled patients, we found there were no differences in age, gender, BMI, ASA and preoperative comorbidity among patients with or without MCI, while educational attainment and neuropsychological test shows statistic difference, which was consistent with our diagnostic criteria for MCI (Table 2). The transcriptional changes of hub genes were detected in the PBMCs from patients with or without MCI by QRT-PCR (Figure 11). The results indicated that the expression levels of TXN was decreased in MCI group in comparison with those in controls, while PRPF19, RUX1, SMARCA4, SRC and TRIM28 were increased in MCI group, which was basically in line with GSE63060 and GSE18309. However, the expression level of The rate of mean annual conversion to dementia in MCI (10%) is far higher than that in the general population (1–2%) (Yan et al., 2022). With the consideration of reversible characteristic of MCI, this stage would provide an optimal window to the early prevention and intervention of dementia. Thus, potential markers for diagnosis and treatment with high efficiency are urgently demanded. In the present study, we identified hub genes related to cellular senescence and developed a diagnostic model for MCI. Gene expression microarray data from GSE18309 and clinical samples were used to further verify the value of this model. We found that 8 biomarkers – SMARCA4, GAPDH, SMARCB1, RUNX1, SRC, TRIM28, TXN and PRPF19 – could identify individuals with MCI with ROC curve in training set (AUC = 0.816) and validation set (AUC = 0.889). Frontiers in Aging Neuroscience 07 frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. A C B D FIGURE 5 Protein–protein interaction (PPI) network construction and the expression of 8 hub-genes. (A) PPI network constructed with the CS-DEGs. (B,C) The significant module identified from the PPI network using the molecular complex detection (MCODE) method. (D) The expressions of 8 hub-genes. p < 0.05, p < 0.01, *p < 0.005. B A B A A C D FIGURE 5 Protein–protein interaction (PPI) network construction and the expression of 8 hub-genes. (A) PPI network constructed with the CS-DEGs. (B,C) The D D D C C FIGURE 5 Protein–protein interaction (PPI) network construction and the expression of 8 hub-genes. (A) PPI network constructed with the CS-DEGs. (B,C) The significant module identified from the PPI network using the molecular complex detection (MCODE) method. 5. Discussion (D) The expressions of 8 hub-genes. p < 0.05, p < 0.01, p < 0.005. B A B FIGURE 6 The ROC curve of diagnostic model. (A) GSE63060 dataset serves as the training set. (B) GSE18309 dataset serves as the validation set. A B B FIGURE 6 The ROC curve of diagnostic model. (A) GSE63060 dataset serves as the training set. (B) GSE18309 dataset serves as the validation se 08 Frontiers in Aging Neuroscience frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. In this study, people with or without MCI were compared to identify differentially expressed genes, and 1,865 genes differentially expressed in key modules were identified based on WGCNA. We identified gene sets group, MCI correlated genes were identified as genes associated with the disease, the expression differences of individual genes are not considered in this analysis, thus, genes differentially expressed between MCI samples A B FIGURE 7 Heatmap of correlation and functional similarity among hub-genes. (A) Correlation matrix for all 8 hub genes. Some hub genes were negatively related, represented in blue, and others were positively related, represented in red. The darker the color, the higher the correlation was (p < 0.05). (B) Functional similarity of all 8 hub genes. A B C D E F G H FIGURE 8 Enrichment analysis of pathway and gene ontology (GO) involved hub genes. (A–H) Gene Set Enrichment Analysis (GSEA) of TRIM28, GAPDH, TXN, PRPF19, RUNX1, SMARCA4, SMARCB1 and SRC. Different colors represent different signaling pathways. A B FIGURE 7 Heatmap of correlation and functional similarity among hub-genes. (A) Correlation matrix for all 8 hub genes. Some hub genes were negatively related, represented in blue, and others were positively related, represented in red. The darker the color, the higher the correlation was (p < 0.05). (B) Functional similarity of all 8 hub genes. A B B A FIGURE 7 Heatmap of correlation and functional similarity among hub-genes. (A) Correlation matrix for all 8 hub genes. Some hub genes were negatively related, represented in blue, and others were positively related, represented in red. The darker the color, the higher the correlation was (p < 0.05). (B) Functional similarity of all 8 hub genes. A B C D E F G H FIGURE 8 Enrichment analysis of pathway and gene ontology (GO) involved hub genes. (A–H) Gene Set Enrichment Analysis (GSEA) of TRIM28, GAPDH, TXN, PRPF19, RUNX1, SMARCA4, SMARCB1 and SRC. Frontiers in Aging Neuroscience 5. Discussion Different colors represent different signaling pathways. C D A B B C D A B C D A D B G F H i (GSEA) f TRIM28 GAPDH TXN E H F E G FIGURE 8 Enrichment analysis of pathway and gene ontology (GO) involved hub genes. (A–H) Gene Set Enrichment Analysis (GSEA) of TRIM28, GAPDH, TXN, PRPF19, RUNX1, SMARCA4, SMARCB1 and SRC. Different colors represent different signaling pathways. In this study, people with or without MCI were compared to identify differentially expressed genes, and 1,865 genes differentially expressed in key modules were identified based on WGCNA. We identified gene sets with similar expression patterns and analyzed the relationship between gene sets and sample phenotypes based on WGCNA analysis of key module genes, MCI was the sample phenotype compared to the control group, MCI correlated genes were identified as genes associated with the disease, the expression differences of individual genes are not considered in this analysis, thus, genes differentially expressed between MCI samples and controls were screened. Then the 1865 DEGs intersect with genes involved in cellular senescence, from the intersection of the DEGs with the genes involved in cellular senescence, DEGs associated with cellular 09 frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. FIGURE 9 Hub gene-drug interaction network. The orange circles represent hub-gene and white rectangles represent small molecule drugs. FIGURE 9 Hub gene-drug interaction network. The orange circles represent hub-gene and white rectangles represent small molecule drugs. FIGURE 9 Hub gene-drug interaction network. The orange circles represent hub-gene and white rectangles represent small molecule drugs. A B FIGURE 10 The miRNA-gene network (A) and transcription factor-gene regulatory network (B). The orange circles represent hub-genes, purple triangles represent miRNAs and blue diamonds represent TFs. B A B A FIGURE 10 The miRNA-gene network (A) and transcription factor-gene regulatory network (B). The orange circles represent hub-genes, purple triangles represent miRNAs and blue diamonds represent TFs. FIGURE 10 The miRNA-gene network (A) and transcription factor-gene regulatory network (B). The orange circles represent hub-genes, purple triangles represent miRNAs and blue diamonds represent TFs. can be affected by DNA damage and the cellular DDR, which would alter cell fate, deregulate intercellular communication, mutations or chromosomal aberrations caused by DNA damage, and then trigger genome instability as well as cellular senescence (Williams and Schumacher, 2016; Mladenov et al., 2018; Petr et al., 2020). 5. Discussion In addition the damage of genes encoding for critical neuronal functions could affect synaptic plasticity, learning as well as memory formation (Kim et al., 2022; Miller et al., 2022; Yoon et al., 2022). Moreover, the high level of ROS as well as subsequent DNA damage would make the brain exposed to oxidative stress and such pathologic changes would increase the accumulation of amyloid-β as well as neurofibrillary tangles, neuronal loss senescence were identified. GO and KEGG enrichment analyses were performed to explore interactions among these 33 CS-DEGs which appeared to be associated with cellular senescence and MCI via DNA damage repair and histone deacetylation. According to the results of GO-BP, CS-DEGs were mainly enriched in regulation of DNA damage response, reactive oxygen species (ROS) response and positive regulation of oxygen species response. ROS are a group of short-lived, highly reactive, oxygen-containing molecules, which are necessary for the communication between nucleus and mitochondria but have detrimental effects at extremely high levels via damaging DNA, activating DNA damage response (DDR) (Valko et al., 2007). Many molecular processes Frontiers in Aging Neuroscience 10 frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. TABLE 2 Demographic data for patients with/without MCI. non-MCI (n=14) MCI (n=14) t/Z/χ2 P value Preoperative data Male sex, n (%) 50.00% (n=7) 50.00% (n=7) 0.000 1.000 Age (yr) 69.50 (64.75, 73.50) 67.50 (66.00, 70.25) −0.554 0.603 BMI (kg/m2) 25.95 (23.94, 27.12) 27.34 (24.28, 28.92) −0.827 0.427 ASA (II/III), n 10/4 9/5 0.164 0.686 Education level (yr) 12.00 (9.00, 12.00) 7.00 (3.75, 9.00) −2.802 0.005 Comorbidity, before operation Hypertension, n (%) 57.14% (n=8) 64.29% (n=9) 0.150 0.699 Diabetes, n (%) 14.29% (n=2) 28.57% (n=4) 0.848 0.357 Coronary Artery Heart Disease, n (%) 14.29% (n=2) 21.43% (n=3) 0.243 0.622 Neuropsychological test MMSE 29.00 (28.00, 29.00) 24.00 (22.75, 25.25) −4.425 0.000* MoCA 25.00 (22.75, 26.25) 20.00 (16.00, 22.00) −4.020 0.000* Descriptive results of continuous variables were presented as the means ± standard deviations, median with inter-quartile range or numbers with percentages. The P-value is calculated by the independent samples T test Kruskal Wallis H test or chi square test respectively p<0 05 compared with group non MCI Descriptive results of continuous variables were presented as the means ± standard deviations, median with inter-quartile range or numbers with percentages. The P-value is calculated by the independent-samples T test, Kruskal-Wallis H test or chi-square test, respectively. p<0.05 compared with group non-MCI. 5. Discussion In term of MF, mounting evidence showed that sequence-specific activators and repressors interact with coregulators, Frontiers in Aging Neuroscience 11 frontiersin.org 10.3389/fnagi.2023.1139789 Ma et al. production and mediate Aβ-induced synaptic deficits and neurotoxicity, in addition, Fyn could also induce tau tyrosine phosphorylation and then form neuroillary tangles (Bhaskar et al., 2005; Yang et al., 2011). The amyloid plaques and neurofibrillary tangles would finally lead to neurotoxicity and cognitive impairment (Haass and Mandelkow, 2010). RUNX1 belongs to master regulators for the age-dependent microglia module and increases the level of G9a through histone lysine methylation, leading to the increase of neuroinflammatory markers such as interleukin-6, tumor necrosis factor-α and then cognitive impairment in rats (Grinan-Ferre et al., 2019; Li et al., 2020). GAPDH is an enzyme known for its propensity to form aggregates when oxidized or complexed with mutant proteins and the amount of soluble or insoluble GAPDH complexes with Aβ in the CSF from patients with various stages of AD directly correlated with the severity of the disease (Gerszon and Rodacka, 2018; Lazarev et al., 2021). Thus, GAPDH is a promising pharmacological target for AD. sites to catalyze H3K56 as well as H4K16 deacetylation when confronting DNA double-strand breaks (DSBs) and stimulate DSB repair through the nonhomologous end joining (NHEJ) pathway. Therefore, it exerts an important regulatory influence on individual learning and memory through the changes of synaptic plasticity, dendritic spine density and synapse number (Fischer et al., 2007; Guan et al., 2009; Gräff et al., 2012; Cummings et al., 2018). The ATP-dependent chromatin remodeling complex BAF, namely SWI/SNF, mainly consists of SMARCB1, SMARCA4, SMARCA2, SMARCE1, ARID1A as well as ARID1B (Santen et al., 2013), and is crucial for the regulation of gene expression and differentiation. It is reported that npBAF is essential for neural development whose subunits have an enormous influence on dendritic morphogenesis, neuronal subtype maturation and learning as well as long-term memory (Alfert et al., 2019). From the CS-DEGs, we identified genes with ≥10 connections in the PPI network analysis as hub genes. 8 hub genes were identified, which included SMARCA4, GAPDH, SMARCB1, RUNX1, SRC, TRIM28, TXN and PRPF19, among which all hub genes were up-regulated in MCI except for TXN. Logistic regression model was constructed to predict the risk of MCI (AUC = 0.816 in training set, 0.889 in validation set), which revealed a high diagnostic value for MCI. 5. Discussion MCI, mild cognitive impairment; BMI, Body Mass Index; ASA, American Society of Anesthesiology; MMSE, mini-mental state examination; MoCA, Montreal Cognitive Assessment. p p q g p gh y independent-samples T test, Kruskal-Wallis H test or chi-square test, respectively. p<0.05 compared with group non-MCI. MCI, mild cognitive impairment; BMI, Body Mass Index; ASA, American Society of Anesthesiology; MMSE, mini-mental state examination; MoCA, Montreal Cognitive Assessment. independent samples T test, Kruskal Wallis H test or chi square test, respectively. p<0.05 compared with group non MCI. MCI, mild cognitive impairment; BMI, Body Mass Index; ASA, American Society of Anesthesiology; MMSE, mini-mental state examination; MoCA, Montreal Cognitive Assessment. FIGURE 11 QRT-PCR validation of the hub genes between controls and MCI. The data were expressed as mean ± SEM (short for standard error of mean). p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, ns means no significance. FIGURE 11 QRT-PCR validation of the hub genes between controls and MCI. The data were expressed as mean ± SEM (short for standard error of mean). p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001, ns means no significance. which in turn either stimulate or inhibit the binding or function (or both) of some transcription complex (Mannervik et al., 1999). As core component of transcriptional corepressor complex, Sin3 plays a transcriptional inhibitory role by binding to histone deacetylase (HDAC) (Kadamb et al., 2013). Sin3/HDAC complex have been implicated in learning and memory, whose dysregulation has been linked to cognitive impairment in brain aging and neurodegenerative diseases (Gräff et al., 2012). with the help of Sin3, HDAC would be promptly recruited to break and microglia activation, resulting in the occurrence and progression of neurodegenerative diseases, such as MCI, AD, among others (Akterin et al., 2006; Ghosh et al., 2015; Buccellato et al., 2021). In terms of the results of GO-MF and GO-CC, most of genes or pathways are principally associated with the regulation of cell cycle and the self-renewal or proliferative capacity of cells (Simone, 2006; Gupta et al., 2020; Braun et al., 2021; Mitra et al., 2022). 5. Discussion Fyn could upregulate amyloid-beta peptides (Aβ) In order to further explore the possibility of these eight hub genes as potential therapeutic targets for MCI, we analyzed the interaction between the hub genes and available therapeutic drugs of MCI and found that numbers of drugs could affect the expression of these hub genes. The DGIdb database was used to identify compounds targeting the proteins encoded by hub genes and 124 drugs were finally found. Natural organic acids may exert beneficial effects to improve cognition through increasing energy metabolism, taking part in antioxidant and anti-inflammatory, and thereby reducing damage and death of neural cells (Kolker et al., 2008; Colin-Gonzalez et al., 2015). Nutritional supplementation of gallic acid preserve the morphological and physiological integrity of the hippocampus against environmental neurotoxins by mopping up free radicals associated with oxidative stress induced AD (Ogunlade et al., 2022). Estradiol, has been widely reported owing to their neuroprotective action because they give rise to a wide range of cell signals and generate effects in genes by means of canonical pathways or through non-conventional mechanisms that are involved in neuronal survival, dendritogenesis and synapse remodeling (Barrera Ocampo et al., 2008). The Src family kinase inhibitor, saracatinib, targeting Fyn as a therapeutic intervention in AD, based on its activation by Aβ via cellular prion protein but also due to its known interaction with tau, uniquely linking the two key pathologies in AD. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS (Imamura et al., 2017). Drugs known to have effects on other neurodegenerative diseases could provide a new direction for MCI research. Micro-RNAs (miRNAs) play a critical role in regulating gene expression. They would bind to specific messenger RNAs (mRNAs) and inhibit their translation as well as related protein synthesis, which is important for many cellular processes, including development, differentiation, and apoptosis. For instance, hsa-miR-212-5p could influence cognition by inhibiting SRC in our study, it is because hsa-miR-212-5p reduces neuronal synaptic plasticity by downregulating BDNF, MECP2, CREB and PTEN. The Inhibition of hsa-miR-212-5p would increase the expression of these proteins and improve cognitive performance, such as learning and long-term memory formation (Li and Cai, 2021). 5. Discussion TXN is involved in many antioxidized and antiapoptotic reactions, at least two isoforms of which have been characterized in mammals, TXN1 (mainly in cytosolic) and TXN2 (mainly in mitochondrial). TXN contributes to the response to intracellular nitric oxide (NO) via the reversible S-nitrosylation of cysteine residues in CASP3, and thereby inhibits caspase-3 activity (Mitchell et al., 2007; Glaser et al., 2008). Besides, TXN-1 is required for nerve growth factor-mediated signal transduction and neurite outgrowth, and is involved in synaptic protein expression induced by BDNF (Bai et al., 2019). Thus, TXN is a promising early biomarker of AD, AD patients’ hippocampus tissue sections displayed abnormal TXN1 immunoreactivity and expression pattern, as promising early biomarkers of AD compared to controls (Arodin et al., 2014). TXN had strong negative correlation with TRIM28, which means TRIM28 would play a protective role in the pathogenesis of MCI. TRIM28 could bind to zinc finger proteins containing KRAB, acts as a transcriptional cofactor and an E3 ubiquitin ligase. The phosphorylation of TRIM28 at S473 promoted association between TRIM28 and cap binding complex dependent translation initiation factor (CTIF), which inhibits aggresome formation (Chang et al., 2021). It has been found that TRIM28 could regulate α-Syn and tau levels via SUMOylation and is highly expressed in neurodegenerative brain tissues (Rousseaux et al., 2016, 2018). As another Ubiquitin-protein ligase, pre-mRNA processing factor 19 (Prpf19/prp19) mainly involved pre-mRNA splicing and plays a neuroprotective role in the DDR. PRPF19 was upregulated during neurodegeneration, for it can antagonize exocyst complex component 7 (Exoc7) to improve neurodegeneration by degrading misfolded proteins in neurons through the ubiquitin-proteasome system, therefore, patients with MCI are often accompanied by higher PRPF19 levels than patients with normal cognitive function (Chen et al., 2021). SRC is a tyrosine- protein kinase whose activity can be inhibited by phosphorylation by c-SRC kinase. c-SRC and other tyrosine-phosphorylating protein kinases with a similar structure to c-SRC gave rise to the concept of Src family kinases (SFKs), SFK stimulation has been associated with microglial activation and neuropathological conditions, including Alzheimer’s and Parkinson’s. Fyn, an Src family non-receptor tyrosine kinase, has been linked to synaptic plasticity, which is a cellular mechanism for learning and memory. 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. Briefly, the cellular senescence-related gene signature can accurately diagnose the occurrence of MCI, they may provide a new avenue for MCI mechanism and treatment. 5. Discussion It has been reported that Frontiers in Aging Neuroscience 12 frontiersin.org Ma et al. 10.3389/fnagi.2023.1139789 nlm.nih.gov/geo/query/acc.cgi?acc=GSE63060 and https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE18309. nlm.nih.gov/geo/query/acc.cgi?acc=GSE63060 and https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE18309. miRNA-455-5p/CPEB1 pathway mediated synaptic and memory deficits in Alzheimer’s Disease through targeting on AMPARs (Xiao et al., 2021). Research has shown that the expression of miR-421 is related to neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and stroke (Yang et al., 2017; Angelopoulou et al., 2019; Ouyang et al., 2022). miR-421 may be involved in the pathogenesis and development of these neurological diseases through mechanisms such as regulating neuron development, metabolism, and immune responses. In neuroscience, TFs are considered important regulatory factors for memory and cognitive function. They can affect the development of neurons, synaptic development, and synaptic plasticity, thereby affecting memory formation and cognitive function. The loss or mutation of transcription factors may be related to neurological disorders and cognitive impairment. The lack of the transcription factor CREB (cAMP response element binding protein) may lead to impaired learning and memory function (Amidfar et al., 2020). The STAT signaling pathway also plays an important role in regulating memory and cognition. STAT3 plays a crucial role in synapse development by controlling axonal guidance and synapse formation, affecting the transmission of neural potentials and memory generation and storage. STAT3 binds to glutamatergic neurotransmitter receptors, regulating glutamate release and function of glutamatergic neurons, thereby affecting memory processing and retrieval (Larsen et al., 2021). Overall, a number of miRNAs as well as TFs were identified that affect expression of these genes, and hence the pathological processes of MCI. The discussion of known effects of targeting these TFs or genetic variations in them in MCI and other neurodegenerative diseases would be helpful and may provide a brand-new research direction for the diagnosis and treatment of MCI and, further, a theoretical basis for the follow-up experimental research on MCI. However, whether MCI patient with over-expression or under-expression of these hub genes could benefit from the regulation of hub genes, or whether these hub genes are promising, therapeutic targets still need further experimental supports including pre-clinical and prospective clinical studies.h Author contributions HW designed the study and revised the manuscript. SM analyzed the data, generated the figures and wrote the manuscript. TX analyzed the data, involved in manuscript writing and patients’ recruitment. XW conducted Qrt-PCR and revised the manuscript. All authors contributed to the article and approved the submitted version. Conflict of interest There are several limitations in our present study. First, we only used the GEO database data and real samples from a single center for internal as well as external validation, and we still need data from other databases or multiple centers for further validation to test the applicability of the predictive signature. Second, when analyzing the DEGs or CS-DEGs, with the consideration of the complexity of datasets in our study, it is difficult to consider some important factors, such as gender, races, regions, among others. Finally, we found the expression changes of eight hub genes in MCI, nonetheless, the detailed mechanism of such changes was not clear. Therefore, more evidences are required to find out the biological foundation. 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. Funding This work was supported by grants from the National Natural Science Foundation of China (82071220), Natural Science Foundation of Tianjin (20JCYBJC01290), and the Science and Technology Foundation of Tianjin Health Commission (MS20013), and Tianjin key Medical Discipline (Specialty) Construction Project (TJYXZDXK-072C). Ethics statement The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the Third Central Clinical College of Tianjin Medical University (approval number: IRB2022-011-02). The patients/participants provided their written informed consent to participate in this study References TRIM28 functions as a negative regulator of aggresome formation. Autophagy 17, 4231–4248. doi: 10.1080/15548627.2021.1909835 Kanehisa, M., and Goto, S. (2000). 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https://openalex.org/W4387019479	https://www.nature.com/articles/s41598-023-43277-6.pdf	English	null	An artificial intelligence model for the radiographic diagnosis of osteoarthritis of the temporomandibular joint	Scientific reports	2,023	cc-by	7,392	An artificial intelligence model for the radiographic diagnosis of osteoarthritis of the temporomandibular joint Wael M. Talaat 1,2,3,4, Shishir Shetty 1,2, Saad Al Bayatti 1, Sameh Talaat 5,6, Louloua Mou Sunaina Shetty 8 & Ahmed Kaboudan 9,10,11 OPEN The interpretation of the signs of Temporomandibular joint (TMJ) osteoarthritis on cone-beam computed tomography (CBCT) is highly subjective that hinders the diagnostic process. The objectives of this study were to develop and test the performance of an artificial intelligence (AI) model for the diagnosis of TMJ osteoarthritis from CBCT. A total of 2737 CBCT images from 943 patients were used for the training and validation of the AI model. The model was based on a single convolutional network while object detection was achieved using a single regression model. Two experienced evaluators performed a Diagnostic Criteria for Temporomandibular Disorders (DC/TMD)-based assessment to generate a separate model-testing set of 350 images in which the concluded diagnosis was considered the golden reference. The diagnostic performance of the model was then compared to an experienced oral radiologist. The AI diagnosis showed statistically higher agreement with the golden reference compared to the radiologist. Cohen’s kappa showed statistically significant differences in the agreement between the AI and the radiologist with the golden reference for the diagnosis of all signs collectively (P = 0.0079) and for subcortical cysts (P = 0.0214). AI is expected to eliminate the subjectivity associated with the human interpretation and expedite the diagnostic process of TMJ osteoarthritis. Osteoarthritis is a chronic inflammatory disease that has serious consequences affecting the quality of life. This common disease leads to pain and dysfunction that negatively impact the quality of sleep and the ability to work1. The consistently increasing prevalence of osteoarthritis is a major cause of concern to the health authorities especially considering the complexities and the high cost of treatment. Osteoarthritis was ranked as the second cause of the “increase in years lived with disability” following diabetes2,3. The etiology of TMJ osteoarthritis is complex and multifactorial, and the disorder is characterized by a progressive cartilage and bone degradation and remodeling4. The inflammatory arthritic condition has been attributed to the increased level of inflamma- tory cytokines that is regulated by monocyte chemoattraction and the decreased biomechanical properties of the disc leading to loss of the adaptive capacity of the TMJ5. It has been shown that the escalated turnover of the subchondral bone accounts for the initiation and progression of osteoarthritis. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah 27272, UAE. 2Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah 27272, UAE. 3Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt. 4Chair, Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah, UAE. 5Department of Orthodontics, Future University in Egypt, Cairo, Egypt. 6Department of Oral Technology, University Clinic, Bonn, Germany. 7Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Beirut Arab University, Tripoli, Lebanon. 8Department of Restorative and Preventive Dentistry, College of Dental Medicine, University of Sharjah, Sharjah 27272, UAE. 9Department of Computer Science, Shorouk Academy, El Shorouk, Egypt. 10Interdisciplinary AI Hub, Future University in Egypt, Cairo, Egypt. 11DigiBrain4 Inc, Chicago, USA. email: wtaha@sharjah.ac.ae Methodologyh The study was approved ethically by the Institutional Review Board at the University of Sharjah (REC-20-09- 21-01). The ethical principles for medical research involving human subjects as mentioned in the Declaration of Helsinki and the “Strengthening the Reporting of Observational studies in Epidemiology” (STROBE)20 guidelines were applied in this study. Patients aged 18–80 years who visited the Oral Diagnosis and Urgent Care Clinic at the University Dental Hospital Sharjah from November 2020 to November 2022 were enrolled in the study and divided into two groups; the TMJ Osteoarthritis and Control groups, respectively. TMJ clinical examination was done for all subjects as part of the initial screening. All subjects were informed about the aim of the study and were requested to sign consent forms before participation. The inclusion criteria for the TMJ Osteoarthritis group involved all the signs and symptoms of osteoarthritis as crepitus, joint pain in the last 30 days, deviation on mouth opening and limitation in opening. Patients were evaluated using the DC/TMD Axis-I assessment instruments as pain screener21, symptom questionnaire22 and demographic questionnaire22 and Axis-II assess- ment instruments as pain drawings, Graded Chronic Pain Scale23, disability score21 and Jaw Functional Limitation Scale21. Only patients with acute or serious dysfunction symptoms according to Helkimo’s clinical dysfunction index (Di)24 were entitled for CBCT exposure. All subjects in the Control group were assessed for the absence of all the clinical and radiographic signs and symptoms of TMD and were recruited when they had CBCT records for reasons other than TMD. Pregnant women, patients who had past TMD treatment and patients who had systemic conditions that could cause joint deformity were excluded. The initial screening procedures were done by a clinical tutor and the DC/TMD assessment was done by an oral and maxillofacial surgeon (W.T.) with more than 20 years’ experience in the management of TMD and an oral radiologist (S.A.) with more than 25 years’ experience and both were blinded to the results of the initial screening. The evaluators were calibrated prior to the start of the study by examining the same 20 patients independently following the DC/TMD protocol. The CBCT examinations were conducted using GALILEOS 3-D X-ray systems (SIRONA Dental Systems, York, PA). www.nature.com/scientificreports/ advanced forms7,8. In addition, TMD-related referred pain obscures the origin of TMD pain, as the pain spreads to other locations that are distant from the joint and the related muscles which aggravates the complexity of the diagnostic procedure9. In spite of the remarkable value of the early diagnosis of TMD in stopping the progression to the severe forms of these disorders, however the process is hindered by the mentioned complexities as well as the subjectivity of the diagnosis that was evident in several studies3,10–12. A reliable diagnostic protocol for TMD was published in 2009 as the Research Diagnostic Criteria for Tem- poromandibular Disorders (RDC/TMD)13, and was updated in 2014 as the Diagnostic Criteria for Temporo- mandibular Disorders (DC/TMD)14. The diagnostic criteria integrates diagnostic decision trees that rely on the clinical and radiographic diagnostic criteria (Axis-I) and the psychosocial condition and pain-related disabilities (Axis-II). The reliability of the DC/TMD for the diagnoses of the different TMD classes has been reported as excellent15. The DC/TMD defines the degenerative joint disease as a disorder that involves degeneration of the articular tissues combined with osseous changes on the condyle and the articular eminence that are detected on computed tomography (CT) and CBCT in the form of subchondral (subcortical) cyst, erosions, sclerosis, flattening and osteophyte16. The interpretation of these signs is highly subjective and there is an urgent need to eliminate this subjectivity to enhance the diagnosis of TMJ osteoarthritis17,18. j y g Deep learning is a category of AI that has been successfully utilized to facilitate diagnosis and enhance deci- sion making in clinical dentistry. The dominant AI model that is used in medical imaging is the neural network applications. When applied to radiomic data, deep learning can identify the complex patterns within the dataset and thus can lead to the objective diagnosis of TMJ osteoarthritis. The AI-supported clinical decision making in the field of TMD may significantly reduce the percentage of cases that progress to the complicated forms of TMD by allowing early diagnosis19. This study aimed to develop and test the performance of an AI model based on neural networks for the diagnosis of TMJ osteoarthritis from CBCT. We hypothesized that there is a high degree of conformity between the expert clinicians and AI in diagnosing TMJ osteoarthritis. Methodologyh The CBCT protocol was: tube voltage: 85 kV, scanning time: 14 s, tube current: 7 mA, effective dose: 75 mSv, voxel size: 150 mm (screen resolution 1366 × 768), field of view: 15 × 15 cm, and slice thickness 1.0 mm. The TMJs were evaluated from the medial pole to the lateral pole in sagittal, coronal and axial planes. The radiographic criteria that were used to confirm the diagnosis of osteoarthritis were subcortical cyst (radiolucent area that may be just below the cortex or in the trabecular bone) (Fig. 1a), flattening of the articular surfaces (disappear- ance of the even convexity or concavity of the articular surfaces) (Fig. 1b), osteophyte (bony outgrowth arising from a mineralized joint surface) (Fig. 1c) and surface erosion (condylar surface irregularities) (Fig. 1d)25. The CBCT radiographs were assessed twice on a 1-week interval by 2 evaluators (W.T. and S.A.) independently. The 2 evaluators then reassessed the records in which there was a disagreement collectively on a third occasion to reach consensus. To ensure a strict blinding protocol, two other researchers (A.K. and S.T.) were responsible for the data entry, training, and testing of the AI model. A total of 2737 images, saved as TIFF files, were used for the training and validation of the AI model, including 1986 cross-sectional images and 751 tangential images. The images used for training and validation were selected from the CBCT records of 943 patients who had 792 osteoarthritic joints and 1094 normal joints (Fig. 2). The images were rescaled to 640 × 640 pixels, and the area of interest was marked in a rectangle and converted to XY coordinates using the LabelImg software (Tzutalin. LabelImg. Git code 2015). The cross-sectional images were used to detect flattening, subcortical cyst and surface erosion, whereas the tangential images were used to detect osteophytes. Development, training and testing of the AI model An artificial intelligence model for the radiographic diagnosis of osteoarthritis of the temporomandibular joint Wael M. Talaat 1,2,3,4, Shishir Shetty 1,2, Saad Al Bayatti 1, Sameh Talaat 5,6, Louloua Mou Sunaina Shetty 8 & Ahmed Kaboudan 9,10,11 OPEN The increased turnover has been attributed to the increased expression of the genes responsible for the osteoclastic activity and increased osteoclastogenesis6.htf g The management of temporomandibular disorders (TMD) often suffers from the inconsistency and controver- sies that surrounds the diagnostic and treatment protocols and the lack of evidence related to the contemporary management principles. This often results in delays in the diagnosis and progression of the disorders to more 1Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah 27272, UAE. 2Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah 27272, UAE. 3Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt. 4Chair, Department of Oral and Craniofacial Health Sciences, College of Dental Medicine, University of Sharjah, Sharjah, UAE. 5Department of Orthodontics, Future University in Egypt, Cairo, Egypt. 6Department of Oral Technology, University Clinic, Bonn, Germany. 7Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Beirut Arab University, Tripoli, Lebanon. 8Department of Restorative and Preventive Dentistry, College of Dental Medicine, University of Sharjah, Sharjah 27272, UAE. 9Department of Computer Science, Shorouk Academy, El Shorouk, Egypt. 10Interdisciplinary AI Hub, Future University in Egypt, Cairo, Egypt. 11DigiBrain4 Inc, Chicago, USA. email: wtaha@sharjah.ac.ae \| https://doi.org/10.1038/s41598-023-43277-6 Scientific Reports \| (2023) 13:15972 www.nature.com/scientificreports/ www.nature.com/scientificreports/ detection using a single regression model from the pixels to the coordinates of the bounding boxes and the class probabilities. The model trains on full images and directly optimizes the detection performance. During object detection, the input image is divided into an S × S grid. If the center of an object falls into a grid cell, that grid cell is responsible for detecting that object. Each grid cell predicts bounding boxes and confidence scores for those boxes (Fig. 3). The YOLO model used in the present study was YOLO5v7 Large with 477 layers, 76,185,580 parameters, 76,185,580 gradients, 110.5 billion floating-point operations per second (GFLOPs). Optimization of the model used the Stochastic Gradient Descent (SGD) with parameter groups 131 weight (decay = 0.0), 135 weight (decay = 0.0005) and 135 bias. The model environment was composed of a central processing unit (CPU) with 12th Generation Intel(R) Core i9-12900K, 3.19 GHz, 128 GB RAM and NVIDIA RTX 3090 Ti, 24,564 GB. The network has convolutional layers that recognize features of the image followed by totally connected lay- ers that undergo prediction of the probabilities27,28. Alternating 1 × 1 convolutional layers reduce the features space from preceding layers. Training of the network was performed for about 135 epochs on the training and validation datasets using a batch size of 64 using Python 3.9.16, PyTorch 1.13.1 and CUDA 11.7. The training hyperparameters were: learning rate = 0.01, momentum = 0.937, weight decay = 0.0005, warmup epochs = 3.0, warmup momentum = 0.8, warmup bias learning rate = 0.1, box = 0.05, cumulative layout shift (cls) = 0.5, cls_ poisitive weight = 1.0, objectness loss gain (obj) = 1.0, objectness positive weight = 1.0, training intersection over union (IoU) threshold = 0.5. The learning rate schedule was 10−3 for the first epochs then raised to 10−2 for 75 epochs, then 10−3 for 30 epochs then 10−4 for the last 30 epochs. A completely separate testing set composed of 350 images (250 cross-sectional images and 100 tangential images) was used for testing the model. The set had a single image for each TMJ of 175 patients. g g p During the testing phase, two experienced evaluators (W.T. and S.A.) performed a DC/TMD-based assess- ment, including the evaluation of the CBCT records, on the 350 patients constituting the testing set. www.nature.com/scientificreports/ The CBCT radiographs were assessed twice on a 1-week interval by the 2 evaluators independently and reassessed in case of disagreement collectively on a third occasion to reach consensus. The concluded diagnosis generated from this assessment was considered as the golden reference. The performance of the AI model in diagnosing osteoarthritis from the CBCT images was then compared to an oral radiologist (S.S.) against the golden reference. The oral radiologist had 15 years of clinical experience and was blinded to the research protocol.h The diagnostic performance measures used for the assessment of the diagnostic performance of the AI model were sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), intersection over union between detected object and ground truth polygon (IoU), test accuracy and the Kappa coefficient of agree- ment. Sensitivity is defined as the proportion of true positive results to all positive results (Sensitivity = true posi- tives/(true positives + false negatives)). Specificity is the proportion of true negative results to all negative results (Specificity = true negatives/(true negatives + false positives)). PPV is the proportion of positive test results which are actually positive, according to the golden reference, to the total positive test results (PPV = true positives/(true positives + false positives)). NPV is the proportion of negative test results which are actually negative, according to the golden reference, to the total negative test results (NPV = true negatives/(true negatives + false negatives)). IoU is the ratio of the overlapping between the predicted bounding box and the ground truth bounding box. Test accuracy is the proportion of all the true test results to all test results (Test Accuracy = (true positives + true negatives)/(sum of All results)). Development, training and testing of the AI model Development, training and testing of the AI model Our model was based on the You Only Look Once (YOLO) system that uses a single convolutional network to concurrently predict several bounding boxes and generate the class probabilities26,27. YOLO achieves object p , g g Our model was based on the You Only Look Once (YOLO) system that uses a single convolutional network to concurrently predict several bounding boxes and generate the class probabilities26,27. YOLO achieves object https://doi.org/10.1038/s41598-023-43277-6 Scientific Reports \| (2023) 13:15972 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The radiographic criteria used to confirm the diagnosis of osteoarthritis. (a) Subcortical cyst (radiolucent area that may be just below the cortex or in the trabecular bone). (b) Flattening of the articular surfaces (disappearance of the even convexity or concavity of the articular surfaces). (c) Osteophyte (bony outgrowth arising from a mineralized joint surface). (d) Surface erosion (condylar surface irregularities). Figure 1. The radiographic criteria used to confirm the diagnosis of osteoarthritis. (a) Subcortical cyst (radiolucent area that may be just below the cortex or in the trabecular bone). (b) Flattening of the articular surfaces (disappearance of the even convexity or concavity of the articular surfaces). (c) Osteophyte (bony outgrowth arising from a mineralized joint surface). (d) Surface erosion (condylar surface irregularities). Figure 2. Sample CBCT images used in the validation of the AI model. Figure 2. Sample CBCT images used in the validation of the AI model. Figure 2. Sample CBCT images used in the validation of the AI model. https://doi.org/10.1038/s41598-023-43277-6 Scientific Reports \| (2023) 13:15972 \| www.nature.com/scientificreports/ Statistical analysis y Statistical analysis was performed by SPSS (version 20). Percentages of agreement were compared using Z test of two proportions. The level of significance was set at P < 0.05. Two Tailed tests were assumed throughout the analysis for all statistical tests. Figure 3. Object detection was achieved using a single regression model from the pixels to the coordinates of the bounding boxes and the class probabilities The input image is divided into an S × S grid. If the center of an object falls into a grid cell, that grid cell is responsible for detecting that object. Each grid cell predicts bounding boxes and confidence scores for those boxes. Figure 3. Object detection was achieved using a single regression model from the pixels to the coordinates of the bounding boxes and the class probabilities The input image is divided into an S × S grid. If the center of an object falls into a grid cell, that grid cell is responsible for detecting that object. Each grid cell predicts bounding boxes and confidence scores for those boxes. Scientific Reports \| (2023) 13:15972 \| https://doi.org/10.1038/s41598-023-43277-6 www.nature.com/scientificreports/ Results According to the DC/TMD-based golden reference, the testing set had 73 condylar flattening, 134 subcortical cysts, 11 surface erosions and 38 osteophytes. The performance measures of the AI model are shown in Table 1. The diagnostic performance measures of the AI model and the oral radiologist against the golden reference are shown in Tables 2 and 3 respectively. The AI diagnosis showed statistically higher agreement with the golden reference compared to the oral radiologist. Cohen’s kappa showed statistically significant differences in the agree- ment between the AI and the oral radiologist with the golden reference for the diagnosis of all signs collectively (P = 0.0079) and for the diagnosis of subcortical cysts (P = 0.0214). The differences in agreement were not signifi- cant for the diagnosis of condylar flattening (P = 0.0595), surface erosion (P = 0.1581) and osteophyte (P = 0.3886). The osteophytes had the lowest agreement whereas the surface erosions showed the highest agreement with the golden reference. Both the AI model and the oral radiologist showed high test accuracy (> 0.95), however the AI model showed equal or better test accuracy compared to the oral radiologist (Table 4). The agreement between the AI model diagnosis and the oral radiologist diagnosis is shown in Table 5. Discussion Osteoarthritis of the TMJ comprises complex pathophysiological processes and necessitates comprehensive evaluations to detect the remodeling and degeneration of the bone and cartilage. Increasing attention has now focused on the early diagnosis of osteoarthritis to stop the disease progression and prevent or decrease the permanent joint damage3. The clinical diagnosis of TMJ osteoarthritis relies on the radiographic features of the joint components that involve early signs of osseous remodeling like flattening and sclerotic changes followed by erosive, attrition, osteophytes and cyst-like lesions. The diagnostic accuracy, safety and reliability of CBCT Table 1. The performance measures of the AI model. PPV positive predictive values, mAP mean average precision, IoU intersection over union. PPV (95% confidence limits) Sensitivity (95% confidence limits) Test accuracy (95% confidence limits) mAP50% IoU Total training time 0.96 (0.93–0.98) 0.98 (0.96–1.00) 0.99 (0.98–0.99) 94% 0.9308 5 h Table 1. The performance measures of the AI model. PPV positive predictive values, mAP mean average precision, IoU intersection over union. Table 2. The diagnostic performance of the AI diagnosis against the golden reference. Statistically highly significant. PPV positive predictive values, NPV negative predictive values. Diagnostic performance Cohen’s kappa Kappa index P value Sensitivity (95% confidence limits) Specificity (95% confidence limits) PPV (95% confidence limits) NPV (95% confidence limits) Test accuracy (95% confidence limits) Condylar flattening 0.96 (0.91–1.00) 1.00 (1.00–1.00) 1.00 (1.00–1.00) 0.99 (0.98–1.00) 0.99 (0.98–1.00) 0.97 Near perfect agree- ment 0.0000 Subcortical cyst 0.99 (0.98–1.00) 0.98 (0.96–1.00) 0.96 (0.93–0.99) 1.00 (0.99–1.00) 0.98 (0.97–1.00) 0.96 Near perfect agree- ment 0.0000 Surface erosion 1.00 (1.00–1.00) 1.00 (1.00–1.00) 1.00 (1.00–1.00) 1.00 (1.00–1.00) 1.00 (1.00–1.00) 1.00 Perfect agreement 0.0000 Osteophyte 0.97 (0.92–1.00) 0.98 (0.97–1.00) 0.86 (0.76–0.96) 1.00 (0.99–1.00) 0.98 (0.97–0.99) 0.90 Near perfect agree- ment 0.0000 All signs 0.98 (0.96–1.00) 0.99 (0.98–1.00) 0.96 (0.93–0.98) 1.00 (0.99–1.00) 0.99 (0.98–0.99) 0.96 Near perfect agree- ment 0.0000 Table 2. The diagnostic performance of the AI diagnosis against the golden reference. Statistically highly significant. PPV positive predictive values, NPV negative predictive values. Table 2. The diagnostic performance of the AI diagnosis against the golden reference. Statistically highly significant. PPV positive predictive values, NPV negative predictive values. i Table 3. The diagnostic performance of the oral radiologist against the golden reference. Statistically highly significant. PPV positive predictive values, NPV negative predictive values. Discussion Several factors may hinder the early diagnosis of osteoar- thritis and these include the variability in the examination methods, diagnostic criteria and taxonomy between different clinical and research centers8. Among these factors is the subjectivity of the radiographic diagnosis of the disorder. The inconsistency in the diagnosis of osteoarthritis was evident in one study that showed that 206 patients consulted an average of 30 providers from 44 different specialties to seek treatment for their TMD31. Another study showed that 101 orofacial pain patients had multiple consultations with 15 different specialties to diagnose and treat their pain32. The urgent need to eliminate the subjectivity in the interpretation of the radiographic signs of osteoarthritis was emphasized in several studies to facilitate and expedite the diagnosis of TMJ osteoarthritis17,33. Thus, an automated and precise diagnostic system is warranted to improve the accuracy of the diagnosis of TMJ osteoarthritis. in detecting the bony changes associated with osteoarthritis have been reported to be superior to other imaging modalities25,29. The primary diagnosis of TMD has been changed after the CBCT assessment in 26.08% of cases in one study25 and in 58% of cases in another study30. Several factors may hinder the early diagnosis of osteoar- thritis and these include the variability in the examination methods, diagnostic criteria and taxonomy between different clinical and research centers8. Among these factors is the subjectivity of the radiographic diagnosis of the disorder. The inconsistency in the diagnosis of osteoarthritis was evident in one study that showed that 206 patients consulted an average of 30 providers from 44 different specialties to seek treatment for their TMD31. Another study showed that 101 orofacial pain patients had multiple consultations with 15 different specialties to diagnose and treat their pain32. The urgent need to eliminate the subjectivity in the interpretation of the radiographic signs of osteoarthritis was emphasized in several studies to facilitate and expedite the diagnosis of TMJ osteoarthritis17,33. Thus, an automated and precise diagnostic system is warranted to improve the accuracy of the diagnosis of TMJ osteoarthritis. g AI aims to match and surpass the cognitive abilities of humans and secure a new era of improved standards of care in medicine. Date-driven AI or deep learning utilizes the data generated by humans to train mathematical models and build a cognitive system that is able to analyze and reach a diagnosis. Discussion Neural networks are a subset of deep learning that is inspired by the nervous system and contain layers of interconnected neurons. Neural networks transfer signals between the different layers of neurons through a convolutional process to learn the features within the data34. Object detection systems in general starts by detecting the potent features in the input images, then use classifiers to identify the objects in space. The classifiers are applied over a sliding window on the image or on particular areas of the image35. In the present study, the AI model was based on YOLO; a detec- tor that is trained to detect a diversity of objects simultaneously. YOLO puts structural constrains on the grid cell proposals which support multiple simultaneous recognitions of the same object. The system suggests only 98 bounding boxes for one image compared to 2000 for other object recognition systems then incorporates the individual components into a single rectified model26,27. The results have shown that the diagnostic performance measures of the AI model were in statistically higher agreement with the golden reference compared to the oral radiologist. Both the AI model and the oral radiologist had high test accuracy over 0.95, however the AI model had always equal or better test accuracy compared to the oral radiologist. Similar results were obtained in a study that compared the performance of dental clinicians and a multilayer perceptron neural network in diagnosis of orofacial pain and TMD and found that the performance of the neural network was significantly higher than the dental clinicians36. Another study used the Karas’ ResNet AI model for the radiographic diagnosis of TMJ osteoarthritis from orthopantomograms and compared the diagnostic performance of the model with an oral radiologist11. Their model lacked in performance in one the categories of TMJ osteoarthritis, however their model exhibited equal sensitivity to that of the oral radiologist and better balance between sensitivity and specificity. A Neural network was used in another study to classify the condylar morphology on CBCT records and evaluate the correlations between groups of biomarkers in patients with TMJ osteoarthritis37. The neural network staging of osteoarthritis as compared to the clinicians’ consensuses had a 91.2% accuracy, which is in accordance with the results of the present study. Discussion Diagnostic performance Cohen’s kappa Kappa index P value Sensitivity (95% confidence limits) Specificity (95% confidence limits) PPV (95% confidence limits) NPV (95% confidence limits) Test accuracy (95% confidence limits) Condylar flattening 0.89 (0.82–0.96) 1.00 (1.00–1.00) 1.00 (1.00–1.00) 0.97 (0.95–0.99) 0.98 (0.96–0.99) 0.93 Near perfect agree- ment 0.0000 Subcortical cyst 0.93 (0.88–0.97) 0.98 (0.96–1.00) 0.96 (0.93–0.99) 0.95 (0.93–0.98) 0.96 (0.94–0.98) 0.91 Near perfect agree- ment 0.0000 Surface erosion 0.91 (0.74–1.00) 1.00 (1.00–1.00) 1.00 (1.00–1.00) 1.00 (0.99–1.00) 1.00 (0.99–1.00) 0.96 Near perfect agree- ment 0.0000 Osteophyte 0.95 (0.88–1.00) 0.98 (0.97–1.00) 0.86 (0.75–0.96) 0.99 (0.98–1.00) 0.98 (0.96–0.99) 0.89 Near perfect agree- ment 0.0000 All signs 0.92 (0.88–0.95) 0.99 (0.98–1.00) 0.96 (0.93–0.98) 0.98 (0.97–0.99) 0.98 (0.97–0.98) 0.92 Near perfect agree- ment 0.0000 Table 3. The diagnostic performance of the oral radiologist against the golden reference. *Statistically highly significant. PPV positive predictive values, NPV negative predictive values. https://doi.org/10.1038/s41598-023-43277-6 www.nature.com/scientificreports/ Table 4. Test accuracy of the AI model compared to the oral radiologist. Statistically significant. AI model Oral radiologist z P value Condylar flattening 0.99 0.98 1.52 0.12863 Subcortical cyst 0.98 0.96 1.99 0.04614* Surface erosion 1.00 1.00 Equal values Osteophyte 0.98 0.98 0.26 0.79408 All signs 0.99 0.98 2.33 0.01984* Table 4. Test accuracy of the AI model compared to the oral radiologist. Statistically significant. AI model Oral radiologist z P value Condylar flattening 0.99 0.98 1.52 0.12863 Subcortical cyst 0.98 0.96 1.99 0.04614 Surface erosion 1.00 1.00 Equal values Osteophyte 0.98 0.98 0.26 0.79408 All signs 0.99 0.98 2.33 0.01984* Table 4. Test accuracy of the AI model compared to the oral radiologist. Statistically significant. Table 5. The agreement between the AI model diagnosis and the oral radiologist diagnosis. Statistically highly significant. Percentage of agreement (95% confidence limits) Cohen’s kappa P value Condylar flattening 98.57% (97.33–99.81%) 0.95 0.0000* Subcortical cyst 97.43% (95.77–99.09%) 0.95 0.0000 Surface erosion 99.71% (99.16–100.00%) 0.96 0.0000 Osteophyte 99.71% (99.16–100.00%) 0.99 0.0000 All signs 98.86% (98.30–99.41%) 0.96 0.0000 Table 5. The agreement between the AI model diagnosis and the oral radiologist diagnosis. *Statistically highly significant. in detecting the bony changes associated with osteoarthritis have been reported to be superior to other imaging modalities25,29. The primary diagnosis of TMD has been changed after the CBCT assessment in 26.08% of cases in one study25 and in 58% of cases in another study30. www.nature.com/scientificreports/ the most universally accepted standardized protocol14. Secondly, the training database used in the present study was relatively increased compared to several previous studies. Finally, the AI model was trained to learn the detection of the radiographic signs that appear on the cross-sectional CBCT views independently of the training used to learn the signs on the tangential CBCT views.hf g g The performance of object detectors is commonly challenged by several factors as the different resolution input compared to that used in training, foreground or background imbalance, smaller sized objects, low com- putational resources, small datasets and imprecise localizations during predictions. In general, the detection accuracy of two-stage object detectors exceed that of single-stage detectors, however with the development of the single-staged YOLO, its accuracy has surpassed several two-staged detectors27. YOLO has several advan- tages over other detection systems. Firstly, as YOLO is built as a single regression problem, it does not require a complex structure like other models and accordingly it is much faster. The YOLO network operates at a speed of 45 frames per second and the fast version at 150 frames per second. The network concomitantly predicts the bounding boxes, extracts the features and performs the circumstantial reasoning. Secondly, YOLO accomplishes at least double the mean average precision of the other object detection systems. Thirdly, YOLO analyzes the whole image while making detections and does not depend on region proposals and sliding windows like other systems. YOLO is trained on generalized presentations of objects so it generates less than half the average number of background errors compared to other systems27. In a recent study, YOLO-V3 has been compared to two other object detection algorithms; Single Shot Detection and Faster Region based Convolutional Neural Networks. The overall performance of YOLO-V3 was superior to the other two algorithms. YOLO-V3 was the fastest while maintaining excellent accuracy39. The main limitation of YOLO is the difficulty of the detection of objects in new and unfamiliar aspect ratios or presentation, however this limitation is not applicable to the diagnosis of osteoarthritis from CBCT records27.hi The timely identification of signs of TMJ osteoarthritis and the detection of patients at risk of progression to advanced forms may facilitate the development of more effective treatment approaches. Conclusionh The AI model used in the present study had equal or better diagnostic performance for TMJ osteoarthritis compared to the human expert. The adoption of AI in the radiographic diagnosis of osteoarthritis is expected to eliminate the subjectivity associated with the human interpretation and expedite the diagnostic process thus reducing the probability of the disease progression. Further studies are required to refine the network capabili- ties and develop a multimodal cognitive system that analyzes the patients’ history, radiographic and clinical examination data to diagnose osteoarthritis and other forms of TMD efficiently. www.nature.com/scientificreports/ This requires the pro- gression of the current binary classifications to multinomial regression functions that recognize the clinical and radiographic findings in addition to the different biomarkers and classify the early, moderate and severe forms of TMJ osteoarthritis. The development of multinomial deep learning classifiers in the field of TMJ osteoarthritis can promote a line of research that investigates the possibility of diagnosis at an early reversible phase40. The present study validated an AI model that diagnosed the early and late radiographic signs of TMJ osteoarthritis and can be considered a base for further research to build such multinomial deep learning models. Data availabilityh Data availability The data that support the findings of this study are available from the authors upon request. y The data that support the findings of this study are available from the authors upon request. Received: 4 June 2023; Accepted: 21 September 2023 Received: 4 June 2023; Accepted: 21 September 2023 References References 1. Vina, E. R. & Kwoh, C. K. Epidemiology of osteoarthritis: Literature update. Curr. Opin. Rheumatol. 30(2), 160–167 (2018). , , p gy p p ( ), ( ) 2. Quicke, J. G., Conaghan, P. G., Corp, N. & Peat, G. Osteoarthritis year in review 2021: Epidemiology and therapy. Osteoarthr. Cartil. 30(2), 196–206 (2022). 3. Bianchi, J. et al. Osteoarthritis of the Temporomandibular Joint can be diagnosed earlier using biomarkers and machine learning Sci. Rep. 10(1), 8012 (2020). p 4. Tanaka, E., Detamore, M. S. & Mercuri, L. G. Degenerative disorders of the temporomandibular joint: Etiology, diagnosis, and treatment. J. Dent. 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Discussion A natural language processing-based model utilizing artificial neural networks was able to diagnose TMD according to the analysis of the patient’s chief complaint and the measurement of the maximal mouth opening38. In the present study, the high test accuracy achieved by the AI model and the excellent agreements between the model and the radiologist can be attributed to several factors. Firstly, the criteria used for the radiographic diagnosis of osteoarthritis from CBCT records were based on the DC/TMD that was reported to have excellent sensitivity, specificity and interexaminer reliability and is considered Scientific Reports \| (2023) 13:15972 \| https://doi.org/10.1038/s41598-023-43277-6 www.nature.com/scientificreports/ References Retrospective examination of the healthcare “journey” of chronic orofacial pain patients referred to oral and maxillofacial surgery. Br. Dent. J. 214(5), E12 (2013).f 33. Ahmad, M. & Schiffman, E. L. 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Rep. 11(1), 19802 (2021). 9. Farook, T. H. & Dudley, J. Automation and deep (machine) learning in temporomandibular joint disorder radiomics: A systematic review. J. Oral Rehabil. 50(6), 501–521 (2023). 0. Vandenbroucke, J. P. et al. Competing interests h p g The authors declare no competing interests. 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. 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https://openalex.org/W2553871839	https://periodicos.uff.br/midiaecotidiano/article/download/9781/6910	Portuguese	null	Do Hiperlocal Bauruense para o Global Criativo: As Novas Marcas de uma Comunicacao a Partir do Empoderamento Colaborativo	Mídia.e.Cotidiano	2,016	cc-by	7,423	2 Pesquisador do Laboratório de Estudos em Comunicação, Tecnologia e Educação Cidadã (Lecotec) da Unesp. Jornalista e Mestre pelo Programa de Pós-graduação em Comunicação da Faculdade de Arquitetura Artes e Comunicação (FAAC) da Unesp, Campus de Bauru. Email: giovani.vieira.miranda@gmail.com 1 Professor do Curso de Jornalismo da Faculdade de Arquitetura, Artes e Comunicação da Universidade Estadual Paulista Júlio de Mesquita Filho. Doutor em Comunicação e Poéticas Visuais (UNESP), mestre em Projeto Arte e Sociedade (UNESP) e graduado em Comunicação Social/Jornalismo (USP). Email: angelo.sottovia.aranha@gmail.com 2 Pesquisador do Laboratório de Estudos em Comunicação, Tecnologia e Educação Cidadã (Lecotec) da Unesp. Jornalista e Mestre pelo Programa de Pós-graduação em Comunicação da Faculdade de Arquitetura Artes e Comunicação (FAAC) da Unesp, Campus de Bauru. Email: giovani.vieira.miranda@gmail.com Revista Mídia e Cotidiano Seção Temática Número 9. Agosto 2016 Aprovado em: 17/07/2016 Revista Mídia e Cotidiano Seção Temática Número 9. Agosto 2016 Aprovado em: 17/07/2016 1 Professor do Curso de Jornalismo da Faculdade de Arquitetura, Artes e Comunicação da Universidade Estadual Paulista Júlio de Mesquita Filho. Doutor em Comunicação e Poéticas Visuais (UNESP), mestre em Projeto Arte e Sociedade (UNESP) e graduado em Comunicação Social/Jornalismo (USP). Email: angelo.sottovia.aranha@gmail.com ARANHA E MIRANDA FROM BAURU’S HYPERLOCAL TO A GLOBAL CREATIVE: NEW TRACES OF COMMUNICATION BASED ON COLLABORATIVE EMPOWERMENT Angelo Sottovia ARANHA1 Giovani Vieira MIRANDA2 RESUMO: A consolidação das novas tecnologias de comunicação e a possibilidade de se produzir conteúdos noticiosos locais, de forma descentralizada e horizontalizada, podem ser vistas como fatores que impulsionam o desenvolvimento jornalístico em ambientes digitais e possibilitam a valorização do local e o reforço de identidades culturais, que passam a configurar como fontes básicas de significados sociais em contraste com o processo de comunicação habitual dos mass media. A partir desses conceitos, sob as óticas da Economia Criativa e do Jornalismo Hiperlocal, é analisada a experiência de comunicação da Casa do Hip Hop de Bauru, arranjo produtivo criativo local de Bauru, cidade do interior de São Paulo, que tem inovado com a criação de um modelo colaborativo e cidadão frente ao da mídia mainstream da cidade, com novas possibilidades para a seleção, captação, edição e difusão de conteúdos informativos. Palavras-Chave: Jornalismo Cidadão; Jornalismo Local; Novas Tecnologias. ABSTRACT: The consolidation of new communication technologies and the ability to produce content for local, decentralized and horizontally, can be analyzed as elements that drive the journalistic development in the digital environment enabling the development of local and strengthening of identities that, start to set as a basic source of social meaning in contrast to the usual process of the media. Using these concepts, from the perspective of the Creative Economy and Hyperlocal Journalism, it analyzes the communication experience of the House of Hip Hop Bauru, creative production arrangement place of Bauru, city in the interior of São Paulo, which has innovated with the construction a collaborative model and citizen against the mainstream media of the city with new possibilities for the creation, capture, production and distribution of informative contents. p Keywords: Citizen Journalism; Local Journalism; New technologies. 1 Professor do Curso de Jornalismo da Faculdade de Arquitetura, Artes e Comunicação da Universidade Estadual Paulista Júlio de Mesquita Filho. Doutor em Comunicação e Poéticas Visuais (UNESP), mestre em Projeto Arte e Sociedade (UNESP) e graduado em Comunicação Social/Jornalismo (USP). Email: angelo.sottovia.aranha@gmail.com 2 Pesquisador do Laboratório de Estudos em Comunicação, Tecnologia e Educação Cidadã (Lecotec) da Unesp. Jornalista e Mestre pelo Programa de Pós-graduação em Comunicação da Faculdade de Arquitetura Artes e Comunicação (FAAC) da Unesp, Campus de Bauru. Email: giovani.vieira.miranda@gmail.com 66 ARANHA E MIRANDA 1. Comunicação e Novas Tecnologias As tecnologias da informação e da comunicação, atreladas à formação de uma economia global têm influenciado as relações humanas ao criar novos processos sociais, econômicos e culturais (CASTELLS, 1996). As mídias se convergem num processo que, segundo Jenkins (2009, p. 29), não deve ser vislumbrado apenas no âmbito tecnológico, mas em meio a “uma transformação cultural, à medida que consumidores são incentivados a [...] fazer conexões em meio a conteúdos midiáticos dispersos”. A informação se tornou um bem disputado e de valor crescente – as pessoas querem produzir e compartilhar conhecimento, em uma cultura cada vez mais participativa. A efetivação das interações mediadas pelo virtual fez com que fossem criadas e ampliadas novas formas de relações sociais e pessoais, com base na proximidade de interesses e identidades, a partir da emergência e consolidação das novas tecnologias de comunicação e informação (PERUZZO, 2003). E a digitalização tem afetado também, intensamente, os espaços dos suportes culturais. A qualidade e a rapidez na transmissão de pacotes de dados em ambientes que permitem a comunicação de forma anônima, e a livre circulação de informações, criaram cenários propícios para a criação e o compartilhamento de conteúdos – por qualquer pessoa; a qualquer tempo e lugar (SILVEIRA, 2010). As novas tecnologias têm afetado as relações humanas de tal maneira que tais relações já não conseguem mais ser completamente entendidas fora de seu “diálogo” com a tecnologia. Entenda-se, nesta reflexão, o termo ‘tecnologia’ com o significado apresentado por Castells (1996, p. 5), ao apontar que “a tecnologia não é somente a ciência e as máquinas: é também tecnologia social e organizativa”. Em outras palavras, a revolução tecnológica está diretamente relacionada às habilidades de uma sociedade para a difusão e a troca de informações, relacionando-as com o restante do mundo. A disseminação das tecnologias digitais criou uma nova ambiência para a captação, edição e difusão de conteúdos de interesse público. Esse processo favorece os agentes criativos, que se apropriam dessa tecnologia para a composição de novas narrativas. Ao se apropriarem dessa nova ecologia digital, 67 e das Tecnologias da Informação e da Comunicação (TIC’s), os grupos vinculados ao jornalismo cidadão produzem conteúdos informativos que rivalizam com as narrativas hegemônicas (ARANHA; XAVIER, 2015, p. 05). e das Tecnologias da Informação e da Comunicação (TIC’s), os grupos vinculados ao jornalismo cidadão produzem conteúdos informativos que rivalizam com as narrativas hegemônicas (ARANHA; XAVIER, 2015, p. 05). 1. Comunicação e Novas Tecnologias Os projetos alicerçados nesses dispositivos digitais (ARANHA; XAVIEIR, 2015) inovam aspectos norteadores do jornalismo e insinuam novas modelagens no fluxo de informações: pautas com foco na cidadania, organização de redações virtuais, processos colaborativos de produção de conteúdo, objetividade da informação compreendida como categoria cognitiva, posicionamento crítico na cobertura, remodelagem do modelo anglo-saxão, experimentalismo formal – estilo, estrutura narrativa e sistema de codificação – e conceitual – pesquisas universitárias – e novas possibilidades de tipos de financiamento (SQUIRRA, 2012). Os consumidores de conteúdos informativos podem se tornar produtores e o caminho contrário também é válido, porque a informação segue fluxos diversos e sofre modificações ao longo do percurso. Isso se justifica, segundo Jenkins (2009), sobretudo pela redefinição do papel e da posição do consumidor midiático: Se os antigos consumidores eram tidos como passivos, os novos consumidores são ativos. Se os antigos consumidores eram previsíveis e ficavam onde mandavam que ficassem, os novos consumidores são migratórios, demonstrando uma declinante lealdade a redes ou a meios de comunicação. Se os antigos consumidores eram indivíduos isolados, os novos consumidores são mais conectados socialmente. Se o trabalho de consumidores de mídia já foi silencioso e invisível, os novos consumidores são agora barulhentos e públicos (JENKINS, 2009, p. 47). Se os antigos consumidores eram tidos como passivos, os novos consumidores são ativos. Se os antigos consumidores eram previsíveis e ficavam onde mandavam que ficassem, os novos consumidores são migratórios, demonstrando uma declinante lealdade a redes ou a meios de comunicação. Se os antigos consumidores eram indivíduos isolados, os novos consumidores são mais conectados socialmente. Se o trabalho de consumidores de mídia já foi silencioso e invisível, os novos consumidores são agora barulhentos e públicos (JENKINS, 2009, p. 47). Essa mudança na posição do consumidor midiático ocorre, principalmente, porque a internet permite a qualquer pessoa criar conteúdos e formatos. Esses conceitos de Jenkins podem ser comparados às indicações de Benkler (2006), para quem a internet representa uma mudança radical nas antigas tendências de comunicação, justamente porque é o primeiro meio de comunicação que consegue expandir o seu alcance ao descentralizar a estrutura de produção e de distribuição de informações, cultura e conhecimento. A maior parte das tecnologias que compõem a internet é baseada em recombinações e está aberta, ou seja, não está sob o controle de patentes ou outras formas que bloqueariam o acesso à rede, o que facilita o compartilhamento e a recombinação de conteúdos. ARANHA E MIRANDA 1. Comunicação e Novas Tecnologias ARANHA E MIRANDA ARANHA E MIRANDA 68 Silveira (2008), em uma interpretação similar à de Benkler, observa que há uma série de práticas socioculturais que reconfiguraram as redes informacionais, transformando-as em um espaço comum: Silveira (2008), em uma interpretação similar à de Benkler, observa que há uma série de práticas socioculturais que reconfiguraram as redes informacionais, transformando-as em um espaço comum: Uma série de práticas socioculturais reconfiguraram as redes informacionais como um terreno comum — commons, no sentido anglo-saxônico — e incentivaram a produção de processos, repositórios e interfaces a partir do ciberespaço ou em seu redor, tais como a música tecno, a Wikipedia, as redes sociais, a blogosfera, o jornalismo open source, o desenvolvimento de softwares livres, [...] as licenças Creative Commons e até o YouTube (SILVEIRA, 2008, P. 86). Em tese, o tipo de comunicação que prospera no ambiente digital está relacionado à livre expressão. “É a transmissão de fonte aberta, a livre divulgação, a transmissão descentralizada, a interação fortuita, a comunicação propositada e a criação compartilhada que encontram sua expressão na internet” (CASTELLS, 2003, p. 165). O ambiente digital permite, portanto, uma nova cultura: a cultura participativa. Os dispositivos das novas tecnologias de comunicação e informação, por sua interatividade e multifuncionalidade, são potenciais para fortalecer o processo democrático; proporcionam trocas de informações, consultas, debates, de maneira direta e rápida, livres de obstáculos burocráticos. Como aponta Correia (2008, p. 83-84), insiste-se num apelo a um novo paradigma que conduziria ao desenvolvimento de uma nova variedade de democracia, cujos traços seriam: a) interactividade – com todos os utilizadores comunicando uns com outros numa base de reciprocidade; b) globalidade – graças à ausência de fronteiras nacionais; c) liberdade de discurso e de associação; d) construção e disseminação de informação não submetida à censura oficial; e) consequente possibilidade de desafiar as perspectivas oficiais, as rotinas oficiais e instaladas. Na tentativa de entender como as novas tecnologias possibilitam novos modelos de comunicação, sobretudo em âmbitos locais, optou-se por analisar, sob a ótica da Economia Criativa, as atividades de comunicação da Casa do Hip Hop Bauru, projeto gerenciado pelo Ponto de Cultura Acesso Hip Hop. ARANHA E MIRANDA 1. Comunicação e Novas Tecnologias A escolha da cidade, bem como desse Arranjo Produtivo Local (APL) justifica-se, como SANTOS e SILVEIRA (2001) apontam, pelo fato de Bauru fazer parte de uma região concentrada, ocupada por próteses tecnológicas digitais e analógicas, que criam uma ecologia criativa facilitadora de projetos inventivos no campo da cultura e comunicação. O município tem hardware ARANHA E MIRANDA 69 e software para a articulação de projetos do campo da economia criativa em seus principais componentes: patrimônio histórico, mídias, artes e inovações técnicas funcionais (PLANO DA SECRETARIA DA ECONOMIA CRIATIVA - 2011-2014)3. 3 http://www.cultura.gov.br/secretaria-da-economia-criativa-sec (Acesso 15/11/2013, às 10h08). p g ç , q g p p ( ) ç q p ç p processos de negociação, em que cada interagente participa da construçã 2. O Colaborativo online O jornalismo colaborativo é definido como jornalismo feito por mais de uma pessoa, pessoas que contribuíram para o resultado final do que é publicado. (FOSCHINI; TADDEI, 2006, p.19). Todas as pessoas, munidas de celular ou câmera, postando em um blog, seriam potencialmente repórteres. O jornalismo, como o conhecemos, estaria, dessa forma, se modificando (MORETZSOHN, 2006, p.63). (...) a revolução digital trouxe, além da facilidade de distribuição da mensagem, a possibilidade de o receptor participar mais ativamente na (re)construção da mensagem, transformando os receptores em autores (CARNIELLO, 2003, p. 126). A hipermídia, portanto, desenvolve um ambiente propício à colaboração participativa de conteúdos por usuários leigos, já que se tornou muito fácil o registro de informações e a captação de imagens, que pode ser feita com um simples celular. Enquanto o jornalismo tradicional de massa transmite informações padronizadas, as informações que trafegam pela internet são tão diversificadas quanto quem as produz e as consome, assegura-nos ALZAMORA (2006, p.11). Entretanto, existe a necessidade de se pensar como essa linguagem hipermidiática – híbrida, processual e interativa – será apropriada de forma criativa pelo Jornalismo. Dessa maneira, ganha força a cultura interativa e participativa definida a partir do desejo do usuário de desempenhar um papel ativo na elaboração de informações, premissa essencial para a democratização da mesma, conforme MORETZSOHN (2007, p.17). Na web, é possível conceber uma comunicação mais complexa e dinâmica do que aquela produzida nas mídias que seguem exclusivamente o modelo broadcast. Configura-se, assim, uma nova forma de interação mútua, diferente e contrária à interação reativa, como aponta Primo: (...) a interação mútua é aquela caracterizada por relações interdependentes e processos de negociação, em que cada interagente participa da construção 3 http://www.cultura.gov.br/secretaria-da-economia-criativa-sec (Acesso 15/11/2013, às 10h08). ARANHA E MIRANDA 70 inventiva e cooperada do relacionamento, afetando-se mutuamente; já a interação reativa é limitada por relações determinísticas de estímulo e resposta (PRIMO, 2007, p.57). inventiva e cooperada do relacionamento, afetando-se mutuamente; já a interação reativa é limitada por relações determinísticas de estímulo e resposta (PRIMO, 2007, p.57). inventiva e cooperada do relacionamento, afetando-se mutuamente; já a interação reativa é limitada por relações determinísticas de estímulo e resposta (PRIMO, 2007, p.57). Com o avanço da tecnologia e o aumento de usuários da web, o número de fontes de notícias se multiplicou. A notícia, até então sob domínio de donos dos meios de comunicação, perdeu a exclusividade e se tornou uma ferramenta pública. 2. O Colaborativo online É nesse contexto que o jornalismo colaborativo na web se desenvolve. Os defensores dessa prática argumentam que a apuração coletiva gera uma mídia mais abrangente e multilateral A apuração dos acontecimentos é feita levando em consideração o princípio jornalístico de que nenhum fato acontece isoladamente e, sendo assim, pode ser visto de diversos ângulos. Nunca são apenas dois lados, ou versões, como defendem teorias anteriores. ARANHA E MIRANDA 3. O Local e Cidadão como elementos do Jornalismo A informação local do terceiro milênio deve promover a experimentação e converter os cenários de proximidade em lugares de comunicação eficiente e lugares de onde possam ser exportadas novas linguagens e formatos para a comunicação mundial (LÓPEZ GARCÍA, 2008, p. 34). a informação local do terceiro milênio tem que ser uma informação de qualidade, plural, participativa, imaginativa, que explique o que acontece no âmbito onde está sediado o veículo de comunicação, para quem informa e que narre o que afeta e interessa os habitantes desse território espacial, inclusive, quando se produz fora. A informação local do terceiro milênio deve promover a experimentação e converter os cenários de proximidade em lugares de comunicação eficiente e lugares de onde possam ser exportadas novas linguagens e formatos para a comunicação mundial (LÓPEZ GARCÍA, 2008, p. 34). Lemos (2011, p.12) aponta o jornalismo digital localizado como uma conjunção de funções pós-massivas e massivas, onde o usuário pode ter informações mais precisas sobre o seu local de interesse a partir de um cruzamento de notícias. A dimensão hiperlocal no jornalismo (já que ele é sempre local) refere-se, em primeiro lugar, a informações que são oferecidas em função da localização do usuário (sobre o bairro, a rua, etc), e em segundo lugar, pelas características pós-massivas desse novo jornalismo onde qualquer um pode ser produtor de informação. Essa é uma das tendências atuais do jornalismo: vinculação de notícias cruzando diversas fontes, oficiais, profissionais e cidadãs à geolocalização. A partir desse entendimento, pode-se observar a aproximação das práticas do jornalismo hiperlocal com as do jornalismo cidadão, em especial no que se refere ao envolvimento das comunidades e suas organizações com relação aos problemas sociais, econômicos e ambientais urbanos que enfrentam e que, sistematicamente, não são noticiados ou são mal noticiados pelo jornalismo tradicional. 3. O Local e Cidadão como elementos do Jornalismo Apesar do cenário globalizado, Barbosa (2003) lembra que as informações de caráter local também sempre foram de grande relevância para o jornalismo. Entretanto, “o dado novo é que, no jornalismo digital, a estrutura das redes permite novas possibilidades para a geração dos conteúdos locais, ampliando o espaço para a sua veiculação” (BARBOSA, 2003, p. 1). Grier (2008) destaca o jornalismo cidadão como sendo aquele que incide sobre histórias locais em contraponto com eventos nacionais; podendo ser produzido tanto por organizações noticiosas estabelecidas quanto por meio daqueles que não estão inseridos, e nem sempre é 'notícia' no sentido tradicional. Operações de mídia Hiperlocal são geograficamente baseadas, voltadas para a comunidade, organizações nativas e originais em reportar notícias para a web e pretendem preencher as lacunas percebidas na cobertura de uma questão ou região e promovem o engajamento cívico (METZGAR ET AL, 2010, p. 7). Para López García (2008), a comunicação local interpreta a realidade pela ótica dos valores compartilhados e tem contribuído para a personificação de um cenário da comunicação atual, no qual a dupla e simultânea tendência do local e do global apresenta novos recursos, cujas referências dos comunicantes são extraídas das duas esferas, que são complementares durante as ações comunicativas. ARANHA E MIRANDA 71 O jornalismo cidadão envolve uma proposta de produção de informação que contribua para que a/o cidadã/cidadão seja livre e independente, satisfatoriamente informado para que possa tomar decisões políticas que fortaleçam a democracia. Esse é um dos pressupostos do jornalismo ocidental, desde o advento da revolução francesa. Sua capacidade de articulação de narrativas sobre a atualidade, a validação de seus conteúdos e análises, a amplitude de suas observações, o foco crítico em setores significativos da realidade factual e a extensão de sua divulgação deram ao jornalismo, ao longo dos últimos duzentos anos, uma base de credibilidade e autoridade social (ARANHA, Â. S.; XAVIER, J. T.; 2015, p. 9). No âmbito do jornalismo digital, o local pode ser compreendido a partir da sobrevivência dos antigos modelos, em sintonia com as apostas e recursos midiáticos dos novos tempos. Nesse ponto, a informação local do terceiro milênio tem que ser uma informação de qualidade, plural, participativa, imaginativa, que explique o que acontece no âmbito onde está sediado o veículo de comunicação, para quem informa e que narre o que afeta e interessa os habitantes desse território espacial, inclusive, quando se produz fora. ARANHA E MIRANDA 4. O Local como recriação do criativo ARANHA E MIRANDA 72 Os denominados territórios criativos são formados por organizações, instituições e espaços de produção de conhecimento cultural. Nesses espaços, são atores os homens na sua ontológica vocação para serem sujeitos em sua autorreflexão e na reflexão sobre seu espaço e seu tempo, são seres de relação (FREIRE, 1967 p.36-39). No espaço urbano, esses territórios são ocupados por ateliers criativos de produtos, processos e serviços de cultura, próteses tecnológicas digitais e analógicas. O geógrafo Milton Santos (2001) vê esse processo de ocupação territorial complexo e contraditório por criar a possibilidade de múltiplas leituras do real. Leituras como fábula, leituras como perversidade e leituras como possibilidades. Nos Círculos de Cultura esses conceitos são revisitados e ressignificados pelos sujeitos aprendentes (XAVIER, J. T. P.; XAVIER, P. A. M., 2015). O estágio da fábula se apresenta como a da visão de mundo que cada sujeito traz para o círculo de diálogo. No estágio da perversidade, as visões de mundo de cada sujeito são problematizadas no coletivo aprendente. No terceiro estágio, como fruto do diálogo e da confrontação de visões de mundo, se revelam como possibilidades as intervenções futuras dos sujeitos aprendentes desse coletivo, caracterizado como Círculo de Cultura. De fato, se desejamos escapar à crença de que esse mundo assim apresentado é verdadeiro, e não queremos admitir a permanência de sua percepção enganosa, devemos considerar a existência de pelo menos três mundos num só. O primeiro seria o mundo tal como nos fazem vê-lo: a globalização como fábula; o segundo seria o mundo tal como ele é: a globalização como perversidade; e o terceiro, o mundo como ele pode ser: uma outra globalização (SANTOS, 2001, p. 18). Conceitos fundamentais da reflexão de SANTOS (2001) podem ser eleitos como temas geradores, e reinventados pelos agentes, tais como “unicidade técnica”, “convergência dos momentos”, “motor único” e “cognoscibilidade do planeta”. Todavia, pode-se pensar na criação de um outro mundo, mediante uma globalização mais humanizada. As bases materiais do período atual são, entre outras, a unicidade da técnica, a convergência dos momentos e o conhecimento do planeta. É nessas bases técnicas que o grande capital se apoia para construir a globalização perversa citada acima. Mas, essas mesmas bases técnicas poderão servir a outros objetivos, se forem postas a serviço de outros fundamentos sociais e políticos. Parece que as condições históricas do fim do século XX apontavam para essa última possibilidade. 4. O Local como recriação do criativo Tais novas condições tanto se dão no plano empírico quanto no plano teórico (SANTOS, 2001, p.20). 73 ARANHA E MIRANDA ARANHA E MIRANDA ARANHA E MIRANDA A unicidade técnica é a “malha de tecnologia” – analógica e digital – que se desenvolve em um determinado espaço territorial (XAVIER, J. T. P.; XAVIER, P. A. M., 2015), e essa “malha” forma uma ecologia digital que permite aos agentes a captação, edição e difusão de elementos culturais e simbólicos em ateliers criativos formados por coletivos de cultura. “As conexões desses ateliers criativos permitem a convergência de momentos virtuais – espaços digitais de trocas de informações com os momentos históricos e espaços reais de construção coletiva de conhecimentos” (XAVIER, J. T. P.; XAVIER, P. A. M., 2015, p.12). Essa articulação entre os dois momentos amplia os espaços aprendentes, com a apropriação dos diversos segmentos sociais das tecnologias digitais capilarizadas, o alargamento dos espaços de leitura, interpretação e intervenção, e a ampliação do diálogo entre os diversos agentes aprendentes. Os investimentos concentrados em tecnologia formam as regiões concentradas (SANTOS, 2001), ocupadas por próteses digitais e analógicas, espaços propícios para a troca de informações de agentes inovadores e a construção coletiva, de forma vertical, mais aprofundada, e de forma horizontal; ampliada. No cenário constituído pelos Círculos de Cultura presencial e digital os sujeitos históricos aprendentes produzem novos conhecimentos de gestão de processos, de pessoas e recursos, gestão de valores materiais e imateriais, nos espaços de intersecção dos diversos territórios criativos de cultura. ARANHA E MIRANDA 5. Ponto De Cultura Acesso Hip Hop Reconhecida como Ponto de Cultura pelo Governo Federal desde 2011, a ONG Ponto de Cultura Acesso Hip Hop tem o objetivo de promover a inclusão social por meio do hip hop. Isso ocorre em consequência de iniciativas que procuram dar a artistas locais oportunidades para que expressem sua criatividade. O Acesso Hip Hop consolidou-se na cidade como um ponto de conexão que se caracteriza como um círculo de cultura, nos seus aspectos fundamentais, com experiências presenciais e digitais. Esse ponto de conexão atua como protagonista na articulação dos arranjos produtivos locais de cultura (APLc) e das cadeias produtivas de produtos, serviços e processos criativos na cidade. Suas operações se estendem para arranjos culturais, em várias ARANHA E MIRANDA 74 linguagens, das mais tradicionais às mais recentes. Como gestores de projetos culturais, os responsáveis por esse ponto de cultura contribuem para o fortalecimento do cenário independente – em diversas linguagens e plataformas culturais -, e para a formação de novos públicos, não apenas os tradicionais do mercado de cultura, aquele mercado coordenado pelos arranjos produtivos locais de cultura e bens simbólicos monopolistas e centralizadores da criação, seleção, edição e difusão de produtos, serviços e processos culturais hegemônicos. Esse projeto contribui para a consolidação de uma leitura crítica sobre a gestão dos processos culturais locais, como se viu nos debates que aqueceram as discussões durante o processo de criação do Conselho Municipal de Cultura, em articulação com o poder público local e com organizações culturais da sociedade civil e de setores organizados focados na cultura. Esse debate estimulou a reflexão coletiva e trouxe à tona novos desafios, como o ingresso no Sistema Nacional de Cultura – SNC -, instrumento de política pública inclusiva do governo federal, que investe na pluralidade cultural, na formação de novos produtos e novos públicos alinhada à visão do Ministério da Cultura e da Secretaria Nacional de Economia Criativa. Uma das principais características dos projetos relaciona-se à formação de novos operadores, produtores culturais e fruidores, em todas as etapas do ciclo criativo: elaboração, planejamento, análises, proposição de planos, execução de funções e mecanismos de mensuração dos resultados. Cada etapa procura desenvolver ferramentas específicas baseadas no conceito de “tecnologias sociais”, processos inovadores construídos de formas coletivas e colocados à disposição de atores sociais. 4 O projeto Ensaio é realizado mensalmente pelo Ponto de Cultura Acesso Hip Hop, em parceria com a Secretaria Municipal de Bauru. Cada mês, o evento acontece em um bairro periférico diferente, e visa a fomentar a cultura hip hop na cidade. A Semana Municipal do Hip Hop é realizada em espaços públicos, periféricos e centrais, em articulação com o poder público local, realizado em vários pontos da cidade. 5 7 Desde 2012, as mulheres que compõem o movimento Hip Hop em Bauru têm se organizado em coletivo para realizar debates, oficinas, saraus e rodas de conversa com o objetivo de discutir o papel da mulher no movimento Hip Hop. 8 A Biblioteca Móvel – Quinto Elemento existe desde 2014, com ações de distribuição e troca gratuita de livros no Calçadão de Bauru, uma das principais vias de comércio popular da cidade. ç p p , p 5 Lançado em 2013, o Rap Hour leva shows de rap de convidados de Bauru e região para o Teatro Municipal da cidade. 6 5 Lançado em 2013, o Rap Hour leva shows de rap de convidados de Bauru e região para o Teatro Municipal da cidade. 6 Projeto que leva os quatro elementos do Hip Hop para as escolas públicas de Bauru e todo o interior de São Paulo. debates, oficinas, saraus e rodas de conversa com o objetivo de discutir o papel da mulher no movimento Hip Hop. 8 A Biblioteca Móvel – Quinto Elemento existe desde 2014, com ações de distribuição e troca gratuita de livros no Calçadão de Bauru, uma das principais vias de comércio popular da cidade. em 2013, o Rap Hour leva shows de rap de convidados de Bauru e região para o Teatro Municipal da cidad ue leva os quatro elementos do Hip Hop para as escolas públicas de Bauru e todo o interior de São Paulo. 4 O projeto Ensaio é realizado mensalmente pelo Ponto de Cultura Acesso Hip Hop, em parceria com a Secretar M i i l d B C d ê b i ifé i dif i f l h O projeto Ensaio é realizado mensalmente pelo Ponto de Cultura Acesso Hip Hop, em parceria com a Secretaria unicipal de Bauru. Cada mês, o evento acontece em um bairro periférico diferente, e visa a fomentar a cultura hip 5. Ponto De Cultura Acesso Hip Hop Esse processo impulsiona a ampliação progressiva de protagonistas em redes horizontais, articuladas e capilarizadas em bairros centrais e periféricos, mais qualificados nos planos intelectuais, conceituais, técnicos e ético-estéticos. Tais protagonistas em rede ampliada qualificam seus processos de comunicação multilaterais: poder público, agentes elaboradores de políticas públicas, produtores culturais independentes e de mercado, organizações sociais e culturais, instituições de ensino e arranjos culturais institucionalizados. ARANHA E MIRANDA 75 Diante das atividades realizadas e da intervenção na cidade, o ponto de Cultura Acesso Hip Hop “lê”, interpreta e intervém no território criativo da cidade de Bauru. Suas ações propositivas imprimem “digitais” em todas as topografias da cidade, por meio de vários projetos culturais. Seus projetos estimulam inovações em gestões de processos, pessoas e recursos, características dos projetos em desenvolvimento no município, como o “Projeto Ensaio4”, o Projeto Rap Hour5, o Combo 5 Elementos6 e o da Semana Municipal de Hip Hop. Já em sua quinta edição, em 2015, a Semana Municipal do Hip Hop se tornou por meio de mobilização social, há dois anos, uma política cultural da cidade após a aprovação da lei 6358/2013. Por determinação dessa lei, parte do orçamento municipal direcionado à cultura é destinado para a realização de um festival que, além de oferecer shows e oficinas para a população, também valoriza a diversidade cultural e o conhecimento sobre questões sociais, raciais e de gênero, discussões que também chegam às escolas e entidades públicas da cidade. Em 2015, ocorreu a inauguração da “Casa do Hip Hop Bauru” com uma ocupação ativa da antiga Estação Ferroviária, localizada no centro antigo de Bauru em um prédio tombado como patrimônio histórico-cultural pela Prefeitura. A Casa também faz parte da Rede Nacional de Economia e Empreendimentos Solidários do Hip Hop, abriga coletivos independentes como a Frente Feminina de Hip Hop de Bauru7 e a Biblioteca Móvel – Quinto Elemento8, e oferece atividades diárias gratuitas, entre oficinas dos quatro elementos básicos da cultura, oficinas de dança, projetos musicais e audiovisuais, cine debate, além de cursinho pré-vestibular. A articulação em rede ampliada mobiliza e qualifica a utilização dos recursos presentes no município. 76 ARANHA E MIRANDA A atividade online de pessoas interessadas em expor ideias, defender suas causas ou simplesmente opinar a respeito de fatos políticos do cotidiano mostra uma vitalidade inédita para o debate político. Mais do que partidos e governos, esse engajamento com questões políticas a partir do uso de mídias talvez seja um dos principais horizontes da democracia. […] Política, neste sentido amplo, diz respeito às possibilidades de ação no espaço público, isto é, à possibilidade de ser quem se é, defender publicamente as ideias que se tem não só sobre governo e administração, mas também sobre modos de pensar e estilos de vida (SÁ MARTINO, 2014, p. 108-109). ARANHA E MIRANDA 6. Por um novo Jornalismo Cidadão e Colaborativo 6. Por um novo Jornalismo Cidadão e Colaborativo Na plataforma online (www.casahiphopbauru.com.br/), a Casa do Hip Hop Bauru produz e gera conteúdos relativos a assuntos de relevância para a cultura Hip Hop no cenário bauruense. Um dos principais focos de atuação desse site objetiva a divulgação e cobertura de eventos culturais independentes, que ocorrem sob a gerência do Ponto de Cultura Acesso Hip Hop. Ao darem visibilidade aos produtores culturais autorais da cidade nesse espaço de comunicação, os comunicadores procuram fomentar o cenário independente de Bauru, bem como destacar determinados aspectos sociais, econômicos e políticos da cidade. Segundo Berger & Luckmann (2004), um meio de comunicação com tais configurações promove uma renovação do espaço público com a inclusão pelo jornalismo de “instituições intermediárias”, aquelas que reconhecem no cidadão o direito de participar da construção de sentido e, consequentemente, da construção social da realidade, o que reduz a crise subjetiva e intersubjetiva de sentido alimentada pela tendência à alienação e anomia que se verifica na modernidade (VIZEU; ROCHA; MESQUITA, 2010). Com isso, há um aumento considerável no potencial de engajamento cívico: A atividade online de pessoas interessadas em expor ideias, defender suas causas ou simplesmente opinar a respeito de fatos políticos do cotidiano mostra uma vitalidade inédita para o debate político. Mais do que partidos e governos, esse engajamento com questões políticas a partir do uso de mídias talvez seja um dos principais horizontes da democracia. […] Política, neste sentido amplo, diz respeito às possibilidades de ação no espaço público, isto é, à possibilidade de ser quem se é, defender publicamente as ideias que se tem não só sobre governo e administração, mas também sobre modos de pensar e estilos de vida (SÁ MARTINO, 2014, p. 108-109). O projeto de comunicação da Casa conta com quatro gestores fixos que dividem entre si funções essenciais para o funcionamento do site. A organização dos textos a serem publicados, a revisão, diagramação para a web e também o auxílio aos demais participantes em coberturas textuais cabe a uma representante da Casa do Hip Hop que acumula a função colaborativa de assessora da Casa do Hip Hop e do Ponto de Cultura. Há também a gestora de imagens/fotos, responsável pela organização das imagens a 77 serem divulgadas no site (e em outras mídias digitais), pela instrução e auxílio aos demais participantes do projeto em coberturas fotográficas. ARANHA E MIRANDA 6. Por um novo Jornalismo Cidadão e Colaborativo O gestor de mídias sociais controla a divulgação do site na internet e é o responsável pela manutenção das páginas de mídias sociais, principalmente as contas no Facebook da Casa (fb.com/Casa-do-Hip- Hop-Bauru-459089607594322) e do Ponto de Cultura (fb.com/Ponto-De-Cultura- Acesso-Hip-Hop-169696546470431). Além de suas funções específicas, todos os gestores auxiliam na criação de pauta e no recrutamento de novos colaboradores, nas instruções à equipe existente e aos novos integrantes, nas reuniões com possíveis e atuais parceiros e também nas coberturas de eventos. Apesar de funções definidas no modelo de comunicação, o diferencial adotado para a manutenção do site é a sua gestão de caráter colaborativo, aberto a todos os cidadãos bauruenses que queiram colaborar. Nesse aspecto, para Varella (2008), o jornalismo-cidadão diz respeito mais a um desejo coletivo de participação na produção de informação do que à ampliação de mecanismos de interação on-line. Não se trata, portanto, de um movimento derivado de um aumento da oferta de meios sociais on-line, distanciando-se de uma explicação mais tecnicista; ao contrário, a oferta dos meios que é condicionada, em termos, por uma demanda crescente de participação social na produção de mídia. Sendo assim, o jornalismo participativo, colaborativo ou cidadão é “uma ação por meio da informação”, porque, segundo Varella (2007, p. 80), o cidadão- repórter informa algo porque quer que algo seja feito, “que seu bairro esteja limpo, que a prefeitura proporcione melhor atendimento, que o professor ensine com mais dedicação ou que a coleta de lixo seja mais organizada e eficiente”. Essa visão localiza o “jornalismo-cidadão” como uma narrativa local. No caso do projeto da Casa do Hip Hop de Bauru, apesar de ser um projeto aberto à livre colaboração, por natureza, a maioria dos colaboradores são estudantes universitários, com boa parte de graduandos dos cursos de Jornalismo da cidade. De acordo com os coordenadores do Ponto de Cultura, as pautas são encaminhadas para uma lista de emails e a produção de conteúdos se dá de acordo com o interesse, disponibilidade e acordo entre os colaboradores e os gestores do projeto. Todas as decisões são discutidas 78 e tomadas em conjunto pelos colaboradores, sendo cada um deles também responsável pela sugestão e produção contínua de pautas, textos, fotos e materiais em diversos formatos. 6. Por um novo Jornalismo Cidadão e Colaborativo Por meio de oficinas e da prática do que se estuda, os colaboradores podem compreender o funcionamento do processo de cobertura colaborativa por meio da troca de tecnologias sociais e experiências envolvidas no processo, desde o planejamento da cobertura até a divulgação do produto final, sempre trabalhando nas plataformas digitais. Em cada cobertura, os colaboradores interessados realizam trabalhos de produção de textos (caracterizando a obra artística vista), entrevistas (colhendo impressões de públicos e artistas), fotografias (registrando os momentos e conceituando-os) e produções em vídeo (exercitando a linguagem audiovisual). As experiências do trabalho colaborativo permitem trocas de conhecimentos entre os membros do grupo, resultando numa formação cidadã, consciente e atuante a partir da prática colaborativa e espontânea. Essa cobertura potencializa a expressão dos eventos, agrega valores às ações realizadas na cidade e estimula a criação de um acervo de registros da “cena atual” que seria, praticamente, ignorado pelo jornalismo convencional. E essa cobertura colaborativa democratiza a cultura ao viabilizar a veiculação de seus produtos, promover iniciativas culturais e cobrir jornalisticamente os acontecimentos em uma plataforma livre, a internet. Deve-se considerar, ainda, que o processo de registro de produções culturais decorrente da cobertura de eventos gera não só incentivo à produção artística local, como também a compilação e preservação da memória cultural. Contribui, portanto, para a criação de uma identidade artística e social para a cidade. Como apontam Aranha e Xavier (2015, p.12), com as novas possibilidades oriundas de um modelo colaborativo e cidadão, o ARANHA E MIRANDA 79 “discurso único” e unidimensional passa a sofrer a concorrência das narrativas organizadas por esses novos Arranjos Produtivos Locais Intensos de Conteúdo Cultural (ApliCs). A fábula informativa agora disputa espaço com as narrativas das perversidades da exclusão na exclusão, e abre espaço para o discurso da possibilidade da mudança. “A tirania da informação e do dinheiro” – que caracteriza o sistema ideológico – é desnudada pelos conteúdos críticos que articulam uma esfera pública radical, precária, alternativa. As trocas de informações e conteúdos nessa esfera pública radical ampliam as percepções da realidade. Aumenta a cognoscibilidade do planeta, “discurso único” e unidimensional passa a sofrer a concorrência das narrativas organizadas por esses novos Arranjos Produtivos Locais Intensos de Conteúdo Cultural (ApliCs). A fábula informativa agora disputa espaço com as narrativas das perversidades da exclusão na exclusão, e abre espaço para o discurso da possibilidade da mudança. 6. Por um novo Jornalismo Cidadão e Colaborativo “A tirania da informação e do dinheiro” – que caracteriza o sistema ideológico – é desnudada pelos conteúdos críticos que articulam uma esfera pública radical, precária, alternativa. As trocas de informações e conteúdos nessa esfera pública radical ampliam as percepções da realidade. Aumenta a cognoscibilidade do planeta, ARANHA E MIRANDA ARANHA E MIRANDA 79 que passa a ser escrutinado de forma intensa e extensa em seus estudos sobre o processo da globalização, assim como a necessidade de superação do pensamento único pela consciência universal. que passa a ser escrutinado de forma intensa e extensa em seus estudos sobre o processo da globalização, assim como a necessidade de superação do pensamento único pela consciência universal. ARANHA E MIRANDA Considerações Finais As mudanças tecnológicas estabelecidas no final do século XX mudaram o ecossistema dos meios de comunicação e tocam, neste início do século XXI, todos os domínios da vida humana. Com o jornalismo, em específico, não foi diferente. A alteração do papel do jornalista, o uso massivo de redes sociais e o crescimento de coberturas colaborativas questionam o modelo industrial de jornalismo que se consolidou no século passado. Além da mudança no processo, as empresas jornalísticas passaram a sofrer para se manterem sustentáveis financeiramente. A valorização da cobertura de âmbito local pode oferecer alguma esperança nesse momento de incerteza do jornalismo. Embora algumas dúvidas se mostrem, o enfoque no local cidadão pode proporcionar um maior envolvimento da audiência. Juntamente com a criatividade, a prática jornalística calcada na localidade pode trazer grandes ganhos ao jornalismo atual. Resta tirar proveito disso e equacionar questões como fidelização, ampliação da audiência e financiamento, para que a inovação propiciada pela criatividade e pela tecnologia possa trazer desenvolvimento econômico e social, não só para o jornalismo. Diante desse exposto, pode-se inferir que o jornalismo hiperlocal prevê iniciativas que contemplam uma mesma finalidade no contexto atual: apresentar a realidade sobre outros pontos de vista, apresentar uma nova visão de mundo e acreditar na pluralidade de informações para formar opiniões dos leitores e internautas. Aliado aos movimentos populares e movimentos sociais, bem como a todos os arranjos produtivos locais de cultura (APLc) e cadeias produtivas de produtos, serviços e processos criativos na cidade, esse pretende construir uma rede de conteúdo contra- hegemônica, que dê as notícias e divulguem as reportagens que não são dadas e divulgadas em todos os outros lugares. Afirma-se que a forma de controle das mídias pela sociedade tenha vindo à tona com a ferramenta da internet: novas fontes de informação com canais cada vez mais acessíveis, portais de notícias, redes sociais, postagens de acontecimentos em tempo 80 80 real, coberturas em vídeo, etc. São milhões de dados a serem comprovados, refutados e discutidos por cada usuário, cada internauta que tenha em suas mãos um dispositivo que pode lhe mostrar verdades não disponíveis nos até então únicos meios confiáveis de informação, os tradicionais meios de comunicação de massa brasileiros. Considerações Finais Ademais, o jornalismo local praticado nos ambientes digitais fornece um importante referencial de sustentação das identidades culturais locais e pode colaborar com a participação cidadã mais qualificada e envolvida com os temas inerentes a cada local, principalmente aqueles em que as mídias tradicionais e hegemônicas costumam excluir da cobertura praticada atualmente, como é o caso operado pela Casa do Hip Hop de Bauru. JENKINS, H. Cultura da convergência. São Paulo: Aleph, 2009. LEMOS, A. Nova Esfera Conversacional. In: Dimas A Künsch, D.A, da Silveira, S.A, et al. Esfera Pública, redes e jornalismo. Rio de Janeiro. Editora. E-papers, 2009. LÓPEZ GARCÍA, Xosé. Ciberperiodismo em la proximidade. Sevilla: Comunicación Social ediciones y publicaciones, 2008. tp://www.portcom.intercom.org.br/pdfs/99061099541813324499037281994858501101.pdf. http://www.portcom.intercom.org.br/pdfs/99061099541813324499037281994858501101.pdf. METZGAR, Emily; KURPIUS, David; ROWLEY, Karen. Defining Hyperlocal Media: Proposing a Framework for Discussion. Annual meeting of the International Communication Association, Suntec Singapore International Convention & Exhibition Centre, Suntec City, Singapore, Jun 22, 2010 Disponível em: <http://citation.allacademic.com/meta/p_mla_apa_research_citation/4/0/4/9/9/p404999_index.ht ml>. Acessado em: 23 fev. 2016. MORETZSOHN, Sylvia. O mito libertário do “jornalismo cidadão”. In Comunicação e Sociedade, volume 9-10, p 63-81, 2006. FREIRE, P. Educação como prática da liberdade. Rio de Janeiro: Ed. Paz e Terra, 1967. FREIRE, P. Educação como prática da liberdade. Rio de Janeiro: Ed. Paz e Terra, 1967. JENKINS, H. Cultura da convergência. São Paulo: Aleph, 2009. REFERÊNCIAS ALZAMORA, Geane. Para além do jornalismo de massa: a diversidade da informação cultural na internet. In: PINTO, Júlio; SERELLE, Márcio. Interações Midiáticas. Belo Horizonte: Autêntica, 2006, pp. 153 –168. ARANHA, A. S.; X, J. T. P.. Jornalismo cidadão e o protagonismo político local: as experiências do jornal “Voz do Nicéia” e do “Portal Participi”. ÂNCORA-Revista Latino- americana de Jornalismo, v. 2, n. 2, 2015. BARBOSA, S. Jornalismo digital e a informação de proximidade: o caso dos portais regionais, com estudo sobre o UAI e o iBahia. Dissertação de Mestrado apresentada ao Programa de Pós-Graduação em Comunicação e Cultura Contemporâneas, na UFBA, Salvador (BA), Novembro de 2002. Disponível em: http://www.bocc.ubi.pt/pag/barbosa-suzana-portais- mestrado.pdf. Acesso em: 31 jun. 2015. BERGER, Peter L.; LUCKMAN, Thomas. A construção social da realidade. Lisboa: Dinalivro, 2004. CARNIELLO, Mônica Franchi. Interatividade na publicidade digital. Revista ciências humanas, Taubaté , v. 9, n. 2, p. 125-128, 2003. CARVALHO, J Maurício.; CARVALHO; A.M.G. Do hiperlocal aos insumos criativos: as mutações do jornalismo na contemporaneidade. In: CARVALHO,J.M; BRONOSKY (Org.) Jornalismo e Convergência. 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https://openalex.org/W4388523514	https://www.frontiersin.org/articles/10.3389/fpls.2023.1306301/pdf?isPublishedV2=False	English	null	Editorial: New insights in small fruit diseases	Frontiers in plant science	2,023	cc-by	2,061	Editorial on the Research Topic New insights in small fruit diseases © 2023 Jevremovic´ and Mavricˇ Plesˇko. 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. Small or berry fruits, known as “superfoods”, are grown on all continents. They play an important role in human nutrition and health due to the nutritional characteristics and bioactive components of their fruits. They are also hosts of a wide range of pathogens that threaten their production. In susceptible species and cultivars, pathogens reduce yield and fruit quality, and occasionally they can even destroy entire orchards. This Frontiers Research Topic comprises ﬁve contributions from researchers exploring various aspects of the plant pathology of berry fruits. In today’s agriculture, it is essential to decrease the use of synthetic pesticides for disease management in order to preserve the environment and human health. Many investigations are being undertaken to determine the efﬁcacy of natural pesticides produced by plants against pathogens and pests. Gray mold is an economically important disease of numerous berry fruits caused by the fungal pathogen Botrytis cinerea. In recent years, many aromatic oils and plant extracts have been evaluated in the ﬁeld of sustainable plant protection (Raveau et al., 2020). Antifungal substances, as an alternative to fungicides, can be used for the control of pathogens of economic relevance (such as B. cinerea). Dene et al. studied the effect of coriander (Coriandrum sativum) seed extracts and essential oils on grey mold. They showed that extracts of coriander seeds expanded the photosynthetic capacity and antioxidant reaction of strawberry plants but had a negative effect on gray mold suppression. The antioxidant response of strawberry plants was, in most cases, activated more by extract than essential oil. The results of the study showed that higher photosynthetic capacity values did not increase the antifungal effect following treatment with essential oils. A phytotoxic impact occurred when higher concentrations of the extract and essential oil of coriander seeds were used. As already stated, the fungus B. cinerea triggers devastating yield losses in many different plants, including strawberries (Fragaria vesca L.). TYPE Editorial PUBLISHED 08 November 2023 DOI 10.3389/fpls.2023.1306301 Darko Jevremovic´ 1* and Irena Mavricˇ Plesˇko 2 Darko Jevremovic´ 1* and Irena Mavricˇ Plesˇko 2 1Department for Fruit Protection and Certiﬁcation of Planting Material, Fruit Research Institute, Cˇ acˇ ak, Serbia, 2Plant Protection Department, Agricultural Institute of Slovenia, Ljubljana, Slovenia KEYWORDS 1Department for Fruit Protection and Certiﬁcation of Planting Material, Fruit Research Institute, Cˇ acˇ ak, Serbia, 2Plant Protection Department, Agricultural Institute of Slovenia, Ljubljana, Slovenia small fruits, diseases, fungi, phytoplasma, sustainable plant protection Editorial: New insights in small fruit diseases OPEN ACCESS EDITED AND REVIEWED BY Giovanni Bubici, National Research Council (CNR), Italy CORRESPONDENCE Darko Jevremovic´ darkoj@ftn.kg.ac.rs RECEIVED 03 October 2023 ACCEPTED 30 October 2023 PUBLISHED 08 November 2023 CITATION Jevremovic´ D and Mavricˇ Plesˇko I (2023) Editorial: New insights in small fruit diseases. Front. Plant Sci. 14:1306301. doi: 10.3389/fpls.2023.1306301 COPYRIGHT © 2023 Jevremovic´ and Mavricˇ Plesˇko. 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. OPEN ACCESS EDITED AND REVIEWED BY Giovanni Bubici, National Research Council (CNR), Italy CORRESPONDENCE Darko Jevremovic´ darkoj@ftn.kg.ac.rs RECEIVED 03 October 2023 ACCEPTED 30 October 2023 PUBLISHED 08 November 2023 CITATION Jevremovic´ D and Mavricˇ Plesˇko I (2023) Editorial: New insights in small fruit diseases. Front. Plant Sci. 14:1306301. doi: 10.3389/fpls.2023.1306301 COPYRIGHT © 2023 Jevremovic´ and Mavricˇ Plesˇko. 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. OPEN ACCESS OPEN ACCESS EDITED AND REVIEWED BY Giovanni Bubici, National Research Council (CNR), Italy *CORRESPONDENCE Darko Jevremovic´ darkoj@ftn.kg.ac.rs RECEIVED 03 October 2023 ACCEPTED 30 October 2023 PUBLISHED 08 November 2023 CITATION Jevremovic´ D and Mavricˇ Plesˇko I (2023) Editorial: New insights in small fruit diseases. Front. Plant Sci. 14:1306301. doi: 10.3389/fpls.2023.1306301 EDITED AND REVIEWED BY Giovanni Bubici, National Research Council (CNR), Italy frontiersin.org Editorial on the Research Topic Current management approaches rely on the application of fungicides before harvest, which is restricted by rigorous legislation. b-aminobutyric acid (BABA), a defensive elicitor, has been shown to produce resistance against many pathogens, including B. cinerea, in a variety of agricultural plants. According to reports, BABA protects a variety of plant species against many pests and pathogens by activating defense pathways dependent or not on salicylic acid (Cohen et al., 2016; Wilkinson et al., 2018). BABA is produced by stressed plants and can also be synthesized chemically. To identify resistance effects of BABA Badmi et al. evaluated how Frontiers in Plant Science 01 frontiersin.org Jevremovic´ and Mavricˇ Plesˇko 10.3389/fpls.2023.1306301 10.3389/fpls.2023.1306301 directly applied BABA, soil-drenched BABA, and untreated strawberry plants responded to B. cinerea infection. Local application of BABA to leaves boosted disease resistance, while systemic application as a soil drench reduced plant growth and increased vulnerability to B. cinerea infection. According to this research, different studied factors (e.g. the tissue type, plant developmental stage, application method, and genotype) can impact the effect of defense elicitors on plant resistance. The ﬁndings of a study on Botrytis cinerea control in strawberries using plant extracts and plant amino acids have highlighted the signiﬁcance of this ﬁeld of study and emphasized the need for more research to fully implement successful application and treatment techniques. phytoplasma infection is associated with variations in genome-wide DNA methylation, Liu et al. studied the methylome and transcriptome patterns of phytoplasma-infected mulberry plants. The authors identiﬁed a signiﬁcant number of genes with varying levels of methylation and expression, and conﬁrmed that phytoplasma infection can positively regulate plant disease resistance through methylation and expression of genes (in this case the Mu-GsSRK gene). The research provided new information about the mulberry-phytoplasma association. When proposed, the aim of this Research Topic was to present the latest research and perspectives on berry fruit diseases. The research presented was conducted in different countries and continents, conﬁrming the severity and distribution of the pathogens causing more or less detrimental diseases in small fruits. The funding data for the published manuscripts showed that all reported studies were ﬁnancially supported by governmental and regional resources. Phytophthora rubi is a devastating pathogen causing the decay of raspberries (Rubus idaeus L.) in numerous countries throughout the world. Raspberry growers in North America are particularly concerned about Phytophthora root rot root disease (Sapkota et al., 2022). Funding The author(s) declare that ﬁnancial support was received for the research, authorship, and/or publication of this article. Contract number 451-03-47/2023-01/200215, Ministry of Science, Technological Development and Innovation of the Republic of Serbia, and P4-0072 funded by Slovenian Research and Innovation Agency. The highbush blueberry (Vaccinium corymbosum L.) and cranberry (V. macrocarpon Ait.) are native to North America but are grown all over the world in production orchards. Blueberry and cranberry yields in orchards can occasionally decrease over time, and the problem reappears even after replanting. In such a scenario, the main reason for reduced productivity is very likely linked to the soil. The diversity of rhizosphere microbiomes (bacteria and fungi) is regarded as a signiﬁcant predictor of soil health (Schloter et al., 2018). The rhizosphere microbiome of cranberry and blueberry crops in New Jersey, USA, was studied by Kawash et al. 2023. When comparing blueberry and cranberry at the phylum level, little variations were observed in the composition of the bacterial microbiome, with Proteobacteria and Acidobacteria being the predominant ones. The fungal communities associated with blueberry and cranberry were quite different, dominated by the phyla Ascomycota and Basidiomycota. These ﬁndings establish a framework for investigating microbiomes that could affect the health of Vaccinium species in the Northeast USA. Acknowledgments The editors would like to thank all the authors and reviewers who participated in this Research Topic. Frontiers in Plant Science Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Author contributions DJ: Writing – original draft. IM: Writing – review & editing. Editorial on the Research Topic In order to comprehend the genetic mechanisms underpinning plant host adaptability, Tabima et al. (2017) reported the full genome of a P. rubi isolate originating from the Netherlands. In their current research, Sapkota et al. sequenced an additional 25 P. rubi isolates from various locations and different cultivars in Canada. According to the research, substantial genotypic diversity and limited gene diversity were found among sequenced isolates. The analysis revealed signiﬁcant genotypic diversity and low gene diversity among sequenced isolates. This in-depth investigation has revealed new information and insight into the P. rubi genome structure, and it serves as an important global resource for future population diversity studies on this pathogen. Obtained results could also be useful for raspberry breeding programs to develop Phytophthora-resistant cultivars. 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. Phytoplasmas are phloem-limited plant pathogenic bacteria associated with numerous diseases in plants. They induce a wide range of growth irregularities that can affect a plant’s appearance and negatively impact fruit production. It is still unclear how phytoplasma affects the host plant’s physiological functioning (Namba, 2019). In order to determine whether the response to 02 frontiersin.org 10.3389/fpls.2023.1306301 Jevremovic´ and Mavricˇ Plesˇko Wilkinson, S. W., Pastor, V., Paplauskas, S., Peétriacq, P., and Luna, E. (2018). Long- lasting b-aminobutyric acid-induced resistance protects tomato fruit against Botrytis cinerea. Plant Pathol. 67 (1), 30–41. doi: 10.1111/ppa.12725 Tabima, J. F., Kronmiller, B. A., Press, C. M., Tyler, B. M., Zasada, I. A., and Grünwald, N. J. (2017). Whole genome sequences of the raspberry and strawberry pathogens Phytophthora rubi and P. fragariae. Mol. Plant-Microbe Int. 30, 767–769. doi: 10.1094/MPMI-04-17-0081-A Sapkota, S., Burlakoti, R. R., Punja, Z. K., Dossett, M., and Gerbrandt, E. (2022). Understanding the root rot and wilting complex of raspberry: current research advances and future perspectives. Can. J. Plant Pathol. 44, 323–344. doi: 10.1080/ 07060661.2021.2011420 Schloter, M., Nannipieri, P., Sørensen, S. J., and van Elsas, J. D. (2018). Microbial indicators for soil quality. Biol. Fertil. Soils 54 (1), 1–10. doi: 10.1007/s00374-017-1248-3 References Cohen, Y., Vaknin, M., and Mauch-Mani, B. (2016). BABA-induced resistance: milestones along a 55-year journey. Phytoparasitica 44 (4), 513–538. doi: 10.1007/ s12600-016-0546-x Cohen, Y., Vaknin, M., and Mauch-Mani, B. (2016). BABA-induced resistance: milestones along a 55-year journey. Phytoparasitica 44 (4), 513–538. doi: 10.1007/ s12600-016-0546-x Namba, S. (2019). Molecular and biological properties of phytoplasmas. Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 95, 401–418. doi: 10.2183/pjab.95.028 Namba, S. (2019). Molecular and biological properties of phytoplasmas. Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 95, 401–418. doi: 10.2183/pjab.95.028 Kawash, J., Oudemans, P. V., Erndwein, L., and Polashock, J. J. (2023). Assessment and comparison of rhizosphere communities in cultivated Vaccinium spp. provide a baseline for study of causative agents in decline. Front. Plant Sci. 14. doi: 10.3389/ fpls.2023.1173023 Kawash, J., Oudemans, P. V., Erndwein, L., and Polashock, J. J. (2023). Assessment and comparison of rhizosphere communities in cultivated Vaccinium spp. provide a baseline for study of causative agents in decline. Front. Plant Sci. 14. doi: 10.3389/ fpls.2023.1173023 Raveau, R., Fontaine, J., and Lounès-Hadj Sahraoui, A. (2020). Essential oils as potential alternative biocontrol products against plant pathogens and weeds: A review. Foods 9, 365. doi: 10.3390/foods9030365 03 03 frontiersin.org Frontiers in Plant Science Frontiers in Plant Science
https://openalex.org/W3004728976	http://darchive.mblwhoilibrary.org/bitstreams/99c450b4-a6fc-5167-9aae-f01805f292bc/download	English	null	Ecophysiology of coral reef primary producers across an upwelling gradient in the tropical central Pacific	PloS one	2,020	cc-by	12,680	Maggie D. JohnsonID¤a, Michael D. Fox¤b, Emily L. A. Kelly¤c, Brian J. Zgliczynski, Stuart A. Sandin, Jennifer E. Smith Maggie D. JohnsonID¤a, Michael D. Fox¤b, Emily L. A. Kelly¤c, Brian J. Zgliczynski, Stuart A. Sandin, Jennifer E. Smith Maggie D. JohnsonID¤a, Michael D. Fox¤b, Emily L. A. Kelly¤c, Brian J. Zgliczynski, Stuart A. Sandin, Jennifer E. Smith Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, University of California San Diego, San Diego, California, United States of America Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, University of Califor San Diego, San Diego, California, United States of America ¤a Current address: Biology Department, Woods Hole Oceanographic Institution, Massachusetts, United States of America ¤b Current address: Geology and Geophysics Department, Woods Hole Oceanographic Institution, Massachusetts, United States of America ¤c Current address: Center for Ocean Solutions, Stanford University, California, United States of America magjohnson@whoi.edu ¤a Current address: Biology Department, Woods Hole Oceanographic Institution, Massachusetts, United States of America ¤b Current address: Geology and Geophysics Department, Woods Hole Oceanographic Institution, Massachusetts, United States of America ¤c Current address: Center for Ocean Solutions, Stanford University, California, United States of America * magjohnson@whoi.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 RESEARCH ARTICLE Abstract Patterson, Australian Bureau of Agricultural and Resource Economics and Sciences, AUSTRALIA Received: September 15, 2019 Accepted: January 15, 2020 Published: February 4, 2020 Copyright: © 2020 Johnson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Heather M. Patterson, Australian Bureau of Agricultural and Resource Economics and Sciences, AUSTRALIA Copyright: © 2020 Johnson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Upwelling is an important source of inorganic nutrients in marine systems, yet little is known about how gradients in upwelling affect primary producers on coral reefs. The Southern Line Islands span a natural gradient of inorganic nutrient concentrations across the equatorial upwelling region in the central Pacific. We used this gradient to test the hypothesis that ben- thic autotroph ecophysiology is enhanced on nutrient-enriched reefs. We measured metab- olism and photophysiology of common benthic taxa, including the algae Porolithon, Avrainvillea, and Halimeda, and the corals Pocillopora and Montipora. We found that tem- perature (27.2–28.7˚C) was inversely related to dissolved inorganic nitrogen (0.46–4.63 μM) and surface chlorophyll a concentrations (0.108–0.147 mg m-3), which increased near the equator. Contrary to our prediction, ecophysiology did not consistently track these pat- terns in all taxa. Though metabolic rates were generally variable, Porolithon and Avrainvillea photosynthesis was highest at the most productive and equatorial island (northernmost). Porolithon photosynthetic rates also generally increased with proximity to the equator. Photophysiology (maximum quantum yield) increased near the equator and was highest at northern islands in all taxa. Photosynthetic pigments also were variable, but chlorophyll a and carotenoids in Avrainvillea and Montipora were highest at the northern islands. Phycobi- lin pigments of Porolithon responded most consistently across the upwelling gradient, with higher phycoerythrin concentrations closer to the equator. Our findings demonstrate that the effects of in situ nutrient enrichment on benthic autotrophs may be more complex than labo- ratory experiments indicate. While upwelling is an important feature in some reef ecosys- tems, ancillary factors may regulate the associated consequences of nutrient enrichment on benthic reef organisms. OPEN ACCESS Citation: Johnson MD, Fox MD, Kelly ELA, Zgliczynski BJ, Sandin SA, Smith JE (2020) Ecophysiology of coral reef primary producers across an upwelling gradient in the tropical central Pacific. PLoS ONE 15(2): e0228448. https://doi. org/10.1371/journal.pone.0228448 org/10.1371/journal.pone.0228448 Editor: Heather M. Patterson, Australian Bureau of Agricultural and Resource Economics and Sciences, AUSTRALIA Received: September 15, 2019 Accepted: January 15, 2020 Published: February 4, 2020 Copyright: © 2020 Johnson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Heather M. Introduction The availability of inorganic nutrients in the environment influences organismal physiology and can have cascading effects on ecosystem-scale processes [1]. Both natural and anthropo- genic sources of nutrients can change community structure and function by facilitating growth of some species over others, in addition to increasing an ecosystem’s energetic foundation, capacity to support greater biomass [2], and trophic complexity [3]. In marine systems, the majority of research on nutrient enrichment has focused on the effects of anthropogenic inputs, including the process of eutrophication, which can overwhelm and fundamentally alter coastal habitats [4]. However, natural inputs are also important in nearshore systems, and can be linked to large-scale oceanographic processes such as upwelling, eddies, and internal tides [5]. Though the effects of coastal upwelling have been relatively well-studied in temperate eco- systems [6], less is known about how inorganic nutrient input from upwelling affects classically oligotrophic systems such as tropical coral reefs. Persistent upwelling caused by long-standing currents [7], or periodic upwelling caused by episodic processes (e.g., internal tides) [8], deliver subthermocline water enriched in dissolved inorganic nitrate, phosphate, and carbon to the ocean surface [5]. These deep, cooler waters mix with warmer surface waters and replenish depleted inorganic nutrients that are important for regulating biological activity [9,10]. Such interactions of environmental variables with organismal function are referred to as ecophysiology. Large-scale oceanographic processes, like upwelling, can influence the ecophysiology of resident organisms, particularly those that require inorganic nutrients for essential biological processes. For example, nutrient availability directly influences autotroph photosynthesis and respiration. Alterations to the nutrient land- scape resulting from natural or anthropogenic enrichment can have cascading effects on broader ecosystem processes by altering primary production [11,12], by favoring growth of some species over others [13], and, ultimately, by affecting how much energy is available to higher trophic levels [3]. Thus, understanding how the delivery of essential nutrients by upwelling can influence autotroph ecophysiology will shed light on how natural oceanographic processes shape ecosystem structure and function. Autotrophs are particularly sensitive to availability of inorganic nutrients, because dissolved inorganic nitrogen (DIN) and phosphorous (DIP) are requisite in the synthesis and use of photosynthetic machinery [11,14]. Though marine photosynthesis is generally not limited by the availability of carbon, due to the abundant supply of inorganic carbon in seawater and car- bon-concentrating mechanisms in many autotrophs [15], it is often limited by the availability of inorganic nutrients [11]. Upwelling effects on coral and algal ecophysiology Competing interests: The authors have declared that no competing interests exist. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by funding from the Moore Family Foundation, the Gordon and Betty Moore Foundation, the Scripps family, and anonymous donors. The funders had no role in study design, data collection and analysis, or preparation of the manuscript. 1 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Introduction For example, nitrate or phosphate limitation decreases productiv- ity and growth in numerous autotrophic taxa [1,16] including phytoplankton [17], microalgae [18], and macroalgae [19]. Accordingly, greater availability of DIN and DIP enhances photo- synthesis and generally increases marine primary production [20], and the input of inorganic nutrients from upwelling consistently increases productivity of phytoplankton communities in the surface ocean [10,21]. Though a positive relationship between autotroph physiology and nutrient availability is well established, there are instances where ancillary abiotic factors, such as iron limitation, can constrain photosynthesis and primary production of algae in nutrient replete conditions [22,23]. Given the fundamental role of nutrients in marine photosynthesis and autotroph physiol- ogy, ecosystems depleted in nutrients and dominated by autotrophs have the potential to respond strongly to nutrient inputs. Tropical coral reefs are typically oligotrophic, with the benthos dominated by highly productive macroalgae and corals that provide the primary source of carbon fixation for the ecosystem [24]. Corals are considered mixotrophs, because their energy comes from consuming organic particulates (i.e., heterotrophy) and from utilizing PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 2 / 22 Upwelling effects on coral and algal ecophysiology carbon fixed by photosynthesis of endosymbiotic microalgae in the family Symbiodiniaceae (i.e., autotrophy). Yet, at an ecosystem scale, they typically function as autotrophs, because the majority (up to 100%) of the daily energetic requirements of most corals is derived from pho- tosynthesis [25, 26]. Hereafter, we consider corals from an autotrophic perspective, grouping them (and their endosymbiotic microalgae) with algal taxa common on coral reefs. In tropical ecosystems with an abundance of autotrophs, such as coral reefs, the availability of DIN and DIP is perhaps the most important factor constraining photosynthesis and primary produc- tion, because light is abundant and temperatures are relatively stable [27]. Coral reefs are, thus, an ideal study system to explore the effects of natural nutrient inputs from upwelling in a typi- cally low nutrient ecosystem. Upwelling has been documented on coral reefs across ocean basins, ranging from the Flor- ida Keys [8] and the Colombian Caribbean [28] to the Seychelles [29] and the Great Barrier Reef [30]. The tropical central Pacific, where coral reefs exist around remote islands and atolls, is another region where upwelling likely plays an important role [31,32]. Introduction Equatorial upwelling is a persistent oceanographic process, caused by divergence of Ekman transport to the equator [7], that creates a region from ~8˚N to 8˚S of the equator [33] where surface-ocean inorganic nutrient concentrations are higher than oligotrophic tropical gyres [34]. The nutrient input from upwelling supports high levels of primary production in equatorial Pacific surface waters, as evidenced by increased surface-ocean productivity closer to the equator [10,21,35]. While patterns of phytoplankton productivity in the equatorial Pacific are well established, less is known about how the associated increase in inorganic nutrients effects the ecophysiology of benthic autotrophs. We used a natural gradient of surface ocean productivity and nutrient availability across the Southern Line Islands (SLI) to explore how upwelling-driven nutrient enrichment influ- enced the ecophysiology of benthic autotrophs common on coral reefs (Fig 1). The goals of this study were to, 1) characterize key environmental characteristics associated with upwelling across the SLI archipelago and, 2) to quantify corresponding changes in the ecophysiology of common benthic autotrophs. We hypothesized that metabolism (i.e., photosynthesis and res- piration) and photophysiology (i.e., maximum quantum yield and photosynthetic pigment content) of abundant coral and algal genera would increase across the SLI with greater expo- sure to upwelling. We further predicted that metabolic rates and pigment concentrations would increase (indicating enhancement), with increasing proximity to the equator. By under- standing patterns in ecophysiology of key benthic autotrophs across the SLI, we can consider how patterns in surface-ocean phytoplankton productivity relate to patterns in benthic pro- ductivity on coral reefs. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Materials and methods Site description This study was conducted on a 2013 expedition to the SLI aboard the M/Y Hanse Explorer under a Scientific Research Permit granted to SAS from the Republic of Kiribati, Environment and Conservation Division (permit #021113). The five uninhabited islands of the SLI span ~900 km from Malden Island in the north to Flint Island in the south (S1 Table). At 4.0˚ and 5.6˚ south of the equator, Malden and Starbuck Islands, respectively, are situated within the equatorial upwelling region that spans ~8˚N to 8˚S of the equator [33]. The remaining atoll and two islands (Millennium Atoll, Vostok Island, Flint Island) lie at increasing distances from the upwelling region (S1 Table), with Flint Island at the southernmost end of the archipelago. The coral reefs of the SLI are characterized by high biomass of herbivorous fish and top preda- tors [36] and are dominated by reef-building coralline algae and corals [37]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 3 / 22 Upwelling effects on coral and algal ecophysiology Fig 1. Coral and algae of the Southern Line Islands. Common genera of corals and algae across the Southern Line Islands in the Republic of Kiribati, central Pacific. (a) The crustose coralline alga, Porolithon sp. and (b) the corals Pocillopora meandrina and (c) Montipora aequituberculata were collected from all five islands. (d) The fleshy green alga Avrainvillea amadelpha was found only at Vostok and Malden, and (e) the calcareous green alga, Halimeda sp., was found at Flint, Millennium and Starbuck. Fig 1. Coral and algae of the Southern Line Islands. Common genera of corals and algae across the Southern Line Islands in the Republic of Kiribati, central Pacific. (a) The crustose coralline alga, Porolithon sp. and (b) the corals Pocillopora meandrina and (c) Montipora aequituberculata were collected from all five islands. (d) The fleshy green alga Avrainvillea amadelpha was found only at Vostok and Malden, and (e) the calcareous green alga, Halimeda sp., was found at Flint, Millennium and Starbuck. https://doi.org/10.1371/journal.pone.0228448.g001 Environmental parameters Water samples were collected for nutrient analyses at each island over three separate sampling intervals. During a cruise to the SLI in 2009, water samples were collected in triplicate from ~ 1 m above the benthos at 1–3 sites per island (referred to as 2009 in situ) [31]. During the 2013 cruise, three water samples were collected from ~1 m above the benthos every 5 m from 5–25 m depth (referred to as 2013 in situ) [40]. There were no differences in nutrient concentrations across depths in 2013, thus all depths were pooled for a more robust estimate of environmental conditions across the reef slope of an island on a given day (n = 9–12, per island). Additionally, triplicate water samples were collected from the bulk seawater used during metabolic incubations at each island (referred to as 2013 incubation). Seawater samples were filtered through 1.2 μM GF/C filters (Whatman) and frozen at -20˚C. Samples from 2009 and 2013 in situ were analyzed at the Univer- sity of Hawaii Hilo EPSCoR analytical laboratory and 2013 incubation samples were analyzed at the University of California Santa Barbara Marine Science Institute Analytical lab. Seawater was analyzed for total DIN, which includes nitrate (NO3 -), nitrite (NO2 -) and ammonium (NH4 +), and for DIP. Replicate samples were averaged by island for each sampling period. To assess in situ temperature we deployed 6–8 autonomous sensors (Manta 2, Eureka Environ- mental Engineering) at 10 m depth for 3–4 days per island (5 min sampling interval). Daily mean temperatures were calculated across sensors to determine daily and overall site means. Photosyn- thetically active radiation (PAR) was measured adjacent to the instrument with a 4π quantum sen- sor (LI-193) attached to a LiCor LI-1400 meter in an underwater housing. We measured PAR every minute for 2–3 days and calculated an average light intensity during the period of maximum irradiance and peak photosynthesis per day (10:00–14:00) [43], and then a daily average per island. All deployments occurred on sunny days, except for one cloudy day at Vostok. every minute for 2–3 days and calculated an average light intensity during the period of maximum irradiance and peak photosynthesis per day (10:00–14:00) [43], and then a daily average per island. All deployments occurred on sunny days, except for one cloudy day at Vostok. Remote sensing To estimate surface-ocean primary production across the SLI, we used the eight-day 0.0417˚ (4 km) spatial resolution product of chlorophyll a (chl a, mg mg-3) derived from the Moderate Resolution Imaging Spectroradiometer (https://modis.gsfc.nasa.gov/about/) (sensu Gove et al. (2013)[38]). Monthly mean chl a data were generated for each island and averaged over the period of September–December 2013 to provide an integrated estimate of surface-ocean pri- mary production around each island over the period of the cruise. The oceanographic condi- tions during this time are most relevant to the ecophysiology of the organisms studied [39] and provide a reliable estimate of annual conditions across the SLI [40]. While these estimates PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 4 / 22 Upwelling effects on coral and algal ecophysiology of surface-ocean production are measured in offshore waters adjacent to the SLI reefs, they can be used to reliably infer reef-scale patterns such as benthic community structure [41] and ecosystem calcification [42]. To confirm that large-scale patterns of surface primary produc- tion track in situ nutrient environments across the SLI, we also collected discrete water sam- ples from above the reef benthos as described below. Maximum quantum yield Maximum quantum yield is a useful proxy for photosynthetic efficiency because it is propor- tional to the rate of electron transport, and it provides a convenient, non-invasive, and instan- taneous estimate of the performance of the photosynthetic machinery in both algae and corals [49]. Maximum quantum yield was measured with a red Pulse Amplitude Modulation Fluo- rometer (Diving PAM, Walz). Samples were dark adapted for 2 hours after respiration mea- surements. To account for variation in fluorescence-based maximum quantum yield (Fv/Fm) measurements within individuals, we calculated an average of three haphazardly selected points on each individual from approximately the same region (~ 2 cm from the tip/edge). Measuring light intensity was minimized to avoid actinic effects (F0 = 300–500) and gain was minimized to avoid amplifying noise [50]. Saturation intensity (8) and saturation pulse width (0.8) were constant for all measurements. Due to logistical difficulties, no measurements were taken at Flint. Specimen collection Two species of coral and up to three genera of algae were collected at 10 m depth from unshaded microhabitats at each island (adjacent to sensors), with four individuals of each taxa (Fig 1 and S1 Table). After collection, samples were held in buckets with ambient seawater under shaded, ambient light until physiological incubations. Water was regularly replenished with new ambient seawater. In general, we sampled the most abundant coral and algal taxa on a given island, but due to biogeographic differences we were unable to collect the same species at all islands (S1 Table). At all islands, we collected similar sized fragments of the branching coral Pocillopora meandrina, the plating coral Montipora aequituberculata, and the crustose coralline alga (CCA) Porolithon sp. (5–8 cm diameter/height). Calcareous algae in the genus Halimeda were present only at Flint, Starbuck, and Millennium. We collected 8 cm long frag- ments of the most abundant species at each of the three islands (Flint: H. opuntia, Starbuck: H. micronesica, Millennium: H. taenicola). We consider Halimeda at the genus level because photophysiology and response to higher nutrient concentrations are similar across species within the genus [44,45]. Abundance of fleshy macroalgae is exceedingly low across the SLI [37], and the fleshy green alga Avrainvillea amadelpha was present only at Malden and Vostok. 5 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Upwelling effects on coral and algal ecophysiology Metabolism Physiological studies were conducted aboard the M/Y Hanse Explorer over 3–4 days at each island, with incubations commencing within 1–2 hrs after sample collection. Photosynthesis and respiration rates were determined by measuring oxygen exchange under saturating irradi- ance and total darkness, respectively, using sealed metabolic incubation chambers and optical dissolved oxygen (DO) probes (Hach Intellical LDO101) following the exact methods of John- son et al. (2017) [46]. All incubations of a given species (n = 4, per species), and a blank con- trol, were run simultaneously in a temperature controlled bath. Each chamber contained one specimen (or seawater control) and a magnetic stir bar. No more than 2 taxa were incubated on a given day. Samples were kept in ambient conditions for 1–3 hrs before the start of incuba- tions, and were acclimated to incubation chambers for 5–10 min before the start of measure- ments. Saturating irradiance (~700 μM quanta m2 s-1) was supplied by two LED light fixtures (Aqua Illumination, Hydra) suspended directly above the incubation chambers. Light levels inside incubation chambers were measured with a submerged 4π quantum sensor (LI-193) and a LiCor LI-1400 meter. Due to a probe malfunction, no oxygen production data were col- lected for Porolithon at Starbuck. Photosynthesis and respiration were calculated as the linear slope of oxygen concentration over the duration of the incubation (45 min), and the blank for each set of incubations was subtracted from the rates of respective replicates to account for background changes in oxygen. Oxygen production in the light was assumed to represent net photosynthesis and oxygen con- sumption in the dark to represent respiration. To estimate total oxygen produced during incu- bations, or gross photosynthesis, respiration rates were added to net photosynthesis. For consistency, rates across all taxa were normalized to surface area and are expressed as μmol O2 cm-2 hr-1. Surface area was determined for coralline fragments by foil-wrapping following March (1970) [47], for corals by wax dipping following Stimson and Kinzie (1991) [48], and for algae by planar image analysis using ImageJ. Photosynthetic pigments Samples were frozen at -20˚C after physiological assessments and transported to Scripps Insti- tution of Oceanography for pigment analyses. Extractions of chl a and carotenoids followed the methods and equations of Moran and Porath (1980) [51] and Wellburn (1994) [52]. In brief, chl a and carotenoids were extracted from three intact subsamples of each Halimeda and Avrainvillea individual in N,N-Dimethylformamide (DMF). Pigments were extracted in total darkness at 4˚C for 24 hrs. The extract was then centrifuged and the supernatant was analyzed PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 6 / 22 Upwelling effects on coral and algal ecophysiology spectrophotometrically with a diode array spectrophotometer (Agilent, UV-vis 8453) with 1 nm resolution. For the coral samples, coral tissue was first removed from the skeleton with an airbush, and the blastate was homogenized. The endosymbiotic algal fraction was isolated from the host animal tissue, and chl a and carotenoids were extracted from the algal fraction in DMF as described above. Two subsamples from each coral individual were analyzed. Pigments were extracted from the coralline alga, Porolithon, with procedures adapted from Payri et al. (2001) [53] and Kursar and Alberte (1983) [54]. Water soluble phycobilin pigments were extracted first using 0.01 M phosphate buffer. Two 1-cm2 punches were collected from each coralline algal sample and then each subsample was ground separately in the dark and over ice using a mortar and pestle. Pigments were extracted in darkness at 4˚C for 24 hrs. Sam- ples were then centrifuged and the supernatant was analyzed for allophycocyanin, phycocya- nin, and phycoerythrin content based on the equations of Kursar and Alberte (1983) [54]. The remaining pellets were then processed for chl a and carotenoids in DMF following the proce- dure above. Subsamples from algal pigment extractions were averaged for each individual. Por- olithon and coral pigments were normalized to subsample surface area and macroalgal pigments were normalized to subsample wet weight. spectrophotometrically with a diode array spectrophotometer (Agilent, UV-vis 8453) with 1 nm resolution. For the coral samples, coral tissue was first removed from the skeleton with an airbush, and the blastate was homogenized. The endosymbiotic algal fraction was isolated from the host animal tissue, and chl a and carotenoids were extracted from the algal fraction in DMF as described above. Two subsamples from each coral individual were analyzed. Pigments were extracted from the coralline alga, Porolithon, with procedures adapted from Payri et al. Photosynthetic pigments (2001) [53] and Kursar and Alberte (1983) [54]. Water soluble phycobilin pigments were extracted first using 0.01 M phosphate buffer. Two 1-cm2 punches were collected from each coralline algal sample and then each subsample was ground separately in the dark and over ice using a mortar and pestle. Pigments were extracted in darkness at 4˚C for 24 hrs. Sam- ples were then centrifuged and the supernatant was analyzed for allophycocyanin, phycocya- nin, and phycoerythrin content based on the equations of Kursar and Alberte (1983) [54]. The remaining pellets were then processed for chl a and carotenoids in DMF following the proce- dure above. Subsamples from algal pigment extractions were averaged for each individual. Por- olithon and coral pigments were normalized to subsample surface area and macroalgal pigments were normalized to subsample wet weight. Statistical analyses All variables met assumptions of normality and homogeneity of variances, determined by visual inspection of residuals using q-q plots and Shapiro-Wilks tests and Levene’s test, respec- tively. Mean environmental parameters were analyzed across islands with a one-way ANOVA with island as a fixed factor. In situ temperatures and remotely sensed chl a were compared using daily and 8-day mean values, respectively. Inorganic nutrient concentrations were ana- lyzed separately for each sampling interval (2009 in situ, 2013 in situ, 2013 incubation). Daily mean light intensity was compared using values between 10:00–14:00. Each response variable was analyzed separately by genus with a one-way ANOVA with island as a fixed factor. Where significant differences were detected, Tukey’s HSD identified differences among islands. All statistical analyses were conducted in R [55]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Environmental parameters https://doi.org/10.1371/journal.pone.0228448.g002 https://doi.org/10.1371/journal.pone.0228448.g002 https://doi.org/10.1371/journal.pone.0228448.g002 incubations (S2 Table). DIN was ~350% higher at Malden (4.63 ± 0.13 μM) versus Flint (0.46 ± 0.01 μM) in 2013, and ~200% higher than at either Vostok (0.93 ± 0.05 μM) or Millen- nium (0.95 ± 0.08 μM) (Table 1). Concentration of DIP was 75–150% higher at the northern islands than at the three southern islands (Table 1). incubations (S2 Table). DIN was ~350% higher at Malden (4.63 ± 0.13 μM) versus Flint (0.46 ± 0.01 μM) in 2013, and ~200% higher than at either Vostok (0.93 ± 0.05 μM) or Millen- nium (0.95 ± 0.08 μM) (Table 1). Concentration of DIP was 75–150% higher at the northern islands than at the three southern islands (Table 1). Environmental parameters Surface chl a concentrations were higher near the equator, and lower farther from the equator (Fig 2 and S2 Table). From Sept—Dec 2013, mean surface chl a was highest at Malden, lowest at Flint, and similar across the three middle islands. Malden surface water chl a concentrations were 10% higher than Starbuck, while Starbuck, Millennium and Vostok were 15–20% higher than Flint (Table 1). Mean daily in situ temperatures decreased significantly across the SLI based on latitude (S2 Table), with coolest temperatures closest to the equator. Temperatures were similarly warm at Vostok and Millennium, at 28.70 ± 0.01˚C and 28.70 ± 0.02˚C, respectively, and decreased to 27.20 ± 0.05˚C at Malden (Table 1). Average daily PAR levels did not vary significantly across islands (S2 Table), though the lowest daily average intensity between the hours of 10:00–14:00 was at Vostok on a cloudy day (428 ± 43) and the highest was at Malden (884 ± 1) (Table 1). In situ water samples from 2009 and 2013, and bulk water samples from incubations in 2013, showed a consistent pattern of increasing ambient DIN and DIP from south to north that mirrors the larger surface chl a and temperature gradients across the SLI (Table 1). Mal- den and Starbuck were significantly enriched in DIN and DIP compared to Flint, Vostok and Millennium, regardless of whether the water was sampled from ~1 m above the benthos in 2009, throughout the water column in 2013, or from the nearshore surface water used in PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 7 / 22 Upwelling effects on coral and algal ecophysiology Fig 2. Surface-ocean chl a during cruise. Average surface-ocean chl a concentrations increased from south to north across the Southern Line Islands over four months encompassing the research expedition. Surface chl a (mg mg-3) was estimated from the eight-day 0.0417˚ (4 km) spatial resolution product derived from the Moderate Resolution Imaging Spectroradiometer, and concentrations ranged from 0.01 (blue) to 0.17 mg mg-3 (red) across the archipelago. Fig 2. Surface-ocean chl a during cruise. Average surface-ocean chl a concentrations increased from south to north across the Southern Line Islands over four months encompassing the research expedition. Surface chl a (mg mg-3) was estimated from the eight-day 0.0417˚ (4 km) spatial resolution product derived from the Moderate Resolution Imaging Spectroradiometer, and concentrations ranged from 0.01 (blue) to 0.17 mg mg-3 (red) across the archipelago. Upwelling effects on coral and algal ecophysiology Table 1. Environmental parameters across the Southern Line Islands, with islands listed from south to north. Island Year Temp (˚C) PAR§ Chl a (mg m-3) DIN (μM) DIP (μM) Flint 2013 in situ 28.50 ± 0.04 608 ± 3 0.108 ± 0.003 0.46 ± 0.01 0.13 ± 0.003 2013 incubation 2009 in situ 1.16 ± 0.16 0.78 ± 0.02 0.20 ± 0.005 0.15 ± 0.003 Vostok 2013 in situ 28.70 ± 0.01 428 ± 43 0.128 ± 0.001 0.93 ± 0.05 0.16 ± 0.01 2013 incubation 2009 in situ 1.62 ± 0.33 1.51 ± 0.35 0.23 ± 0.02 0.16 ± 0.003 Millennium 2013 in situ 28.70 ± 0.02 647 ± 4 0.125 ± 0.004 0.95 ± 0.08 0.16 ± 0.01 2013 incubation 2009 in situ 1.18 ± 0.13 2.03 ± 0.06 0.22 ± 0.02 0.21 ± 0.01 Starbuck 2013 in situ 28.30 ± 0.01 798 ± 12 0.133 ± 0.001 3.86 ± 0.05 0.42 ± 0.03 2013 incubation 2009 in situ 4.50 ± 0.04 3.59 ± 0.38 0.35 ± 0.02 0.23 ± 0.01 Malden 2013 in situ 27.20 ± 0.05 884 ± 1 0.147 ± 0.003 4.63 ± 0.13 0.44 ± 0.01 2013 incubation 2009 in situ 5.15 ± 0.06 3.87 ± 0.32 0.51 ± 0.07 0.27 ± 0.02 I l d l ( SE) i t l t th S th Li I l d I it t t d h t th ti ll ti di ti (PAR) d Table 1. Environmental parameters across the Southern Line Islands, with islands listed from south to north. Island-scale mean (± SE) environmental parameters across the Southern Line Islands. In situ temperature and photosynthetically active radiation (PAR) were measured with autonomous sensors for 2–4 days per island. Average PAR at peak irradiance (between 1000–1400) is presented, as these values most closely resemble intensities used in lab incubations. Chl a concentrations were derived from satellite data spanning a four-month window around the expedition (Sept—Dec 2013). In situ water samples were collected for analysis of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) from one site per island in 2013 (n = 9–12), and in triplicate from 3–6 sites per island during a research cruise in 2009. §μmol quanta-1 m-2 s-1. Island-scale mean (± SE) environmental parameters across the Southern Line Islands. Metabolism Metabolic rates of all genera were variable across the SLI archipelago, and although there was some evidence of enhancement in ecophysiology with increasing proximity to the equator, the trend was not consistent across all taxa (Fig 3 and Table 2). PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 8 / 22 In situ temperature and photosynthetically active radiation (PAR) were measured with autonomous sensors for 2–4 days per island. Average PAR at peak irradiance (between 1000–1400) is presented, as these values most closely resemble intensities used in lab incubations. Chl a concentrations were derived from satellite data spanning a four-month window around the expedition (Sept—Dec 2013). In situ water samples were collected for analysis of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) from one site per island in 2013 (n = 9–12), and in triplicate from 3–6 sites per island during a research cruise in 2009. §μmol quanta-1 m-2 s-1. https://doi.org/10.1371/journal.pone.0228448.t001 Photosynthesis (net and gross) and respiration of the CCA Porolithon were highest at Mal- den near the equator (Fig 3A and Table 2), but did not vary significantly across the three southern islands, Flint, Vostok, and Millennium (S3 Table). Some of the variability in gross photosynthesis is related to different patterns of response in net photosynthesis versus respira- tion, because gross photosynthesis is the sum of both. In Porolithon, the significant increase in gross photosynthesis at Malden is linked to a 211% increase in net photosynthesis from Flint to Malden, but only a 24% increase in respiration. In Halimeda, gross photosynthesis was con- sistent at the three islands where it was found (Flint, Millennium, Starbuck) (Fig 3C). How- ever, this pattern obscures a marginally significant 45% increase in net photosynthesis at the southernmost island (Flint) (S3 Table), and a significant 173% increase in respiration rates at the northern islands (Table 2). These opposing trends in net photosynthesis and respiration across the islands are negated in the estimates of gross photosynthesis. In Avrainvillea, gross photosynthesis was 54% higher at Malden than Vostok (Fig 3E and Table 2), which was driven by significantly higher net photosynthesis (131%) but no difference in respiration from Vostok to Malden (S3 Table). Coral metabolism was highly variable across the SLI. Gross photosynthesis of Pocillopora varied among islands but did not clearly relate to proximity to the equator and the associated increase in nutrient concentrations (Fig 3G and Table 2). These differences were associated with variable, but not significantly different, rates of net photosynthesis and respiration (S3 Table). The plating coral, Montipora, also had variable photosynthesis (net and gross) and res- piration rates across the archipelago (Fig 3I and S3 Table), with highest rates at Vostok and lowest rates at Millennium (Table 2). PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Island-scale mean (± SE) environmental parameters across the Southern Line Islands. In situ temperature and photosynthetically active radiation (PAR) were measured with autonomous sensors for 2–4 days per island. Average PAR at peak irradiance (between 1000–1400) is presented, as these values most closely resemble intensities used in lab incubations. Chl a concentrations were derived from satellite data spanning a four-month window around the expedition (Sept—Dec 2013). In situ water samples were collected for analysis of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) from one site per island in 2013 (n = 9–12), and in triplicate from 3–6 sites per island during a research cruise in 2009. §μmol quanta-1 m-2 s-1. 9 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Upwelling effects on coral and algal ecophysiology Fig 3. Coral and algal metabolism and photosynthetic efficiency. Box plots of gross photosynthesis and maximum quantum yield of (a-b) Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and (i-j) Montipora aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and missing bars indicate where a given species was not present at an island. Due to logistical difficulties, there were no maximum quantum yield measurements at Flint or photosynthesis measurements from Porolithon at Starbuck. Significant differences between islands were determined by Tukey’s HSD, different letters represent significant differences at p < 0.05. Gross photosynthetic rates for Porolithon and corals were normalized to surface area, and are expressed as μmol O2 cm-2 hr-1. Rates for the algae, Halimeda and Avrainvillea, were normalized to fresh weight, thus for those taxa the units are μmol O2 mg-2 hr-1. Fig 3. Coral and algal metabolism and photosynthetic efficiency. Box plots of gross photosynthesis and maximum quantum yield of (a-b) Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and (i-j) Montipora aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and missing bars indicate where a given species was not present at an island. Due to logistical difficulties, there were no maximum quantum yield measurements at Flint or photosynthesis measurements from Porolithon at Starbuck. Significant differences between islands were determined by Tukey’s HSD, different letters represent significant differences at p < 0.05. Gross photosynthetic rates for Porolithon and corals were normalized to surface area, and are expressed as μmol O2 cm-2 hr-1. Rates for the algae, Halimeda and Avrainvillea, were normalized to fresh weight, thus for those taxa the units are μmol O2 mg-2 hr-1. https://doi.org/10.1371/journal.pone.0228448.g003 Upwelling effects on coral and algal ecophysiology Table 2. Photosynthesis and respiration rates. Genus Island Net Photosynthesis (μmol O2 cm-2 hr-1) Respiration (μmol O2 cm-2 hr-1) Porolithon Flint 0.19 ± 0.16 0.91 ± 0.23 Vostok 0.05 ± 0.13 0.94 ± 0.04 Millennium 0.40 ± 0.15 0.96 ± 0.09 Starbuck - 0.44 ± 0.06 Malden 0.59 ± 0.13 1.13 ± 0.05 Halimeda Flint 0.61 ± 0.07 0.11 ± 0.03 Millennium 0.43 ± 0.05 0.28 ± 0.01 Starbuck 0.42 ± 0.03 0.30 ± 0.04 Avrainvillea Vostok 0.15 ± 0.01 0.32 ± 0.03 Malden 0.35 ± 0.05 0.36 ± 0.05 Pocillopora Flint 0.69 ± 0.10 0.49 ± 0.03 Vostok 0.34 ± 0.23 0.52 ± 0.06 Millennium 0.66 ± 0.09 0.62 ± 0.04 Starbuck 0.43 ± 0.13 0.49 ± 0.03 Malden 0.49 ± 0.09 0.55 ± 0.02 Montipora Flint 0.68 ± 0.17 0.62 ± 0.12 Vostok 0.80 ± 0.09 0.90 ± 0.10 Millennium 0.58 ± 0.05 0.45 ± 0.02 Starbuck 0.93 ± 0.19 0.58 ± 0.02 Malden 0.77 ± 0.08 0.60 ± 0.03 Mean (± SE) net photosynthesis and dark respiration rates of corals and algae surveyed across the SLI, n = 4 per genus at each island. Rates for Porolithon and corals were normalized to surface area. Rates for the algae, Halimeda and Avrainvillea, were normalized to fresh weight, thus, the units for those taxa are μmol O2 mg-2 hr-1. Table 2. Photosynthesis and respiration rates. Mean (± SE) net photosynthesis and dark respiration rates of corals and algae surveyed across the SLI, n = 4 per genus at each island. Rates for Porolithon and corals were normalized to surface area. Rates for the algae Halimeda and Avrainvillea were normalized to fresh weight thus the units for those taxa are μmol O mg-2 hr-1 quantum yield did not differ across islands for Halimeda (Fig 3D and S3 Table), and was sig- nificantly higher at Malden than at Vostok for Avrainvillea (Fig 3F and S3 Table). Maximum quantum yield of the coral Pocillopora was significantly higher at Malden than at the southern islands, which did not differ from each other (Fig 3H and S3 Table). In Monti- pora, maximum quantum yield was significantly higher at Starbuck than at Malden, and higher at Malden than at Vostok and Millennium (Fig 3J and S3 Table). quantum yield did not differ across islands for Halimeda (Fig 3D and S3 Table), and was sig- nificantly higher at Malden than at Vostok for Avrainvillea (Fig 3F and S3 Table). Maximum quantum yield of the coral Pocillopora was significantly higher at Malden than at the southern islands, which did not differ from each other (Fig 3H and S3 Table). In Monti- pora, maximum quantum yield was significantly higher at Starbuck than at Malden, and higher at Malden than at Vostok and Millennium (Fig 3J and S3 Table). Maximum quantum yield Maximum quantum yield increased across the SLI with increasing proximity to the equator for all taxa, though the magnitude of change varied by genus. Porolithon maximum quantum yield was up to 65% higher at Malden than at the southern islands (Fig 3B), where yield did not differ significantly from Vostok and Millennium to Starbuck (S3 Table). Maximum 10 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Photosynthetic pigments Porolithon and coral pigment concentrations were normalized to surface area, and rates are expressed as μg cm-2. Halimeda and Avrainvillea pigment concentrations were normalized to fresh weight, thus units for those taxa are μg mg-2. Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b) https://doi.org/10.1371/journal.pone.0228448.g004 Photosynthetic pigments Photosynthetic pigment concentrations generally followed the predicted pattern of increasing with proximity to the equator (Fig 4 and S3 Table). Chl a concentrations tended to increase from south to north for all taxa but Porolithon and Halimeda. However, in Pocillopora, chl a concentrations were lower than expected at Starbuck, but higher than expected at Millennium (Fig 4G). In Montipora, chl a concentrations increased by 115–198% from Flint to Malden (Fig 4I). Carotenoids showed little pattern across taxa or islands due to high levels of variation among individuals (Fig 4). Phycobilin pigment concentrations, including allophycocyanin, phycocyanin, and phycoer- ythrin of the coralline red alga Porolithon were variable across the SLI (Fig 5), with island-spe- cific significant differences for phycoerythrin only (S3 Table). Phycoerythrin concentrations increased by 150% from Flint to Malden (Fig 5). Though there were similar trends for phyco- cyanin, including a 185% increase from Flint to Malden, these differences were not significant, likely due to high variability among individuals within an island. 11 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Upwelling effects on coral and algal ecophysiology Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b) Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and (i-j) Montipora aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and missing bars indicate where a given species was not present at an island. Significant differences between islands were determined by Tukey’s HSD, different letters represent significant differences at p < 0.05. Porolithon and coral pigment concentrations were normalized to surface area, and rates are expressed as μg cm-2. Halimeda and Avrainvillea pigment concentrations were normalized to fresh weight, thus units for those taxa are μg mg-2. https://doi.org/10.1371/journal.pone.0228448.g004 Upwelling effects on coral and algal ecophysiology Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b) Fig 4. Photosynthetic pigments of corals and algae. Box plots of chl a and carotenoid concentrations in (a-b) Porolithon sp., (c-d) Halimeda sp., (e-f) Avrainvillea amadelpha, (g-h) Pocillopora meandrina, and (i-j) Montipora aequituberculata. Islands are ordered from south to north across the x-axis (from left to right), and missing bars indicate where a given species was not present at an island. Significant differences between islands were determined by Tukey’s HSD, different letters represent significant differences at p < 0.05. Discussion The isolated and remote SLI represent a unique study system to evaluate how variability in exposure to equatorial upwelling, and thus availability of inorganic nutrients, influences coral and algal ecophysiology. Results of our in situ measurements, discrete water samples, and remote sensing of surface chl a illustrate that the SLI are increasingly exposed to high levels of surface primary production and inorganic nutrients with proximity to the equator. We hypothesized that the ecophysiology of benthic autotrophs (i.e., metabolism, maximum PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 12 / 22 Upwelling effects on coral and algal ecophysiology Fig 5. Phycobilin pigment concentrations. Mean (± SE) phycobilin pigment concentrations normalized to surface area for the crustose coralline alga Porolithon sp. across the Southern Line Islands. Islands are ordered from south to north. Allophycocyanin (light pink), phycocyanin (pink), and phycoerythrin (dark pink) were measured on 2 subsamples of each individual (n = 4) per island. Data points are means of subsamples per individual. Significant differences in pigment concentrations across islands were determined for each pigment with Tukey’s HSD. Different letters represent significant differences at p < 0.05. https://doi.org/10.1371/journal.pone.0228448.g005 Fig 5. Phycobilin pigment concentrations. Mean (± SE) phycobilin pigment concentrations normalized to surface area for the crustose coralline alga Porolithon sp. across the Southern Line Islands. Islands are ordered from south to north. Allophycocyanin (light pink), phycocyanin (pink), and phycoerythrin (dark pink) were measured on 2 subsamples of each individual (n = 4) per island. Data points are means of subsamples per individual. Significant differences in pigment concentrations across islands were determined for each pigment with Tukey’s HSD. Different letters represent significant differences at p < 0.05. https://doi.org/10.1371/journal.pone.0228448.g005 https://doi.org/10.1371/journal.pone.0228448.g005 quantum yield, pigment concentrations) would reflect this pattern of nutrient enrichment. Although in some instances photophysiology was enhanced at the more nutrient-enriched islands, the overall patterns were variable across taxa and did not indicate a clear trend of phys- iological enhancement. The lack of a clear relationship between exposure to upwelling and the ecophysiology of pri- mary producers is notable because greater availability of inorganic nutrients generally has a predictable, positive effect on autotrophs. While our findings corroborate well-established pat- terns of increasing surface ocean primary production near the equator [10,56,57], they indicate that provisioning of nutrients from equatorial upwelling does not necessarily enhance auto- troph physiology on the benthos, as we observed in the SLI. Discussion The ambiguous relationship between nutrient availability and ecophysiology suggests that other abiotic or biotic factors may be contributing to benthic biological processes in the central Pacific. These results indi- cate that nutrients may not be the primary drivers of benthic primary production on some coral reefs. Environmental parameters The equatorial islands of Starbuck and Malden were cooler and more nutrient-rich than the southern islands of Flint, Vostok, and Millennium, which became incrementally warmer and depleted in nutrients with proximity to the equator. Our measurements of environmental con- ditions were constrained to discrete samples, short-duration in situ measurements, and short- term remote sensing of chl a. With these data, alone, our scope for interpretation is temporally limited. However, our findings corroborate a plethora of existing long-term data sets and a PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 13 / 22 Upwelling effects on coral and algal ecophysiology rich history of oceanographic research that, collectively, improve our understanding of long- term upwelling patterns in the remote equatorial Pacific. Decades of research have character- ized equatorial upwelling as a persistent and predictable oceanographic feature driven by the Equatorial Undercurrent (EUC) [7,10]. The EUC delivers cool, nutrient rich water to the ocean surface on a west-to-east pathway [33], and causes net accumulation and temporal per- sistence of high nitrate and phosphate around the equator [34]. Higher DIN and DIP fuels phytoplankton growth and, thus, increases surface chl a concentrations. This trend is evident in our 4-month chl a data, and is even more conspicuous in the climatology from 2004–2015 [40]. Further, the same patterns have been described in numerous other studies in the same region [21,35,58]. The coupling of upwelling with oceanic production is well established, but much less is known about potential cascading effects on primary production of the benthos, particularly in coral reef habitats. Coral reefs are typically considered to exist in oligotrophic waters that are often likened to marine deserts [24], and on average DIN and DIP concentrations in the Caribbean and Pacific are estimated to be ~0.4 μM and ~0.2 μM, respectively [59]. However, our findings demon- strate that nutrient concentrations on some reefs can well exceed these global averages. At the most equatorial site, Malden, the conditions are more akin to the nutrient regime of a temper- ate kelp forest in southern California, where nitrate concentrations can increase up to 5 μM during wind-driven upwelling season [60], than the archetypal oligotrophic coral reef. Environmental parameters Similar patterns of natural enrichment have been documented in other upwelling-influenced coral reef systems, such as on Conch Reef in the Florida Keys, where episodic pulses driven by inter- nal tidal bores can increase ambient DIN to 1–4 μM and DIP to 0.1–0.3 μM [61]. Elevated lev- els of DIN and DIP often have negative implications for overall community structure on coral reefs, because excess nutrients can fuel the dominance of fleshy macroalgae [4]. Yet, the ben- thos on nutrient-enriched islands of Malden and Starbuck is dominated by reef-building corals and calcifying algae, where reef builders cover up to ~80% of the benthos (as opposed to fleshy and turf algae that account for ~15%) [37]. Our findings indicate that coral reef ecosystems in the central Pacific can thrive in nutrient conditions typically considered detrimental to reef- building corals. Indeed, coral cover is also high on Jarvis Island, which is just north of Malden and at the epicenter of equatorial upwelling, where DIN concentrations can naturally fluctuate up to 16 μmol [62]. Though inorganic nutrients can influence benthic dynamics, the relative dominance of reef-builders on these islands likely is also linked to high biomass of herbivorous fishes [36]. Given the persistence of an extreme gradient in inorganic nutrient concentrations across coral reefs of the central Pacific, there is a clear need to explore the role of naturally nutrient variability in shaping biological processes and the resulting implications for the asso- ciated benthic reef communities. Metabolism Evaluating the relationship between autotroph metabolism and inorganic nutrient availability provides insight into the potential effects of equatorial upwelling on benthic biological pro- cesses. Metabolic rates indicate the physiological status of corals and algae, and photosynthetic rates are directly proportional to rates of primary production. Given the potential for nutrient availability to influence rates of primary production in microalgae [63] and macrophytes [64], we hypothesized that photosynthesis of corals and algae would generally increase across the SLI corresponding to DIN and DIP availability. However, our measurements of coral and algal metabolism did not show a strong pattern of enhancement across the SLI, except in the CCA, Porolithon. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 14 / 22 Upwelling effects on coral and algal ecophysiology Porolithon metabolic rates generally tracked with the nutrient gradient. Of the benthic macroalgae, metabolism of the green alga, Avrainvillea, was higher at the site closer to the equator, while Halimeda was largely unchanged across sites. These data should be interpreted cautiously as each macroalgal genera was found at only 2 and 3 of the 5 islands, respectively. Metabolic rates of the corals, Pocillopora and Montipora, were variable across all islands with no particular pattern. The lack of a clear metabolic response to nutrient enrichment across all taxa in this study may indicate that other environmental factors influence metabolism, or may be a result of high variability associated with the methodology. Further, gross metabolic rates may not fully capture the capacity for autotrophs to respond to nutrient availability at the time-scale of the present study. In contrast to photosynthetic efficiency (maximum quantum yield) and photosynthetic pig- ment content, which quantify the functioning of cellular light-harvesting machinery, meta- bolic rates constitute a net organismal response. Comparing and contrasting the metabolic responses of corals and macroalgae across the same gradient may thus be encumbered by inherent differences between strictly autotrophic algae and mixotrophic corals [65]. While algal metabolism represents cellular processes (e.g., respiration and photosynthesis) of only the autotrophic alga, coral metabolism is a net response of the coral holobiont, which encompasses the heterotrophic coral host, the autotrophic endosymbionts and the associated microbial community [66]. Estimating photosynthesis through oxygen production, particularly with cor- als, may be too coarse a metric to resolve finer-scale trends underlying genera-specific responses. Metabolism The physiological effects of nutrient enrichment on corals may thus be more effec- tively assessed by pigment content and maximum quantum yield [67]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Photosynthetic pigments Pigment concentrations in algae and corals are flexible and highly responsive to prevailing environmental conditions [73], particularly to irradiance and nutrient concentrations [74]. A positive photosynthetic pigment-nutrient relationship has been demonstrated in a suite of photosynthetic taxa and across a range of environmental conditions. This relationship gener- ally manifests as an increase in chl a and carotenoid concentrations in response to excess avail- ability of DIN and DIP [14,75]. The predictability of this relationship makes algal pigment content a useful metric in understanding the abiotic conditions of an ecosystem because pig- ments in coral reef algae [76] and corals [67,77] can be used as bioindicators for nutrient pollu- tion and water quality. Given the strength and consistency of this relationship, we expected that pigment concentrations would increase across the SLI with increasing proximity to the equator. In most taxa studied, the primary photosynthetic pigments, chl a and carotenoids, were highest at the most northern islands, with some variability across the middle islands of the archipelago. Chl a generally increased with greater exposure to nutrient availability in the green algae and the corals, and patterns of carotenoids mirrored those of chl a, but with more variability. Porolithon showed the least consistent pattern with respect to the primary pig- ments, but the accessory phycobilin pigments demonstrated the strongest response to the nutrient gradient. There was a positive, incremental increase in phycoerythrin and phycocya- nin concentrations at islands that corresponded to increasing availability of ambient DIN and DIP, though this trend was significant only for phycoerythrin. The phycobilin complexes in crustose corallines, and other red algae, act as accessory pigments that broaden the spectrum of light available for photosynthesis [78]. Phycobilins are also particularly responsive to DIN, in part because algae can use phycobilin pigment complexes to store excess nitrogen [54]. Nitrogen storage for luxury consumption by algae is an ecological adaptation to nutrient limi- tation and provides a source of DIN that can be later metabolized to fuel growth when nutri- ents are limiting [79]. The increase in Porolithon phycobilins with greater exposure to inorganic nutrient availability may indicate that these algae can store the excess DIN delivered by equatorial upwelling in pigment-protein complexes. Maximum quantum yield Maximum quantum yield is a direct measure of the functioning of chl a pigment molecules, and provides a proxy for the photosynthetic efficiency of autotrophs. At the organismal scale, a higher photosynthetic efficiency indicates that, when all reaction centers are engaged, the auto- troph can fix more carbon per quanta through the light-dependent reactions of photosynthesis [68]. Because photosynthetic efficiency is directly coupled with pigment content, and there is a predictable and well-established relationship between pigment content and nutrients, we hypothesized that maximum quantum yield would increase with increasing availability of DIN and DIP. Maximum quantum yield followed the predicted latitudinal pattern for corals and algae across the SLI, though the magnitude of response was taxon-specific. Fluorescence measurements of photosystem II provide valuable insight into the photosyn- thetic performance of autotrophs and their response to varying nutrient regimes [69,70]. Indeed, photosynthetic efficiency of coral and algal taxa was highest at most northern islands sampled, and there was an overall trend of increasing photosynthetic efficiency with increasing exposure to equatorial upwelling. These results indicate that long-term exposure to ample DIN and DIP availability may increase the number, function, or efficiency of photosystem reaction centers. Our data corroborate a wide body of work demonstrating that DIN and DIP availabil- ity directly impacts the ability of algae to build and maintain properly functioning photosystem complexes that are essential for photosynthesis [20]. Enhanced photosynthetic efficiency could have ecological ramifications because efficiency is positively correlated with primary produc- tion [71,72]. Thus, greater exposure to inorganic nutrient availability may indicate higher potential for primary production and a potential increase in the capacity of benthic autotrophs to channel more energy to higher trophic levels. However, future experiments should confirm the consistency of these patterns by incorporating larger sample sizes and greater replication across natural nutrient regimes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 15 / 22 Upwelling effects on coral and algal ecophysiology Photosynthetic pigments The increase in some of the photosyn- thetic pigments with increased availability of DIN and DIP indicates that equatorial upwelling could fuel primary production in benthic autotrophs by facilitating the development of more pigments, which supports greater capacity to harvest light and generate energy. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Effects of equatorial upwelling on ecophysiology The lack of a strong relationship between benthic autotroph ecophysiology and nutrient avail- ability in the SLI is an unexpected result, and indicates that DIN and DIP may not have been the only limiting factor for metabolism and photophysiology in the taxa studied. Other poten- tial abiotic drivers of autotroph ecophysiology across islands could include temperature, light, and other nutrients. For many algae, maximum photosynthesis occurs over a range of temper- atures, thus the 1.5˚C change in temperature from south to north did not likely contribute to metabolic rates [80]. Similarly, light can be ruled out as a confounding factor because there was no significant difference in the amount of available light across the SLI, despite the 4˚ change in latitude. Biotic factors could also have influenced ecophysiology as well, such as prior damage from grazing or exposure to competition or disease, though we attempted to minimize these effects by selecting individuals that appeared healthy. The limited temporal scope of both our physiological and environmental measurements are also important to consider. Due to the logistical constraints associated with field work at remote islands, physiological measurements could only be taken once, and thus represent a PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 16 / 22 Upwelling effects on coral and algal ecophysiology snapshot of ecophysiology at one point in time. The inorganic nutrient gradient resulting from upwelling is persistent through time [40], but could be magnified at different times of the year, depending on the strength of prevailing winds. Our environmental measurements are likewise temporally limited, though we have bolstered our discrete samples with historical perspective and satellite data that encompasses several months around the expedition. Furthermore, we quantified only DIN and DIP in our water samples, using discrete samples at one time point over two different expeditions. Thus, we cannot conclude what other nutrients may have influ- enced autotroph ecophysiology. We can speculate that iron limitation of photosynthesis may have contributed to some of the observed patterns in photosynthesis (and photophysiology). In areas of high nitrate and low surface chl a, iron availability can limit photosynthesis and growth of phytoplankton [81]. However, our satellite data illustrate that there was high sur- face-ocean production at the equatorial islands, and, thus, do not provide evidence that phyto- plankton primary production was iron limited. Effects of equatorial upwelling on ecophysiology Future work should explore the relationship of coral and algal ecophysiology in response to equatorial upwelling in more detail, the potential for seasonal responses to inorganic nutrient availability, and the role of additional abiotic fac- tors in constraining benthic photosynthesis in nutrient replete conditions. These data provide an interesting perspective on the role of inorganic nutrients and upwell- ing in shaping benthic biological processes. Despite the potential for nutrient availability to elicit strong photosynthetic responses, we found only moderate, and sometimes inconsistent, physiological enhancement with increasing exposure to equatorial upwelling in the taxa stud- ied. This lack of a clear pattern contrasts with the conspicuous gradient in surface ocean pro- ductivity and inorganic nutrient availability. Our findings agree with one other study to explore ecophysiology of coral reef benthic taxa in response to upwelling. Eidens et al. (2014) similarly documented substantial variation in benthic autotroph metabolic responses across an upwelling gradient in Colombia [82]. Using comparable methodologies, they found that upwelling increased photosynthetic rates in turf algae, decreased rates in corals and inconsis- tently affected photosynthesis in macroalgae and coralline algae [82]. Though surface ocean productivity is key in the biophysical coupling of large-scale oceanography with benthic com- munities, it does not appear to be directly linked to patterns in benthic autotroph ecophysiol- ogy, at least in short-term physiological assessments. Future experiments should incorporate growth rates, net community metabolism, and other more integrated measures of benthic pro- duction to better understand the relationship between surface ocean production and benthic ecosystem processes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Conclusions Here we document, for the first time, the ecophysiology of abundant benthic autotrophs on coral reefs of the SLI in the remote central Pacific. We illustrate that the SLI span a conspicu- ous gradient of increasing surface ocean production and inorganic nutrient availability (DIN, DIP) that indicate the SLI become increasingly exposed to upwelling with decreasing distance to the equator (i.e., latitude). Though we predicted that coral and algal metabolism (photosyn- thesis, respiration) and photophysiology (maximum quantum yield, photosynthetic pigments) would increase with greater exposure to upwelling, we found evidence of this in only some parameters. The CCA, Porolithon, generally responded positively with greater exposure to nutrient availability, particularly with respect to metabolism and phycobilin pigment content. The green algae and corals also showed some indications of photophysiological enhancement, but with more variability. The lack of a strong and consistent physiological response in all taxa across this upwelling gradient indicates that other factors may be limiting photosynthesis and photophysiology of benthic autotrophs in the SLI. PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 17 / 22 Upwelling effects on coral and algal ecophysiology The effects of nutrients on organismal physiology may have broader ecosystem implica- tions, if physiological enhancement (or lack thereof) is indicative of primary production. Our results would then suggest that benthic primary production may not always track with patterns in surface ocean productivity. A limited number of studies on upwelling in coral reefs indicate that inorganic nutrient delivery via upwelling or internal tides can increase surface ocean pro- ductivity of oceanic islands and atolls [32], biomass of fish [2], and heterotrophy in corals [40]. However, the link between increased surface productivity due to upwelling and benthic pro- ductivity remains unclear. Unraveling the role of natural nutrient sources in shaping coral reefs may be one key to improving our understanding of how coral reef community structure and function will change as humans continue to alter natural nutrient landscapes. Supporting information S1 Table. Coordinates, land area, and benthic taxa sampled at the five Southern Line Islands in the Republic of Kiribati. Islands closest to the equator have higher inorganic nutri- ent concentrations due to equatorial upwelling. In all tables and figures, islands are listed from south to north in order of increasing proximity to the equator. (DOCX) S3 Table. ANOVA table of island effects on ecophysiology by genera. Significance at p < 0.05 is noted in bold. (DOCX) S4 Table. Raw physiological data of corals and algae across the Southern Line Islands. Raw physiological data of coral and algae replicates from incubations, quantum yield measure- ments, and pigment content. S4 Table. Raw physiological data of corals and algae across the Southern Line Islands. Raw physiological data of coral and algae replicates from incubations, quantum yield measure- ments, and pigment content. (XLSX) Acknowledgments We are grateful to the Environment and Conservation Division of the Republic of Kiribati for allowing us to conduct research in the Southern Line Islands. We thank the captain and crew of the M/Y Hanse Explorer for logistical support and field assist. We also thank U. Abeysekera, A. Emanuel, E. Jacobs, E. Juhlin, J. Le, and A. Vawter for assistance in the lab, as well as A. Andersson, J. Kohn, and J. Leichter for feedback on an earlier draft of the manuscript. We are grateful to the support and assistance of F. Rohwer during the cruise and for his contributions to an earlier draft of the manuscript. We thank the Moore Family Foundation, the Gordon and Betty Moore Foundation, the Scripps family, and the anonymous donors who helped make this research possible with their financial support. Author Contributions Conceptualization: Maggie D. Johnson, Michael D. Fox, Emily L. A. Kelly, Jennifer E. Smith. Data curation: Maggie D. Johnson, Michael D. Fox. Formal analysis: Maggie D. Johnson, Michael D. Fox. Funding acquisition: Stuart A. Sandin, Jennifer E. Smith. 18 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0228448 February 4, 2020 Upwelling effects on coral and algal ecophysiology Investigation: Maggie D. Johnson, Michael D. Fox, Emily L. A. Kelly, Brian J. Zgliczynski, Stu- art A. Sandin, Jennifer E. Smith. Methodology: Maggie D. Johnson, Michael D. Fox, Jennifer E. Smith. 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https://openalex.org/W1974083396	https://www.ccsenet.org/journal/index.php/mas/article/download/46790/25232/	English	null	Empirical Study of the Psychology of the Winner (On the Example of the Republic of Mordovia Athletes)	Modern applied science	2,015	cc-by	6,040	Modern Applied Science; Vol. 9, No. 5; 2015 ISSN 1913-1844 E-ISSN 1913-1852 Published by Canadian Center of Science and Education Modern Applied Science; Vol. 9, No. 5; 2015 ISSN 1913-1844 E-ISSN 1913-1852 Published by Canadian Center of Science and Education Modern Applied Science; Vol. 9, No. 5; 2015 ISSN 1913-1844 E-ISSN 1913-1852 Published by Canadian Center of Science and Education The study receives funds from the Ministry of Education and Science of the Russian Federation in the framework of the project 2.1.2. Solution to complex problems of formation of professional competence of the teacher and psychologist in continuing education programs of the Institute of Strategic Development for 2012-2016. The study receives funds from the Ministry of Education and Science of the Russian Federation in the framework of the project 2.1.2. Solution to complex problems of formation of professional competence of the teacher and psychologist in continuing education programs of the Institute of Strategic Development for 2012-2016. Abstract The paper describes the results of empirical studies on the psychological traits of the winning athletes of international and European tournaments, with the title of "Honoured Master of Sports" and "International Master of Sports" in sports such as athletics and combat sports. The study used the following psychological and acmeological diagnostic methods: author's questionnaire named "Psychology of the winner", which includes questions relating to the biographical information of athletes, intellectual and personal traits, motivation and value constructs, emotional and volitional components of personality, etc..; the "Mini-Mult" questionnaire (a shortened version of the Minnesota multidimensional personality test, MMPI), which assesses personal traits (tendency to asthenoneurotic, sociopathic, anxiety and mistrust, hypertemic options of personal development, touchiness, susceptibility to affective reactions doubt, emotional detachment degree, complexity of social interaction, activity, and excitability); the Cattell test 16 PF (Form A), which measures the traits and properties of the human person as character, temperament, intelligence, emotional, volitional, moral, communication, and other features. In addition, there were interviews conducted in a free form, as well as handwriting analysis was applied to signatures of the winning athletes. The authors justified the position that outstanding winning athletes are the acmeological objects. The authors develop a systematic description of the psychological traits of the winning athletes. The paper demonstrates the psychological traits of the winning athletes specialising in athletics and combat sports. The paper formulates conclusions and the urgency of further study of the psychology of the winner. Keywords: psychological training in sports, psychology of the winner, sport psychology, psychology of professional, winning athletes, acmeology, acmeological object Received: December 24, 2014 Accepted: December 29, 2014 Online Published: March 25, 2015 doi:10.5539/mas.v9n5p211 URL: http://dx.doi.org/10.5539/mas.v9n5p211 Received: December 24, 2014 Accepted: December 29, 2014 Online Published doi:10.5539/mas.v9n5p211 URL: http://dx.doi.org/10.5539/mas.v9n5p211 Empirical Study of the Psychology of the Winner (On the Example of the Republic of Mordovia Athletes) Diana Valerievna Zhuina1, Vladimir Georgievich Zazykin2 & Ludmila Gennadyevna Maydokina3 1 Department of Psychology and defectology, Department of Applied Psychology and Special Mordovian State Pedagogical Institute Named after M. E. Evseviev, Saransk, Russia Diana Valerievna Zhuina1, Vladimir Georgievich Zazykin2 & Ludmila Gennadyevna Maydokina3 Department of Psychology and defectology, Department of Applied Psychology and Special Mordo Pedagogical Institute Named after M. E. Evseviev, Saransk, Russia Department of Occupational Psychology of professional activity Institution Russian Academy o Economy and Public Administration under the President of the Russian Federation, Moscow, Russia 3 Faculty of Physical Education, Department of Theory and methods of physical culture and sports Mordovian State Pedagogical Institute Named after M. E. Evseviev, Saransk, Russia Correspondence: Diana Valerievna Zhuina, Department of Psychology and defectology, Department of Applied Psychology and Special Mordovian State Pedagogical Institute Named after M. E. Evseviev, Saransk, Russia. Tel: 7-891-7693-6031. Accepted: December 29, 2014 Online Published: March 25, 2015 URL: http://dx.doi.org/10.5539/mas.v9n5p211 1. Introduction Priority in the state policy of the Russian Federation is paid to the development of professional sports. Across the country, including in the Republic of Mordovia, there are conditions for the effective training of professional athletes in the chosen sport. In particular, the Government of the Republic of Mordovia signed an agreement on cooperation with the Ministry of Sport of the Russian Federation, which pursued clarification the powers and responsibilities in the implementation of the Federal Target Program "Development of physical culture and sports in Russia for 2006-2015" and had a positive impact on efficient implementation of state policy in physical culture and sports (The Federal Target Program, 2006). 211 Modern Applied Science Vol. 9, No. 5; 2015 www.ccsenet.org/mas According to statistics, as of January 01, 2014, the Republic of Mordovia has 42 sports schools, including 36 youth sports schools and 6 specialized youth sports school of Olympic reserve included in the list of the Ministry of Sports of the Russian Federation, as well as the Olympic Training Centre Republic of Mordovia for Race Walking named after V. M. Chegin, the School of highest sports art, the Republican Centre for Sports Training for Teams of the Republic of Mordovia, which involved a total of more than 29,300 people. These figures indicate the coordinated work of the regional authorities to implement the Strategy for the Development of Physical Culture and Sports in the Russian Federation for the period up to 2020 approved by the Federal Government as of August 07 2009 (Regulation of the Government of the Russian Federation, 2009). The Republic of Mordovia has all the required conditions for psycho-acmeological, medical and methodological support for training highly skilled athletes able to achieve high sports results. Over the last 12 years, the Republic of Mordovia was represented in the national team of the Russian Federation by athletes involved in athletics and combat sports. Among all, during the 2004 Olympic Games in Athens, Mordvinian athletes won 1 gold and 1 silver medal, during the 2008 Olympics in Beijing, athletes were awarded by 2 gold and 1 bronze medals, during the 2012 competitions in London, the treasury of the Russian Federation got 2 gold, 1 silver, and 1 bronze medals from the Mordvinian athletes. In addition, the athletes of Olympic Training Centre in race walking named after V. M. Chegin and Specialized youth sports school of Olympic reserve named after A. V. 1. Introduction Mishin gained high achievements at the World Championships and European tournaments. Victories of Mordovia athletes at the Olympics 2004-2012 demonstrated the dynamics of sports achievements serving as the basis for a study aimed at investigating the psychology of winning athletes. 2. Literature Review Approach to the problem of the winner's psychology is impossible without the analysis of the theoretical and methodological foundations of the problems on establishing a professional, which is reflected in the works of local teachers and psychologists, such as E. F. Zeer, E. A. Klimov, L. M. Mitin, N. S. Pryazhnikov, S. A.Chistyakov et al. The most complete reflection of the essential individual traits of a professional, the regularities of formation and development of such a structural component of the personality as career orientation, can be found in the psychological and acmeological scientific theories and concepts, such as: psychological theory of personality (Antsyferova, L. I., Myasischev, V. N., Platonov et al.); psychological theories of abilities (Druzhinin, V. N., Il’in, E. P., Teplov et al.); general psychological concepts of the developing individual's potential (Altschuller, G. S., Guilford, G., Jackson, F., Markov, V. N., Petlenko et al.), the concept of researching careers (I. P. Lotova, A. К. Markov, et al.); theory of career progression (Egorshin, A. P., Mall, E. G., Pryazhnikov, N. S., Mogilevkin et al.), and career strategy (Romanov, V. L.); theory of professional orientation of a person (Gonobolin, F. N., Dodonov, V. I., Zabrodin, Yu. M., Sosnowsky, B. A., , Klimov, E. A, Kuzmina, N. V., Krutetsky, V. A., Lomov, B. F., Markova, A. К.); theory, revealing the features of career orientation of a person (Zhdanovich, A. A., Kiselyova, E. V., Ovseychik, N. V., Shane, E.); theory of career orientation (Kanamatova, A. K., Mokritskaya, A. O., Shchelokova, E. G,) (Mokritskaya, A. O., 2011). After analysing all the available research in the field of psychology and acmeology, one can conclude that at the initial stage, psychological and acmeological support will be required in the form of the structure of sustained support for becoming a professional. A promising option of the psychological support may be the psycho-acmeological model of career orientation for the individual, developed by D. V. Zhuina, Comprising of 5 sections (Zhuina, D. V., 2014): 1) theoretical and methodological; 2) target; 3) meaningful; 4) organizational and developmental; and 5) assessment and effective sections. In order to confirm the scientific validity of the developed psycho-acmeological model of career orientation for the individual within the theoretical and methodological section, the main approaches, theories, concepts and methodological principles forming the basis of the model, are covered. 2. Literature Review The target section of psycho-acmeological developed model has a set goal to optimize the process of formation and development of career orientation for the individual. To achieve this goal, it was necessary to define the very essence of the concept of "career orientation of the individual". In order to do this in the meaningful section, we defined the main components of the career orientation of the individual with their definition. Thus, analysing the psychological and acmeological literature on the issue of the research, we identified a number of studies on the definition of value-semantic (Shchelokova, E. G.) and socio-psychological (Mokritskaya, A. O.) content of the career orientation concept (Mokritskaya, A. O., 2011), (Schelokova, E. G., 2011). Nevertheless, we believe that this phenomenon is poorly understood in terms of ontology, content, and structure of the concept. The general concept of career orientation for the individual is not developed. There is no theoretical model of psychological and acmeological career orientation; 212 Vol. 9, No. 5; 2015 Modern Applied Science www.ccsenet.org/mas the psycho-acmeological factors of managers' career are unexplored; methodological tools are not developed; the main components of the career orientation are not described. the psycho-acmeological factors of managers' career are unexplored; methodological tools are not developed; the main components of the career orientation are not described. We have developed a new definition of career orientation for the individual, by which we mean a complex structural component of the overall orientation of the person, representing a multidimensional process orientation and meaning to human activities in the professional field, integrative traits including stable career image, formed career orientation and career potential, stable career motivation, mediated by the professionalization process. The developing organizational section represents peculiarities of psycho-acmeological support of the career orientation for the individual in terms of activity of the Acmeological centre, being the innovative structure, which is part of the Centre for collective use "Mordovia basic centre of teacher education" (the city of Saransk). The purpose of the Centre is to provide sociocultural and psychological-pedagogical support to professional and personal development of subjects in different fields, and promotion of human resources in the region. 3. Method Currently, the psychological science pays sufficient attention to research on the study of the psychological training of athletes during the training sessions and competitive activities (Maydokina, L. G., Kudashkina, O. V., 2014, 2011). Psychological factors in the training process and training the athlete for competition are great importance both in the papers by the experts in the field of sport theory (V. N. Platonov, N. G. Ozolin, Zh. K. Kholodov, L. P. Matveev, V. S. Kuznetsov et al.), and in the works of sports psychologists (A. V. Alekseev, G. D. Babushkin, G. D. Giessen, G. D. Gorbunov, V. N. Nepopalov, A. Ts. Puni, A. V. Rodionov, G. I. Savenkov, V. F. Sopov, O. A. Chernikova, et al.) (E. P. Il’in, 2011; V. A. Kurashvili, 2008; V. F. Sopov, 2010). А. V. Rodionov believes that the psychological training of an athlete means an organized, controlled process of realization of athlete's potential mental capabilities in the objective results adequate to these capabilities (Psychology of Physical Culture and Sports, 2010). G. D. Gorbunov defines the psychological training as the process of formation and improvement of significant sports personality traits and their underlying mental qualities. The task of psychological training in everyday activities becomes more narrow and specific - to create athlete's mental state favourable for the success of training and competition (G. D. Gorbunov, 2012). According to N. N. Setyaeva, the psychological training helps to maximize the use of the athlete's potential to improve the efficiency and reliability of their competitive activities. The effectiveness and reliability of competitive activity of an athlete is determined by their not only diverse preparedness, but also readiness to a specific competition (N. N. Setyaeva, A. V. Fursov, 2010). G. D. Gorbunov indicates that the psychological training is primarily an educational process aimed at personal development by establishing an appropriate system of relationships. This allows transferring fragile mental state into a stable one, i. e. into an individual trait. In this case, the psychological training of the athlete to a long training process is carried out, firstly, due to the continuous development and improvement of the grounds for sports training and, secondly, by creating favourable attitudes towards various aspects of the training process (G. D. Gorbunov, 2012). According to V. R. 2. Literature Review In accordance with the purpose, one of the main tasks of the Centre is to support complex formation and development of career orientation for subjects of educational space at pedagogical university, scientific and methodological support of career guidance and employment assistance, career planning and development (development, testing and introduction of modern professional orientation, socio-adaptation technologies and planning and career development technologies) (Zhuina, D. V., 2011). 3. Method According to the author, it is not possible to carry out psychological training without the knowledge of the athlete's individuality, without regard certain psychological qualities (congenital or acquired during life) possessed by the athlete. 3. The general psychological training of an athlete as a basis of formation of personality in the process of sports training. The structure of this type of training is based on moral, volitional, emotional, intra-group (communicative) and intellectual types of training. Each of these types solves specific problems. 3. The general psychological training of an athlete as a basis of formation of personality in the process of sports training. The structure of this type of training is based on moral, volitional, emotional, intra-group (communicative) and intellectual types of training. Each of these types solves specific problems. 4. Psychological training for specific training as a system of psychological and methodological activities related to the following: 4. Psychological training for specific training as a system of psychological and methodological activities related to the following: a) the formation of the target goal for the given training; a) the formation of the target goal for the given training; b) the organization of the athlete's attention, which is aimed on physical actions performed by the athlete with the program of consciousness orientation; c) the psychological analysis of the performed training. 5. Precompetitive psychological training of the athlete as a system of measures for the mobilization of the athlete's readiness to a specific competition. 6. Pre-start psychological training is aimed at organizing the life and activity of the athlete, their attention to the pre-start period for competitive activity, the formation of a competitive spirit, and setting to achieve high results. 7. Directly competitive psychological training associated with the development of the athlete's ability to think tactically and operationally in the highly competitive struggle, the ability to regulate arbitrarily their emotional states, reactions, and relationships. 8. Post-competition psychological training aimed at the analysis of successful and unsuccessful competitive activity by individual parties for controlling mental and motor actions, for solving challenges and super tasks assigned to the athlete (G. I. Savenkov, 2006). The analysis of the problem of psychological training in sports demonstrated that science developed theoretical and methodological basis for the psychological support of an athlete. However, the problems of the winner’s psychology are understudied. 3. Method Malkin, the psychological work with athletes at the initial stage of sports training involves the formation of a stable motivation in sports, building self-esteem, forming an optimal aspiration level, goals for manifesting volitional effort during physical stress, forming the ability to concentrate and develop operative memory (V. R. Malkin, 2008). We shall agree with V. N. Nepopalov and A. A. Magomedov that psychological training is an important component for athlete winning the competition. The work of coaches shall study and take into account the individual psychological traits of athletes and environment. In addition, coaches shall maintain contact with the environment in their work. The ability of an athlete to overcome timely the aroused difficulties is largely due to the high development of the athlete in all aspects of their psyche, especially thinking, memory, will, attention, and imagination. Therefore, the continuous improvement of these traits shall be the goal of psychological support (V. N. Nepopalov, A. Magomedov, 2009). 213 Vol. 9, No. 5; 2015 Modern Applied Science www.ccsenet.org/mas G. I. Savenkov includes the following elements into the structure of psychological training of an athlete: G. I. Savenkov includes the following elements into the structure of psychological training of an athlete: 1. Psychological passport of the chosen sport, the system requirements for the psyche and personality of an athlete engaged in the sport determining the achievement of high sports results. This structure element is characterized by the traits of personality and psyche, to which one shall strive in the process of sports perfection. 1. Psychological passport of the chosen sport, the system requirements for the psyche and personality of an athlete engaged in the sport determining the achievement of high sports results. This structure element is characterized by the traits of personality and psyche, to which one shall strive in the process of sports perfection. 2. Psychological traits of an athlete as a factor in assessing mental qualities and personality traits exhibited by the athlete. According to the author, it is not possible to carry out psychological training without the knowledge of the athlete's individuality, without regard certain psychological qualities (congenital or acquired during life) possessed by the athlete. 2. Psychological traits of an athlete as a factor in assessing mental qualities and personality traits exhibited by the athlete. 3. Method 8) List the professionally important qualities of a coach, contributing to achievement of high sports 19) List the qualities of a coach, preventing achievement of high sports results. 20) Name the conditions, which, in your opinion, contribute to the achievement of certain results in sports activities 20) Name the conditions, which, in your opinion, contribute to the achievement of certain results in sports activities 3. Method 8) Rate from 1 to 10 the value of psychological training of a highly skilled athlete. 9) What is the content of psychological training of the athlete in the sports training system? 10) Is there anybody to carry targeted psychological training for you in the sports training system? 11) Name the content of psychological training of the athlete in the sports training system. 12) List the qualities of personality, character traits that, in your opinion, are in demand in your sport. 13) List the qualities that, in your opinion, contribute to achieving high results in your sport. 14) List the ways of development the psychological qualities required for your sport. 15) Suggest psychological description of the winner (3 5 sentences) 3) Experience in sports activities. 4) What was the first step of your sports career? 5) Who influenced on, helped the development of your sports career? 6) Do you know what does the psychological training of the athlete imply? 7) What role does the psychological training of the athlete play in the sports training system? 8) Rate from 1 to 10 the value of psychological training of a highly skilled athlete. 9) What is the content of psychological training of the athlete in the sports training system? 10) Is there anybody to carry targeted psychological training for you in the sports training system? 11) Name the content of psychological training of the athlete in the sports training system. 12) List the qualities of personality, character traits that, in your opinion, are in demand in your sport. 13) List the qualities that, in your opinion, contribute to achieving high results in your sport. 14) List the ways of development the psychological qualities required for your sport. 15) Suggest psychological description of the winner (3-5 sentences) 16) Are you satisfied with the terms of training sessions? 17) Rate the satisfaction degree with the school classes from 1 to 5. 18) List the professionally important qualities of a coach, contributing to achievement of high sports results. 19) List the qualities of a coach, preventing achievement of high sports results. 20) Name the conditions, which, in your opinion, contribute to the achievement of certain results in spor activities 7) Rate the satisfaction degree with the school classes from 1 to 5. 3. Method The analysis of the problem of psychological training in sports demonstrated that science developed theoretical and methodological basis for the psychological support of an athlete. However, the problems of the winner’s psychology are understudied. In order to answer the question on what is characteristic of the winner's psychology, one shall conduct a psycho-acmeological study of the winner's psychology. These studies were initiated based scientific and practical innovation "Acmeological centre" at Mordovia State Pedagogical Institute. The prerequisite of this study is the following: the traits of psychology of the winner can be determined by studying the psychological traits of those who are eligibly called the winners (A. A. Derkach, V. G. Zazykin, 2003). These are primarily the Heroes of Russia, heroes awarded by the Order of "Courage", and athletes-champions. An empirical study was carried out for this purpose in the form of psycho-acmeological survey of 15 winners of major international tournaments in athletics and martial arts with sports titles "Honoured Master of Sports", "International Master of Sports", and having solid sports experience from 14 to 25 years, indicating that their sports victories are not a matter of chance. Athletes-winners are acmeological subjects since they achieved outstanding results by achieving peaks in their personal and professional development. This results in the fact that such a sample is quite representative from acmeological point of view, as outstanding athletes in any sport comprise about five per cent of all those who practice it. The study of winning athletes' psychology used the following methods: author's questionnaire named "Psychology of the winner"; the questionnaire "Mini-Mult" (a shortened version of the Minnesota multidimensional personality test, MMPI); the Cattell test 16 PF (Form A). In addition, there were interviews conducted in a free form, as well as handwriting analysis was applied to signatures of the winning athletes (V. G. Zazykin, 2010). Particularly informative is the author's questionnaire named "Psychology of the winner", which includes the following questions: 1) Full name, age. 2) Sporting achievements, awards. 214 Vol. 9, No. 5; 2015 Modern Applied Science www.ccsenet.org/mas 3) Experience in sports activities. 4) What was the first step of your sports career? 5) Who influenced on, helped the development of your sports career? 6) Do you know what does the psychological training of the athlete imply? 7) What role does the psychological training of the athlete play in the sports training system? 4. Results In order to study personality traits and features, reflecting the relatively stable ways of human interaction with the world and oneself, the method of "The 16-factor Cattell test" (Form A) was applied. According to the data obtained in the analysis of test results, the following results were received (see Table 2). Table 2. Individual traits of the winning athlete winning by the 16-factor Cattell test Factor Combat athletes, % Track and field athletes, % “+" pole “-" pole “+" pole “-" pole A 0 2 (40%) 0 1 (10%) B 0 0 5 (50%) 0 C 0 0 0 0 E 2 (40%) 0 0 0 F 0 0 0 0 G 3 (60%) 0 3 (30%) 0 H 0 0 2 (20%) 0 I 0 3 (60%) 0 1 (10%) L 0 0 0 0 M 0 0 0 8 (80%) N 0 0 0 0 O 0 0 0 0 Q1 0 0 0 0 Q2 0 0 0 0 Q3 0 0 0 0 Q4 0 0 0 0 Table 2. Individual traits of the winning athlete winning by the 16-factor Cattell test dual traits of the winning athlete winning by the 16-factor Cattell test Table 2 based on the personality profile (positive profile meaning), demonstrates the predominance of the G factor (high super-ego) among combat athletes - 60% of the subjects. These people have strong character, honesty, perseverance, poise, responsibility, insistence, persistence, determination, composure, sense of duty, perseverance in achieving the goal, accuracy, and are business oriented. 40% of the athletes (2 people) demonstrate prevalence of the E factor (dominance). They are characterized by domination, authoritativeness, obstinacy, self-confidence, assertiveness, aggressiveness, stubbornness, conflict, wilful, instability, independence, and inflexibility. Track and field athletes revealed the predominance of the B factor (high intelligence) by positive personality profile - 5 people (50%). This factor is characterized by such personality traits as self-discipline, intelligence, abstract thinking, vision, and short reaction time. 3 people (30%) have predominant G factor (high super-ego). 2 people (20%) have dominant H factor (Parmia), which is characterized by such traits as courage, enterprise, social boldness, risk tolerance, sociability, activity, and responsiveness. Negative personality profile of combat athletes have dominant I factor (Harria) - 3 people (60%). These people have low sensitivity, severity, rationality, realism judgments, practicality, some brutality, courage, self-confidence, and responsibility. 4. Results The scientific and practical centre "Acmeological centre", being the innovative structure of Mordovia basic teacher training centre at Mordovia State Pedagogical Institute named after M. E. Evseviev, conducted an empirical study of the psychology of the winner. The study involved 30 athletes of different age - winners of international and European tournaments in athletics and martial art, having sports titles of "Honoured Master of Sports" and "International Master of Sports». The results of the diagnostic based on the "Mini-Mult" questionnaire (a shortened version of the Minnesota multidimensional personality test, MMPI) are presented in Table 1. Table 1. Properties of the individual winning athlete by the Mini-Mult questionnaire Table 1. Properties of the individual winning athlete by the Mini-Mult questionnaire Basic scales Track and field athletes, % Combat athletes, % Hypochondriasis (Hs) 0 0 Depression (D) 2 (20%) 1 (20%) Hysteria (Hy) 0 0 Psychopathy (Pd) 0 0 Paranoia (Pa) 0 0 Psychasthenia (Pt) 0 0 Schizoidness (Se) 0 0 Hypotension (Ma) 8 (80%) 4 (80%) According to Table 1, such individual feature as hypotension dominates among track and field athletes (8 people - 80%), manifesting in high spirits regardless of the circumstances, characterized by energy, activity, vigour, and vitality. Such personalities like to work with often changes, and readily contact with people. The individual traits found in track and field athletes extremely rarely include depression (2 people - 20%) inherent in diligent, conscientious, moral and dutiful people, not able to decide on their own, having no self-confidence, and who fall into despair at the slightest failure. Similar results were obtained on the sample of combat athletes. They are also characterized by hypotension (4 people – 80%). Rarely individual traits include depression (1 person – 20%). 215 Vol. 9, No. 5; 2015 Modern Applied Science www.ccsenet.org/mas Perhaps these results are explained by great physical and emotional stress experienced by the athletes during training. These conditions tend to lead to lower mood, loss of the ability to experience pleasure, loss of interest in usual activities, and as a consequence - to depression. Perhaps these results are explained by great physical and emotional stress experienced by the athletes during training. These conditions tend to lead to lower mood, loss of the ability to experience pleasure, loss of interest in usual activities, and as a consequence - to depression. 4. Results 2 people (40%) are characterized by the A factor (Schizothymia), distinct with invisibility, isolation, criticality, inflexibility, defending own ideas, and objectivity. In terms of negative personality profile, track and field athletes demonstrate dominating M factor (Praxernia) - 8 people (80%). These people are characterized by practicality, strength, honesty, integrity, and management of the objective reality. 1 person in each group (10%) manifested the A factor (Schizothymia) and the I factor (Harria). Thus, the personal traits of athletes definitely include strong character, integrity, perseverance, balance, responsibility, perseverance, persistence, determination, persistence in achieving goals, practicality, strength, and honesty. These qualities determine the winners in different sports. 5. Discussion A significant proportion of athletes (13 people - 86.7%) are noted for the presence of emphasizing arcs and strokes at the end of their signatures, indicating a desire to stand out, to draw attention to them. Some of the respondents (3 people - 20%) are distinguished with undecipherable, complex signature with lots of wavy and complicate lines, which is the evidence of variability, the desire to live for oneself, perhaps selfishness. Sometimes it is a manifestation of inflated self-esteem, emphasizing their status, "statehood". The signature of 2 people (13.3%) is characterized by sharp, energetic vertical lines, which is a sign of dominance. Thus, the results of the "Signature" revealed the winner, primarily, as having the desire to dominate, to emphasize own importance, the desire to stand out, to draw attention. The empirical study will define the main personal qualities inherent in the winning athletes: activity, energy, communication, diligence, integrity, strong character, perseverance, responsibility, perseverance, persistence, determination, perseverance in achieving the goal, the desire to dominate, and the desire to stand out. The prospects for further psychological and acmeological studies of winners determine the object and the methodical expansion of space, identification of the factor structure of the individual winner, and the definition of correlations. 5. Discussion The results of the survey allowed formulation of the following conclusions. The majority of respondents began their career with training in the sports section (15 people – 100 %). A significant part of the participants (12 216 Modern Applied Science www.ccsenet.org/mas Vol. 9, No. 5; 2015 people - 80%) note the influence of the coach on the formation of their sports career. 3 people (20%) claim that career advancement was influenced by their loved ones (family, friends, etc.). people - 80%) note the influence of the coach on the formation of their sports career. 3 people (20%) claim that career advancement was influenced by their loved ones (family, friends, etc.). The question "Do you know what the psychological training of the athlete implies?" the majority of respondents answered positively (13 people – 86.6 %). All the participants noted the importance of psychological training in the training sessions. At the same time, a significant portion of respondents answered the question "Please rate the value of psychological training for a highly skilled athlete on a scale from 1 to 10" put the highest marks: 2 people (13.3%) - 8 points; 2 people - 9 (13.3%) points; 11 people (73.4%) - 10 points). The questionnaire offered the participants to reveal the contents of psychological training of the athlete in the sports training, which caused some difficulties. According to them, the psychological training of the athlete comes to motivation to win, self-control, and willpower. A series of questions was offered relating to the description of the personal traits of the winner. The most common responses are the following: "perseverance, persistence," "commitment," "hard work", "patience", "fortitude", "mental stability". In addition, the subjects offered psychological traits of the winner, among they often encountered: "hard work", "courage, the ability to go to the end, never give up", "win at all costs", "ability to sacrifice everything to achieve this goal", "thirst for victory", "patience", "self-confidence", "continuous self-work", and "belief in success". Analysing the results of the "Signature" methodology the following data was obtained: the majority of athletes (11 people - 73.3%) demonstrated in their signature the initial letter of their name, which indicates an effort to emphasize the importance, and somewhat high self-esteem. The signature is sprawling, occupying a lot of space, which characterizes the desire to emphasize their status, their capabilities, and even expand them. 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https://openalex.org/W3017128574	http://jcopolicy.uobaghdad.edu.iq/index.php/jcopolicy/article/download/140/112	Chinese	null	(ممانعات اعادة القدرة الاستراتيجية العراقية بعد عام 2003 (رؤية تحليلية	Al-ʻulūm al-siyāsiyyāẗ/Mağallaẗ al-ʿulūm al-siyāsiyyaẗ	2,018	cc-by	22,047	       : ﺍﳌﻠﺨـﺺ ﻛﺎﻥ ﻭﻣﺎ ﻳﺰﺍﻝ ﻣﻮﺿﻮﻉ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﻌﺮﺍﻕ ﺑﻌﺪ ﺍﻻﺣﺘﻼﻝ ﻭﺍﻟﻐﺰﻭ ﺍﻷﻣﺮﻳﻜﻲ ﻋﺎﻡ ٣٠٠٢ ﻡ ﺃﺣﺪ ﺍﳌﺴﺎﺋﻞ ﺍﳌﺜﲑﺓ ﻟﻠﺠﺪﻝ ﻭﺍﻟﺴﺠﺎﻝ ﻭﺍﻟﻨﻘﺎﺵ ﺍﳊﺎﺩ ، ﺇﺫ ﻳـﺜﲑ . ﲨﻠﺔ ﺍﺷﻜﺎﻻﺕ ﺳﻴﺎﺳﻴﺔ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻷﻭﱃ ، ﻭﺍﺟﺘﻤﺎﻋﻴﺔ ﻭﺍﻗﺘﺼﺎﺩﻳﺔ ﻭﻋﺴـﻜﺮﻳﺔ ﻭﺃﻣﻨﻴـﺔ ﻭﻧﻘﻮﻝ ﺳﻴﺎﺳﻴﺔ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻷﻭﱃ ﻷﻥ ﺍﳌﻨﺎﺧﺎﺕ ﻭﺍﻷﺣﻮﺍﻝ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺗـﺆﺩﻱ ﺩﻭﺭﺍﹰ ﻛـﺒﲑﺍﹰ : ﻭﻓﺎﻋﻼﹰ ﺇﱃ ﺟﺎﻧﺐ ﻋﻮﺍﻣﻞ ﺃﺧﺮﻯ ﻣﻜﻤﻠﺔ ﳍﺎ . ﻭﺍﻟﺴﺆﺍﻝ ﺍﳌﻬﻢ ﺍﻟﺬﻱ ﻳﻄﺮﺡ ﻫﻨـﺎ ، ﻫـﻮ ﻛﻴﻒ ﳝﻜﻦ ﻟﻠﻜﺘﻞ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺍﳊﺎﻛﻤﺔ ﺍﻻﻧﺘﻘﺎﻝ ﺇﱃ ﻣ ﺮﺣﻠﺔ ﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ﺑﺪﻭﻥ ﲢﻔﻈـﺎﺕ؟ ، ﻭﻣﻦ ﺍﺟﻞ ﺍﻹﺟﺎﺑﺔ ﻋﻦ ﻫﺬﺍ ﺍﻟﺘﺴﺎﺅﻝ ﻗﺴﻤﻨﺎ ﺍﻟﺪﺭﺍﺳﺔ ﺇﱃ ﺛﻼﺛﺔ ﳏﺎﻭﺭ ﺃﺳﺎﺳﻴﺔ، ﺇﺫ ﻳﺘﻨـﺎﻭﻝ ﺍﻷﻭﻝ : ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ﻭ ﺃﻧﻮﺍﻋﻬﺎ . ﻭﻳﺘﻨﺎﻭﻝ ﺍﶈﻮﺭ ﺍﻟﺜﺎﱐ : ﻋﻼﻗﺔ ﺍﻟﻘـﺪﺭﺓ ﺑﺎﻹﺳـﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺃﺑﻌﺎﺩﻫﺎ. ﰲ ﺣﲔ ﻳﺘﻨﺎﻭﻝ ﺍﶈﻮ ﺭ ﺍﻟﺜﺎﻟﺚ : ﺍﳌﻤﺎﻧﻌﺎﺕ )ﺍﻟﺪﺍﺧﻠﻴﺔ، ﻭﺍﳋﺎﺭﺟﻴﺔ ( ﺍﻟﱵ ﺗﻮﺍﺟـﻪ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢. ﻡ ﻭﺗﻨﺘﻬﻲ ﺍﻟﺪﺭﺍﺳﺔ ﺑﺎﺳﺘﻨﺘﺎﺟﺎﺕ ﺗﺘﻀﻤﻦ ﺗﻮﺻـﻴﺎﺕ ﻟﻠﻨـﻬﻮ ﺽ ﺑﺎﻟﻘـﺪﺭﺓ ﺍﻹﺳـﺘﺮ ﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ. : ﺍﳌﻘـﺪﻣـﺔ () .ﺟﺎﻣﻌﺔ ﺍﻟﻨﻬﺮﻳﻦ / ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ ـ ﻗﺴﻢ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ                : ﺍﳌﻘـﺪﻣـﺔ ﺭﻫﺎ ﻋﻠﻰ ﺍﻛﻤﻞ ﻭﺟﻪ ﻭﲪﻞ ﻟﻨﺎ ﻋﻨﻮﺍﻥ ﺍﻟﺪﺭﺍﺳﺔ ﺃﺷﻜﺎﻟﻴﺔ ﻛﺒﲑﺓ ﺍﻛﺘﻈﺖ ﺑﺘﺴﺎﺅﻻﺕ ﻋﺪﺓ ، ﻭﺃﺛﺎﺭﺕ ﺍﳉـﺪﻝ ﰲ ﳏﺎﻭﻟﺔ ﻟﻼﺟﺎﺑﺔ ﻋﻨﻬﺎ ، ﻭﻣﻦ ﻫﺬﻩ ﺍﻟﺘﺴﺎﺅﻻﺕ : ﻣﺎ ﻫﻲ ﺍﻟﻘﺪﺭﺓ ﻭﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ؟ ﻭﻣﺎ ﺃﺑﺮﺯ ﺃﺑﻌﺎﺩ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ؟ ، ﻭﻣﺎ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻮﺍﺟﻪ ﺇﻋـﺎﺩﺓ ﺑﻨـﺎﺀ ﻗـﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﻌﺪ ﺍﻻﺣﺘﻼﻝ ﺍﻷﻣﺮﻳﻜﻲ ﻋﺎﻡ ٣٠٠٢ ﻡ ﺩﺍﺧﻠﻴﺎﹰ ﻭﺇﻗﻠﻴﻤﻴﺎﹰ ﻭﺩﻭﻟﻴﺎﹰ ؟ ، ﻭﻫـﻞ ﺍﻟﺘﻮﺻﻴﺎﺕ ﺍﳌﻄﺮﻭﺣﺔ ﻛﺎﻓﻴﺔ ﻭﻗﺎﺩﺭﺓ ﻋﻠﻰ ﻃﻤﺄﻧﺔ ﻣﺎ ﺗﺘﻌﺮﺽ ﻟـﻪ ﺍﻟﻘـﺪﺭﺓ ﺍﻟﻌﺮﺍﻗﻴـﺔ ﻣـﻦ ﺍﺭﺗﺒﺎ ﻛﺎﺕ ﻭﺍﺧﺘﻼﻻﺕ ﺑﻨﺎﺋﻴﺔ ﺗﺪﻓﻊ ﲦﻨﻬﺎ ، ﻓﻀﻼﹰ ﻋﻤ ﺎ ﻳﻨﺴﺠﻪ ﺿﺪﻫﺎ ﺍﻵﺧﺮﻭﻥ ﻣﻦ ﺧﺎﺭﺟﻪ؟. ﻻ ﺑﺪ ﻣﻦ ﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ، ﻣﻦ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻓﺎﻋﻠﺔ ﺗﻨﺘﻈﻢ ﻓﻴﻬﺎ ﺍﻟﻘﺪﺭﺍﺕ ﲝﺎﺿـﻨﺔ ﻭﻃﻨﻴـﺔ ﻭﺍﺿﺤﺔ ، ﺑﻞ ﻭﺃﻥ ﺗﻨﺸﻂ ﺫﺍﻛﺮﺓ ﺍﻟﻘﺎﺋﻤﲔ ﻋﻠﻴﻬﺎ، ﲟﺎ ﻳﻜﻔﻞ ﳍﻢ ﺍﻟﻘﺪﺭﺓ ﻭﺍﻻﻣﻜﺎﻧﻴـﺔ ﻋﻠـﻰ ﺍﺳﺘﺜﻤﺎ. ﺭﻫﺎ ﻋﻠﻰ ﺍﻛﻤﻞ ﻭﺟﻪ ﻭﲪﻞ ﻟﻨﺎ ﻋﻨﻮﺍﻥ ﺍﻟﺪﺭﺍﺳﺔ ﺃﺷﻜﺎﻟﻴﺔ ﻛﺒﲑﺓ ﺍﻛﺘﻈﺖ ﺑﺘﺴﺎﺅﻻﺕ ﻋﺪﺓ ، ﻭﺃﺛﺎﺭﺕ ﺍﳉـﺪﻝ ﰲ ﳏﺎﻭﻟﺔ ﻟﻼﺟﺎﺑﺔ ﻋﻨﻬﺎ ، ﻭﻣﻦ ﻫﺬﻩ ﺍﻟﺘﺴﺎﺅﻻﺕ : ﻣﺎ ﻫﻲ ﺍﻟﻘﺪﺭﺓ ﻭﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ؟ ﻭﻣﺎ ﺃﺑﺮﺯ ﺃﺑﻌﺎﺩ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ؟ ، ﻭﻣﺎ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻮﺍﺟﻪ ﺇﻋـﺎﺩﺓ ﺑﻨـﺎﺀ ﻗـﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﻌﺪ ﺍﻻﺣﺘﻼﻝ ﺍﻷﻣﺮﻳﻜﻲ ﻋﺎﻡ ٣٠٠٢ ﻡ ﺩﺍﺧﻠﻴﺎﹰ ﻭﺇﻗﻠﻴﻤﻴﺎﹰ ﻭﺩﻭﻟﻴﺎﹰ ؟ ، ﻭﻫـﻞ ﺍﻟﺘﻮﺻﻴﺎﺕ ﺍﳌﻄﺮﻭﺣﺔ ﻛﺎﻓﻴﺔ ﻭﻗﺎﺩﺭﺓ ﻋﻠﻰ ﻃﻤﺄﻧﺔ ﻣﺎ ﺗﺘﻌﺮﺽ ﻟـﻪ ﺍﻟﻘـﺪﺭﺓ ﺍﻟﻌﺮﺍﻗﻴـﺔ ﻣـﻦ ﺍﺭﺗﺒﺎ ﻛﺎﺕ ﻭﺍﺧﺘﻼﻻﺕ ﺑﻨﺎﺋﻴﺔ ﺗﺪﻓﻊ ﲦﻨﻬﺎ ، ﻓﻀﻼﹰ ﻋﻤ ﺎ ﻳﻨﺴﺠﻪ ﺿﺪﻫﺎ ﺍﻵﺧﺮﻭﻥ ﻣﻦ ﺧﺎﺭﺟﻪ؟. : ﺍﳌﻘـﺪﻣـﺔ ﻧﺘﻴﺠﺔ ﺍﻟﺘﻐﻴﲑ ﺍﳉﺬﺭﻱ ﺍﻟﺬﻱ ﺣﺼﻞ ﰲ ﺍﳌﻌﺎﺩﻟﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﰲ ﺍﻟﻌﺮﺍﻕ ﺑﻌـﺪ ﺍﻻﺣـﺘﻼﻝ ﻭﺍﻟﻐﺰﻭ ﺍﻷﻣﺮﻳﻜﻲ ﻋﺎﻡ ٣٠٠٢ ﻡ ، ﻭﻓﻘﺪﺍﻥ ﺍﻟﻌﺮﺍﻕ ﻟﺴﻴﺎﺩﺗﻪ ﺍﻟﻮﻃﻨﻴﺔ ، ﻭﻣﺎ ﺭﺍﻓﻘﻪ ﻣﻦ ﺗﺪﻣﲑ ﻟﻠﺒﲎ ﺍﻟﺘﺤﺘﻴﺔ ﻧﺘﻴﺠﺔ ﺍﻟﻌﻤﻠﻴﺎﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﻣﻦ ﺳﺮﻕ ﻭﺣﺮﻕ ﻭﺗﺪ ﻣﲑ ﻣﻨﻈﻢ ﺷﺎﺭﻛﺖ ﻓﻴـﻪ ) ﺍﻃﺮﺍﻑ ﺩﺍﺧﻠﻴﺔ، ﻭﺩﻭﻝ ﺇﻗﻠﻴﻤﻴﺔ(، ﺩﻭﻟﻴﺔ ، ﻭﺍﻻﻧﻘﻼﺏ ﻋﻠﻰ ﺍﻟﻨﻤﻂ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺍﳊﻴﺎﺓ ﺍﻟﻌﺎﻣﺔ () .ﺟﺎﻣﻌﺔ ﺍﻟﻨﻬﺮﻳﻦ / ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ ـ ﻗﺴﻢ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ١٧٣       ﺍﻟﱵ ﻛﺎﻧﺖ ﺳﺎﺋ ﺪﺓ ﻗﺒﻞ ﻋﺎﻡ ٣٠٠٢(ﻡ ، ﻣﺮ ﺍﻟﻌﺮﺍﻕ ﺑـ)ﻣﺮﺣﻠﺔ ﺍﻟﻼﺍﺳﺘﻘﺮﺍﺭ ، ﺃﺛﺮﺕ ﺑﺸﻜﻞ ﻣﺒﺎﺷ ﺮ ﻭﻛﺒﲑ ﻋﻠﻰ ﻗﺪﺭﺍﺗﻪ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ . ﻭﻛﺎﻥ ﻣﻦ ﺍﻟﻀﺮﻭﺭﻱ ﻋﻠﻰ "ﺍﻟﻜﺘـﻞ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﺍﳊﺎﻛﻤﺔ ﺍﳉﺪﻳﺪﺓ" ﺍﻟﱵ ﺟﺎ ﺀﺕ ﺃﻥ ﺗﻌﻤﻞ ﻋﻠﻰ ﻭﺿﻊ ﻣﺸﺮﻭﻉ ﺃﻭ ﺧﺎﺭﻃﺔ ﻃﺮﻳﻖ ﻟﻠﻮﺻـﻮﻝ ﺇﱃ ﺍﻷﻫﺪﺍﻑ ﺍﻟﱵ ﳚﺐ ﺍﻟﻮﺻﻮﻝ ﺍﻟﻴﻬﺎ . ﻟﻜﻦ ﰲ ﻇﻞ ﺍﳉﺪﻝ ﺍﻟﺪﺍﺋﺮ ﺣﻮﻝ ﺍﳌﺸﺎﻛﺴـﺎﺕ ﻭﺍﻟﺘﻨﺎﻗﻀﺎﺕ ﺑﲔ "ﺍﻟﻜﺘﻞ ﺍﳊﺎﻛﻤﺔ" ﱂ ﺗﺘﻤﻜﻦ ﺍﻷﺧﲑﺓ ﻣﻦ ﺭﺳﻢ ﻣﻼﻣﺢ ﺍﻟﻌﺮﺍﻕ ﺍﳉﺪﻳﺪ ، ﺇﺫ ﻻ ﺗﻮﺟﺪ ﻫﻨﺎﻙ ﺧﺮﻳﻄﺔ ﻟﻠﻤﱰﻝ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﻌﺮﺍﻗﻲ ﺍﳉﺪﻳﺪ ﻟﻴﺘﺴﲎ ﳍﺎ ﻣﻌﺮﻓﺔ ﻣﻮﻗﻊ ﻛﻞ ﻏﺮﻓﺔ ﰲ ﻫﺬﺍ ﺍﳌﺨﻄﻂ، ﻭﺍﺑﻌﺎﺩﻫﺎ ﻭﻣﺎ ﺗﺘﻄﻠﺒﻪ ﻣﻦ ﻣﻮﺍﺩ ﻹﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ﻭﻭﺿﻊ ﺍﻷﺳﺲ ﺍﻟﺮﺻﻴﻨﺔ ﻣﻦ . ﺃﺟﻞ ﺇﻋﺎﺩﺓ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻳﺴﻌﻰ ﻫﺬﺍ ﺍﻟﺒﺤﺚ ﺇﱃ ﳏﺎﻭﻟﺔ ﺑﻠﻮﺭﺓ ﺭﺅﻳﺔ ﲢﻠﻴﻠﻴﺔ ﺣﻮﻝ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻮﺍﺟـﻪ ﺑﻨـﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴ ﺔ . ﻛﻮﻥ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﺍﻧﻄﻠﻘﺖ ﻣﻦ ﲢﻠﻴﻞ ﺍﻟﻮﺿﻊ ﺍﻟﻌﺮﺍﻗـﻲ ﻭﻓﻖ ﺍﳌﻌﻄﻴﺎﺕ ﻭﺍﻟﺘﺪﺍﻋﻴﺎﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﻷﻣﻨﻴـﺔ ﻭﺍﻻﺟﺘﻤﺎﻋﻴـﺔ . ﻭﺑﻄﺮﻳﻘﺔ ﻋﻠﻤﻴﺔ ﺣﻴﺎﺩﻳﺔ ﺍﺧﺘﻔﻰ ﺑﲔ ﺛﺎﻧﻴﺎﻫﺎ ﻣﺸﻬﺪ ﻳﺘﻌﻘﺪ ﺣﻴﻨﺎﹰ ﻭﻏﺎﻟﺒﺎﹰ ، ﻭﻳﻨﻔـﺮﺝ ﻧـﺎﺩﺭﺍﹰ ﻓﺎﻟﻌﺮﺍﻕ ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﻡ ﻭﺍﺟﻪ ﻋﻤﻠﻴﺔ ﺗﻠﻜﺆ ﰲ ﺍﻟﺒﻨﺎﺀ ﺑﺸﻜﻞ ﺳﻠﱯ ، ﻧﺘﻴﺠﺔ ﻏﻴﺎﺏ ﺍﳍﻤﻢ ﺍﻟﻮﻃﻨﻴﺔ ﻭﺍﻟﻐﲑﺓ ﺍﻹﻧﺴﺎﻧﻴﺔ ، ﻭﺍﺭﺗﺒﺎﻁ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﺯﻣﺎﺕ ﻣﻊ ﺳﻠﺴﻠﺔ ﻋﻘﺪ ﻭﻣﻌﻀـﻼﺕ ﻋﻠﻰ ﺃﻛﺜﺮ ﻣﻦ ﺻﻌﻴﺪ ﻭﳎﺎﻝ ﺩﻭﻥ ﺇﳚﺎﺩ ﺍﳊﻞ ﺍﳌﻨﺎﺳﺐ ﺃﻭ ﺍﳌﻄﻠﻮﺏ . ﻭﺑﺬﻟﻚ ﻏﺎﺏ ﺍﻟﻌﺮﺍﻕ ﻭﻏﺎﺏ ﺍﺳﺘﻘﻼﻟﻪ ﺍﻟﻮﻃﲏ ﻭﺑﺮﻭﺯ ﻇﺎﻫﺮﺓ ﺍﻟﱰﻋﺎﺕ ﺍﻟﺪﻳﻨﻴﺔ ﻭﺍﻟﻄﺎﺋﻔﻴﺔ ﺍﳌﺘﻄﺮﻓﺔ ﻭﺍﳌﺘﻌﺼ ﺒﺔ ﻭﺍﻟﱵ . ﺘﻤﻊ ﺍﻟﻌﺮﺍﻗﻲﱂ ﻳﺄﻟﻔﻬﺎ ﺍ ﻣﻦ ﻫﻨﺎ ﻭﰲ ﻇﻞ ﺍﻷﻭﺿﺎﻉ ﺍﶈﻠﻴﺔ ﻭﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ ﺍﻟﺮﺍﻫﻨﺔ ، ﺃﺻﺒﺢ ﻋﻠﻰ ﺍﳉﻤﻴـﻊ ﺍﺩﺭﺍﻙ ﺍﻥ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻫﻮ ﺍﳌﻔﺘﺎﺡ ﺍﻟﻮﺣﻴﺪ ﻟﻠﺘﻌﺎﻣﻞ ﻣﻊ ﺍﻟﺘﻬﺪﻳـﺪﺍﺕ ﻭﺍﳌﺨﺎﻃﺮ ، ﺳﻮﺍﺀ ﺃﻛﺎﻧﺖ ﺗﻠﻚ ﺍﳌﺨﺎﻃﺮ ) ﺩﺍﺧﻠﻴﺔ ﺃﻡ ﺧﺎﺭﺟﻴﺔ ( ، ﻭﺑ "ﺪﻭﻥ ﺗﻠﻚ "ﺍﻟﻘﺪﺭﺍﺕ ﻻ ﳝﻜﻦ ﺍﳊﺪﻳﺚ ﻋﻦ ﲢﻘﻴﻖ ﺍﻷﻣﻦ ﺍﻟﻮﻃﲏ ، ﻭﻻ ﳝﻜﻦ ﺗﻌﺰﻳﺰ ﻣﻜﺎﻧـﺔ ﺍﻟﺪﻭﻟـﺔ ﺍﻹﻗﻠﻴﻤﻴـﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ . ﻟﺬﺍ ﻓﻠﻦ ﻳﻜﻮﻥ ﻣﻦ ﺍﳌﻤﻜﻦ ﺍﳊﺪﻳﺚ ﻋﻦ ﻣﻮﺍﺟﻬﺔ ﺗﻠﻚ ﺍﻟﺘﺤﺪﻳﺎﺕ ﻭﺍﳌﺨـﺎﻃﺮ ، ﺩﻭﻥ ﻗﺮﺍﺀﺓ ﻭﺍﺿﺤﺔ ﻭﺗﺮﺗﻴﺒﺎﺕ ﺗﻘﻮﺩ ﳓﻮ ﻣﻌﺮﻓﺔ ﺍﻟﺼﻮﺭﺓ ﺍﻟﻮﺍﺿﺤﺔ ﻟﻨﻘﺎﻁ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻀﻌﻒ ﻭﻫﻮ ﻣﺎ ﻳﻌﺰﺯ . : ﺍﳌﻘـﺪﻣـﺔ ﻣﻦ ﺃﳘﻴﺔ ﺍﻷﻣﺮ ﻭﺇﺩﺭﺍﺟﻪ ﺿﻤﻦ ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺪﻭﻟﺔ ٢٧٣       : ﺃﳘﻴـﺔ ﺍﻟـﺪﺭﺍﺳـﺔ ، ﺇﻥ ﻣﺸﻜﻠﺔ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﳍﺎ ﺣﻀﻮﺭ ﻃﺎﻍ ﻋﻠﻰ ﺍﻟﺴﺎﺣﺔ ﺍﻟﺪﺍﺧﻠﻴـﺔ ﺎ ، ﺣﱴ ﻏﺪﺕ ﺍﳌﺸﻜﻠﺔ ﺍﻷﻛﱪ ﺍﻟـﱵ ﺗﻮﺍﺟـﻪ ﺇﺫ ﺗﺘﺼﻞ ﺑﻜﻞ ﺷﻲﺀ ، ﻭﻳﺘﺼﻞ ﻛﻞ ﺷﻲﺀ ﺍﻟﻨﻈﺎﻡ ﺍﻟﺴﻴﺎﺳﻲ ﺑﻌﺪ ﻋ ﺎﻡ ٣٠٠٢ ﻡ ﻭﻋﻠﻰ ﻛﺎﻓﺔ ﺍﳌﺴـﺘﻮﻳﺎﺕ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﻭﺍﻻﺟﺘﻤﺎﻋﻴـﺔ . .. ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﻷﻣﻨﻴﺔ ﻭﻣﻦ ﻫﻨﺎ ﺗﻨﺒﻊ ﺃﳘﻴﺔ ﺍﻟﺪﺭﺍﺳﺔ ﻣﻦ ﺗﻨﺎﻭﳍﺎ ﻟﻠﻤﻤﺎﻧﻌﺎﺕ ) ﺍﻟﺪﺍﺧﻠﻴﺔ ، ﻭﺍﳋﺎﺭﺟﻴـﺔ ( ﺍﻟـﱵ ﺗﻮﺍﺟﻪ ﺇﻋﺎﺩﺓ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺪﻭﻟﺔ ، ﺇﺫ ﻓﺸﻠﺖ ﻛﻞ ﺍﳊﻜﻮﻣﺎﺕ ﺍﳌﺘﻌﺎﻗﺒﺔ ﻣﻨﺬ ﻋـﺎﻡ ٣٠٠٢ ﻡ ﻭﳊ ﺪ ﺍﻵﻥ ﻋﻦ ﺍﻟﺘﻌﺎﻣﻞ ﻣﻌﻬﺎ ، ﻭﺑﻘﻴﺖ ﲤﺜﻞ ﻋﺎﻣﻞ ﻭﻫﻦ ﺭﺋﻴﺲ ﰲ ﺑﻨـﺎﺀ ﺍﻟﺪﻭﻟـﺔ ﻭﲢﻘﻴﻖ ﺍﺳﺘﻘﺮﺍﺭﻫﺎ ، ﺍﻷﻣﺮ ﺍﻟﺬﻱ ﻳﺘﻄﻠﺐ ﻓﺤﺼﺎﹰ ﺩﻗﻴﻘﺎﹰ ﻟﺘﻠﻚ ﺍﳌﺸﻜﻠﺔ ﻭﻣﻼﺣﻈﺔ ﺍﻵﺛﺎﺭ ﺍﻟﱵ ﺗﺘﺮﻛﻬﺎ ﻋﻠﻰ ﺍﻟﻌﺮﺍﻕ ﻛﺪﻭﻟﺔ ، ﻭﺍﻟﻌﺮﺍﻗﻴﲔ ﻛﺸﻌﺐ ، ﻭﳏﺎﻭﻟﺔ ﺗﻘﺪﱘ ﺑﻌـﺾ ﺍﻟﺘﻮﺻـﻴﺎﺕ ﰲ ﳏﺎﻭﻟﺔ ﻹﺳﻘﺎﻁ ﺍﳌﻤﺎﻧﻌﺎﺕ ﻭﺍﻟﱵ ﺗﺴﻠﻂ ﺍﻟﻀﻮﺀ ﻋﻠﻰ ﺍﳊﻠﻮﻝ ﺇﱃ ﻧﻮﺍﺡ ٍ ﻋﺪﻳﺪﺓ ، ﻭﺗﻮﻇﻴﻒ . ﺎ ﰲ ﳎﺮﻯ ﻭﺍﺣﺪﳐﺮﺟﺎ ﺔ ﺔﺍﻟﺪﺍ ﺇﺷﻜﺎﻟ ، ﺇﻥ ﻣﺸﻜﻠﺔ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﳍﺎ ﺣﻀﻮﺭ ﻃﺎﻍ ﻋﻠﻰ ﺍﻟﺴﺎﺣﺔ ﺍﻟﺪﺍﺧﻠﻴـﺔ ﺎ ، ﺣﱴ ﻏﺪﺕ ﺍﳌﺸﻜﻠﺔ ﺍﻷﻛﱪ ﺍﻟـﱵ ﺗﻮﺍﺟـﻪ ﺇﺫ ﺗﺘﺼﻞ ﺑﻜﻞ ﺷﻲﺀ ، ﻭﻳﺘﺼﻞ ﻛﻞ ﺷﻲﺀ ﺍﻟﻨﻈﺎﻡ ﺍﻟﺴﻴﺎﺳﻲ ﺑﻌﺪ ﻋ ﺎﻡ ٣٠٠٢ ﻡ ﻭﻋﻠﻰ ﻛﺎﻓﺔ ﺍﳌﺴـﺘﻮﻳﺎﺕ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﻭﺍﻻﺟﺘﻤﺎﻋﻴـﺔ . .. ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﻷﻣﻨﻴﺔ ﻭﻣﻦ ﻫﻨﺎ ﺗﻨﺒﻊ ﺃﳘﻴﺔ ﺍﻟﺪﺭﺍﺳﺔ ﻣﻦ ﺗﻨﺎﻭﳍﺎ ﻟﻠﻤﻤﺎﻧﻌﺎﺕ ) ﺍﻟﺪﺍﺧﻠﻴﺔ ، ﻭﺍﳋﺎﺭﺟﻴـﺔ ( ﺍﻟـﱵ ﺗﻮﺍﺟﻪ ﺇﻋﺎﺩﺓ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺪﻭﻟﺔ ، ﺇﺫ ﻓﺸﻠﺖ ﻛﻞ ﺍﳊﻜﻮﻣﺎﺕ ﺍﳌﺘﻌﺎﻗﺒﺔ ﻣﻨﺬ ﻋـﺎﻡ ٣٠٠٢ ﻡ ﻭﳊ ﺪ ﺍﻵﻥ ﻋﻦ ﺍﻟﺘﻌﺎﻣﻞ ﻣﻌﻬﺎ ، ﻭﺑﻘﻴﺖ ﲤﺜﻞ ﻋﺎﻣﻞ ﻭﻫﻦ ﺭﺋﻴﺲ ﰲ ﺑﻨـﺎﺀ ﺍﻟﺪﻭﻟـﺔ ﻭﲢﻘﻴﻖ ﺍﺳﺘﻘﺮﺍﺭﻫﺎ ، ﺍﻷﻣﺮ ﺍﻟﺬﻱ ﻳﺘﻄﻠﺐ ﻓﺤﺼﺎﹰ ﺩﻗﻴﻘﺎﹰ ﻟﺘﻠﻚ ﺍﳌﺸﻜﻠﺔ ﻭﻣﻼﺣﻈﺔ ﺍﻵﺛﺎﺭ ﺍﻟﱵ ﺗﺘﺮﻛﻬﺎ ﻋﻠﻰ ﺍﻟﻌﺮﺍﻕ ﻛﺪﻭﻟﺔ ، ﻭﺍﻟﻌﺮﺍﻗﻴﲔ ﻛﺸﻌﺐ ، ﻭﳏﺎﻭﻟﺔ ﺗﻘﺪﱘ ﺑﻌـﺾ ﺍﻟﺘﻮﺻـﻴﺎﺕ ﰲ ﳏﺎﻭﻟﺔ ﻹﺳﻘﺎﻁ ﺍﳌﻤﺎﻧﻌﺎﺕ ﻭﺍﻟﱵ ﺗﺴﻠﻂ ﺍﻟﻀﻮﺀ ﻋﻠﻰ ﺍﳊﻠﻮﻝ ﺇﱃ ﻧﻮﺍﺡ ٍ ﻋﺪﻳﺪﺓ ، ﻭﺗﻮﻇﻴﻒ . ﺎ ﰲ ﳎﺮﻯ ﻭﺍﺣﺪﳐﺮﺟﺎ : ﺇﺷﻜـﺎﻟﻴـﺔ ﺍﻟـﺪﺭﺍﺳـﺔ : ﺇﺷﻜـﺎﻟﻴـﺔ ﺍﻟـﺪﺭﺍﺳـﺔ ﻻ ﺑﺪ ﻣﻦ ﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ، ﻣﻦ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻓﺎﻋﻠﺔ ﺗﻨﺘﻈﻢ ﻓﻴﻬﺎ ﺍﻟﻘﺪﺭﺍﺕ ﲝﺎﺿـﻨﺔ ﻭﻃﻨﻴـﺔ ﻭﺍﺿﺤﺔ ، ﺑﻞ ﻭﺃﻥ ﺗﻨﺸﻂ ﺫﺍﻛﺮﺓ ﺍﻟﻘﺎﺋﻤﲔ ﻋﻠﻴﻬﺎ، ﲟﺎ ﻳﻜﻔﻞ ﳍﻢ ﺍﻟﻘﺪﺭﺓ ﻭﺍﻻﻣﻜﺎﻧﻴـﺔ ﻋﻠـﻰ ﺍﺳﺘﺜﻤﺎ. ٣٧٣ ٣٧٣       : ﻓـﺮﺿﻴـﺔ ﺍﻟـﺪﺭﺍﺳـﺔ ﺗﻨﻄﻠﻖ ﺍﻟﺪﺭﺍﺳﺔ ﻣﻦ ﻓﺮﺿﻴﺔ ﻣﻔﺎﺩﻫﺎ : ﺃﻥ ﺍﻟﻘﻮﻯ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻣﻨﺬ ﻋﺎﻡ ٣٠٠٢ ، ﻡ ﻓﻀﻼﹰ ﻋﻦ ﺍﳌﺆﺳﺴﺎﺕ ﱂ ﺗﻌﻤﻞ ﻋﻠﻰ ﺑﻨﺎﺀ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﻃﻨﻴﺔ ﺭﺻﻴﻨﺔ ﻭﺣﻜﻢ ﺭﺷﻴﺪ ، ﺗﻔﻀـﻲ ﺇﱃ ﺇﻋﺎﺩﺓ ﻭﺗﻌﺰﻳﺰ ﻭﺗﻨﻤﻴﺔ ﻛﺎﻓﺔ ﻣﻘﻮﻣﺎﺕ ﺍﻟﻘﺪﺭﺓ ﺣﺎﺿﺮﺍﹰ ﻭﻣﺴﺘﻘﺒﻼﹰ ، ﻓﻤﻊ ﻛﻞ ﻣﺎ ﺗﻌﺮﺿﺖ ﻟﻪ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻣﻦ ﺍﺳ ﺘﺒﺎﺣﺔ ﻣﻦ ﺍﻟﺪﺍﺧﻞ ﻭﺍﳋﺎﺭﺝ ، ﻭﻣﻊ ﻛﻞ ﻣﺎ ﰎ ﻓﻌﻠﻪ ﳌﻮﺍﺟﻬﺔ ﺗﻠـﻚ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻌﻴﻖ ﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ، ﺍﻻ ﺃﻥ ﺍﳉﻬﻮﺩ ﱂ ﺗﻜﻦ ﺗﺰﻝ ﻏﲑ ﻣﻘﻨﻌﺔ ، ﺧﺎﺻﺔ ﰲ ﻇـﻞ ﺍﻣﺘﻼﻙ ﻛﻞ ﻣﻘﻮﻣﺎﺕ ﺍﻟﻘﻮﺓ ، ﻟﺘﻐﺪﻭ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﳌﻠﻤﺢ ﺍﻷﻫﻢ ﳍﺬﺍ ﺍﻟﺘﺪﻫﻮﺭ ﰲ ﺇﻋﺎﺩﺓ ﺗﻠـﻚ . ﺍﻟﻘﺪﺭﺍﺕ ﺍﶈـﻮﺭ ﺍﻷﻭﻝ / ﻣﻔﻬـﻮﻡ ﺍﻟﻘـ ﺪﺭﺓ ﻭﺃﳘﻴﺘﻬـﺎ : ﻟﻔﻬﻢ ﻣﻮﺿﻮﻉ ﺍﻟﻘﺪﺭﺓ ﻭﺃﳘﻴﺘﻬﺎ ، ﰎ ﺗﻘﺴﻴﻢ ﻫﺬﺍ ﺍﶈﻮﺭ ﺇﱃ ﻣﻄﻠﺒﲔ ﳘﺎ - . ( ﺍﳌﻄﻠﺐ ﺍﻷﻭﻝ : ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ) ﻟﻐﺔ ، ﻭﺍﺻﻄﻼﺣﺎﹰ ﺍﶈـﻮﺭ ﺍﻷﻭﻝ / ﻣﻔﻬـﻮﻡ ﺍﻟﻘـ ﺪﺭﺓ ﻭﺃﳘﻴﺘﻬـﺎ : ﻟﻔﻬﻢ ﻣﻮﺿﻮﻉ ﺍﻟﻘﺪﺭﺓ ﻭﺃﳘﻴﺘﻬﺎ ، ﰎ ﺗﻘﺴﻴﻢ ﻫﺬﺍ ﺍﶈﻮﺭ ﺇﱃ ﻣﻄﻠﺒﲔ ﳘﺎ - . ( ﺍﳌﻄﻠﺐ ﺍﻷﻭﻝ : ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ) ﻟﻐﺔ ، ﻭﺍﺻﻄﻼﺣﺎﹰ - . ﺍﳌﻄﻠﺐ ﺍﻟﺜﺎﱐ : ﺃﻧﻮﺍﻉ ﺍﻟﻘﺪﺭﺓ ﰲ ﺍﻟﻠﻐﺔ ﺍﻟﻌﺮﺑﻴﺔ ، ﺗﻌﲏ ﺍﻟﻘﺪﺭﺓ ) ﺍﻟﻄ ﺎﻗﺔ ﻭﺍﻟﻘﻮﺓ ﻋﻠﻰ ﺍﻟﺸﻲﺀ ( ﲟﻌﲎ ﺍﻟـﺘﻤﻜﻦ ﻣﻨـﻪ ﺃﻱ ). ﻗﺪﺭ ﻋﻠﻴﻪ ﻭﺍﻟﺘﻤﻜﻦ ﻣﻨﻪ ١ ﻑ ) ﻟﺴﺎﻥ ﺍﻟﻌﺮ( ﻛﻤﺎ ﻋﺮ ﺏ ( ﺍﻟﻘﺪﺭﺓ ﻣـﻦ ﻟﻔـﻆ ﺍﻟﻘـﺪﻳﺮ ﺍﻟﻘﺎﺩﺭ ، ﻭﻫﻲ ﻣﻦ ﺻﻔﺎﺕ ﺍﷲ ﺗﻌﺎﱃ ﰲ ﻗﻮﻟﻪ : ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ :) ﺇﻥ ﺍﷲ ﻋﻠﻰ ﻛـﻞ )، ( ﺷﻲﺀ ﻗﺪﻳﺮ ٢ ( ﻭﻗﻮﻟﻪ ﺗﻌﺎﱃ: : ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ ) ﺗﺒﺎﺭﻙ ﺍﻟﺬﻱ ﺑﻴـﺪﻩ ﺍﳌﻠـﻚ ). ( ﻭﻫﻮ ﻋﻠﻰ ﻛﻞ ﺷﻲﺀ ﻗﺪﻳﺮ ٣( ﻭﻟﺘﺪﺍﺧﻞ ﻣﺼﻄﻠﺤﻲ ) ﺍﻟﻘﺪﺭﺓ ﻭﺍﻟﻘﻮﺓ( ﻓﺮﻕ ﺍﻟﻘـﺮﺃﻥ ﺍﻟﻜﺮﱘ ﺑﻴﻨﻬﻤﺎ ، ﻗﻮﻟﻪ ﺗﻌﺎﱃ : ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ ) ﻭﻣﺎ ﻛﺎﻥ ﺍﷲ ﻟﻴﻌﺠﺰﻩ ﻣﻦ ﺷـﻲﺀ ﰲ ( ﺍﻟﺴﻤﺎﻭﺍﺕ ﻭﻻ ﰲ ﺍﻷﺭﺽ ﺇﻧﻪ ﻛﺎﻥ ﻋﻠﻴﻤﺎﹰ ﻗﺪﻳﺮﺍﹰ)،٤ ( ﻭﱂ ﻳﻘﻞ ﻗﻮﻳﺎﹰ : ﻷﻥ ﺍﻟﻘﺪﺭﺓ ﺿـﺪ ﺍﻟﻌﺠﺰ ، ﺑﻴﻨﻤﺎ ﺍﻟﻘﻮﺓ ﰲ ﻗﻮﻟﻪ ﺗﻌﺎﱃ : ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ : ) ﺍﷲ ﺍﻟﺬﻱ ﺧﻠﻘﻜـﻢ ﻣـﻦ )، ( ﺿﻌﻒ ﰒ ﺟﻌﻞ ﻣﻦ ﺑﻌﺪ ﺿﻌﻒ ﻗﻮﺓ ﰒ ﺟﻌﻞ ﻣﻦ ﺑﻌﺪ ﻗﻮﺓ ﺿﻌﻔﺎﹰ ﻭﺷﻴﺒﺔ ٥ ( ﻫﺬﻩ ﺗـﺪﻝ ٤٧٣       ﻋﻠﻰ ﺃﻥ ﺿﺪ ﺍﻟﻘﻮﺓ ﺍﻟﻀﻌﻒ ، ﻓﺎﻟﻘﻮﺓ ﻫﻲ ﺃﻥ ﻳﻔﻌﻞ ﺍﻟﺸﻲﺀ ﺑﻼ ﺿﻌﻒ ، ﺑﻴﻨﻤﺎ ﺍﻟﻘﺪ ﺭﺓ ﻫﻲ ). ﻓﻌﻞ ﺍﻟﻔﺎﻋﻞ ﺑﻼ ﻋﺠﺰ ، ﻳﻌﲏ : ﺃﻥ ﻳﻔﻌﻞ ﺍﻟﻔﺎﻋﻞ ﺍﻟﺸﻲﺀ ﺑﻼ ﻋﺠﺰ ٦( ﹰ ﻄﻼ ﻟ ﻋﻠﻰ ﺃﻥ ﺿﺪ ﺍﻟﻘﻮﺓ ﺍﻟﻀﻌﻒ ، ﻓﺎﻟﻘﻮﺓ ﻫﻲ ﺃﻥ ﻳﻔﻌﻞ ﺍﻟﺸﻲﺀ ﺑﻼ ﺿﻌﻒ ، ﺑﻴﻨﻤﺎ ﺍﻟﻘﺪ ﺭﺓ ﻫﻲ ). ٣٧٣ ﻓﻌﻞ ﺍﻟﻔﺎﻋﻞ ﺑﻼ ﻋﺠﺰ ، ﻳﻌﲏ : ﺃﻥ ﻳﻔﻌﻞ ﺍﻟﻔﺎﻋﻞ ﺍﻟﺸﻲﺀ ﺑﻼ ﻋﺠﺰ ٦( ٢ ﹰﺎ ﺍﻟﻘﺪﺓﺍﻄﻼ ، ﺇﻥ ﺍﻟﻘﺪﺭﺓ ﺑﺘﻔﺴﲑﻫﺎ ﺍﻟﻮﺍﺳﻊ ﻫﻲ : ) ﳎﻤﻮﻉ ﺍﳌﻮﺍﺭﺩ ﻭﺍﻟﻄﺎﻗﺎﺕ ﺍﻟﱵ ﲤﺘﻠﻜﻬﺎ ﺍﻟﺪﻭﻟـﺔ ـﺪﻑ ﲢﻘﻴـﻖ ﻭﻭﺿﻌﻬﺎ ﳏﻞ ﺍﻻﺳﺘﺨﺪﺍﻡ ﻣﻦ ﺍﺟﻞ ﺍﻟﺘﺤﺮﻙ ﰲ ﺍﳌﺴﺮﺡ ﺍﻟﺪﻭﱄ ﻭﺍﻹﻗﻠﻴﻤﻲ ﺍﳌﺼﺎﱀ ﺍ). ( ﻟﻘﻮﻣﻴﺔ ٧ ( ﲟﻌﲎ ﺁﺧﺮ ﻫﻲ : ) ﻛﺎﻓﺔ ﺍﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ﺍﻟﱵ ﲤﻠﻜﻬﺎ ﺍﻟﺪﻭﻟـﺔ ، ﺃﻱ ﻛﺎﻓﺔ ﻣﺼﺎﺩﺭ ﺍﻟﻘﻮﺓ ﺳﻮﺍﺀ ﺃﻛﺎﻧﺖ ﺳﻴﺎﺳﻴﺔ ، ﺍﺟﺘﻤﺎﻋﻴﺔ ، ﻋﺴﻜﺮﻳﺔ ، ﺍﻗﺘﺼﺎﺩﻳﺔ ...(. ﻭﻫﺬﺍ ﺍﻷﻣﺮ ﺟﻌﻞ ﺍﻟﻜﺜﲑ ﻣﻦ ﺍﳌﻔﻜﺮﻳﻦ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﲔ ﻳﻨﻈﺮﻭﻥ ﺇﱃ ﺍﻟﻘـﺪﺭﺓ ﻻﺳـﻴﻤﺎ ﰲ ﲢﺪﻳـﺪ ﻓﺎﻋﻠﻴﺔ ﺍﻟﺪﻭﻟﺔ ﻭﻣﻜﺎﻧﺘﻬﺎ ﻭﻣﺎ . ﺗﺆﺩﻳﻪ ﻣﻦ ﻭﻇﺎﺋﻒ ﻭﺃﺩﻭﺍﺭ ﻬﻭ ﻭﻭ ﻴ ﻒﻭﻭﺭ ﻦﻭ ﺆﻳ ﺃﻣﺎ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻘﻮﻣﻴﺔ ﻟﻠﺪﻭﻟﺔ ، ﻓﻬﻲ : ) ﻣﻘﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﻋﻠﻰ ﺍﺳﺘﻐﻼﻝ ﻛﺎﻓﺔ ﻗﻮﻯ ﺩﻓﺎﻋﻬـﺎ ﺍﻟﻮﻃﲏ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﻻﺟﺘﻤﺎﻋﻴﺔ ﻭﺍﳉﻐﺮﺍﻓﻴﺔ ﻭﺍﻟﻌﻠﻤﻴـﺔ ﻭﺍﳌﻌﻨﻮﻳـﺔ ﺎ ﺍﳋﺎﺭﺟﻴﺔ ﻭﺍﻟﺪﺍﺪﻳﺪﺍ ﻭﺍﻟﺘﻘﻨﻴﺔ ﰲ ﻇﻞ ﺇﺭﺍﺩﺓ ﻭﻗﻴﺎﺩﺓ ﻭﻃﻨﻴﺔ ﰲ ﻣﻮﺍﺟﻬﺔ). ( ﺧﻠﻴﺔ ٨( ﻛﻤﺎ ﻭﻳﺮﺗﺒﻂ ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ﺑﺎﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ، ﺇﺫ ﺗﺴﺘﻨﺪ ﺍﻟﻘـﺪﺭﺓ ﻋﻤﻮﻣـﺎﹰ ﻣـﻦ ﻭﺟﻬﺔ ﻧﻈﺮ ﻣﻔﻜﺮﻱ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺇﱃ : ) ﺍﻣﺘﻼﻙ ﺍﻟﺪﻭﻟﺔ ﺇﻣﻜﺎﻧﻴﺎﺕ ﻭﺧﺼـﺎﺋﺺ ﻭﻣـﻮﺍﺭﺩ ﻭﻣﺆﺳﺴﺎﺕ ﻣﻌﻴﻨﺔ ﺗﺸﻜﻞ ﲟﺠﻤﻠﻬﺎ ﻣﻘﻮﻣﺎﺕ ﺍﻟﻘﻮﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ، ﺇﺫ ﺍﻥ ﺿـﻌﻒ ﺃﻱ ﺟﺎﻧﺐ ﻣﻦ ﺗﻠﻚ ﺍﳌﻘﻮﻣﺎﺕ ﻳﻌﲏ ﻋﺪﻡ ﺍﻟﺘﻤﻜﻦ ﻣﻦ ﺣﺸﺪ ﺍﻟﻘﻮﺓ ﺍﻟﺸﺎﻣﻠﺔ ﻭﺑﺎﻟﺘـﺎﱄ ﺑﻨـﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﰲ ﻣﻮﺍﺟﻬﺔ ﺍﻟﺘﺤﺪﻳﺎﺕ ﺍﻟﺪﺍﺧﻠﻴﺔ ﻭﺍﳋﺎﺭﺟﻴﺔ ﰲ ﻇﻞ ﺃﻭﺿـﺎﻉ ﻫﺸـﺔ . ( ﺪﻳﺪ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﺗﻘﻮﺩ ﺇﱃ)٩( ﻟﺬﺍ ﻓﻬﻲ ﺇﺣﺪﻯ ﺍﻟﻨﺘﺎﺋﺞ ﺍﻹﳚﺎﺑﻴـﺔ ﻟﺘﻔﻌﻴـﻞ ﺍﻷﺩﺍﺀ ﺍﻹﺳﺘﺮ ﺎ، ﺇﺫ ﺍﻥ ﺍﻷﺩﺍﺀ ﲢﻜﻤـﻪ ﻗـﺪﺭﺍﺕ ﺍﻟﺪﻭﻟـﺔﺍﺗﻴﺠﻲ ﻟﻠﺪﻭﻟﺔ ﰲ ﲢﻘﻴﻖ ﺃﻫﺪﺍﻓﻬﺎ ﻭﻏﺎﻳﺎ . ﺍﻟﺸﺎﻣﻠﺔ ) ﻣﻦ ﺟﺎﻧﺐ ﺁﺧﺮ ، ﻳﺮﻯ ﺍﺳﺘﺎﺫ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻗﺤﻄﺎﻥ ﺃﲪﺪ ﺳـﻠﻴﻤﺎﻥ ( ﰲ ﻛﺘﺎﺑـﻪ ) ﺎ ﺃﴰﻞ ﻣﻦ ﻛـﻞ ﺃﻧـﻮﺍﻉﺍﻷﺳﺎﺱ ﰲ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ ( ﺃﻥ : ) ﺍﻟﻘﺪﺭﺓ ﺗﺸﻤﻞ ﺍﻟﻘﻮﺓ ﻷ ﺍﻟﺘﺄﺛﲑ ، ﻓﺎﻟﻘﺪﺭﺓ ﻫﻲ ﺍﻹﻣﻜﺎﻧﻴﺔ ﺍﻟﻘﺎﺋﻤﺔ ﻋﻠﻰ ﻛـﻞ ﺍﳉﻮﺍﻧـﺐ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﻭﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﻟﺜﻘﺎﻓﻴﺔ ﻭﺍﻟﻌﻠﻤﻴﺔ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴﺔ ، ﻭﺣﱴ ﺍﻟﻘﺪﺭﺓ ﺍﻟﺸﺨﺼﻴﺔ ﻟﻠﺤـﺎﻛﻢ ﻋﻠـﻰ ﳑﺎﺭﺳﺔ ﺍﻟﺘﺄﺛﲑ ﻭﺍﻟﻀﻐﻂ ﺩﺍﺧﻞ ﺍﻟﺪﻭﻟﺔ ، ﻭﻫﻲ ﻋﻠﻰ ﺍﻟﺼﻌﻴﺪ ﺍﳋـﺎﺭﺟﻲ ﺇﻣﻜﺎﻧﻴـﺔ ﺍﻟﺘـﺄﺛﲑ ٥٧٣       ﻭﺍﻟﺮﺩﻉ ، ﻭﺍﻟﺘﻬﺪﻳﺪ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻌﻨﻒ ﰲ ﺣﺎﻟﺔ ﻋﺪﻡ ﺍﻻﻧﺼﻴﺎﻉ ﻹﺭﺍﺩﺓ ﻃﺮﻑ ﻣﻘﺘﺪﺭ ﻋﻠﻰ ﻃﺮﻑ ﺃﻗﻞ ﺍﻗﺘﺪﺍﺭﺍﹰ. ﻭﺇﱃ ﺟﺎﻧﺐ ﺍﻟﻘﺪﺭﺓ ﺍﳌﺎﺩﻳﺔ ﻫﻨﺎﻟﻚ ﺍﻟﻘﺪﺭﺓ ﺍﳌﻌﻨﻮﻳـﺔ ، ﺃﻱ ﻗـﺪﺭﺓ ، ﺍﻟﻔﺮﺩ ﻋﻠﻰ ﺍﻟﺘﺄﺛﲑ ﲝﻜﻢ ﺷﺨﺼﻴﺘﻪ ، ﻭﲤﻜﻨﻪ ﻣﻦ ﺇﻗﻨﺎﻉ ﺍﻵﺧـﺮﻳﻦ، ﺃﻭ ﲢﻴﻴـﺪ ﺃﻋﻤـﺎﳍﻢ ). ٣٧٣ ( ﻛﺮﺟﺎﻝ ﺍﻟﺪﻳﻦ ﻭﺍﻟﻘﺎﺩﺓ ﻭﺍﳊﻜﺎﻡ ٠١( ﻭﻫﻨﺎﻙ ﻣﻦ ﻳﻌﺮﻓﻬﺎ ﻋﻠﻰ ﺎﺍ : ) ﳑﺎﺭﺳ ﺔ ﻋﻤﻠﻴﺔ ﻣﻦ ﺧـﻼﻝ ﺍﻟﺘﻮﻇﻴـﻒ ﻭﺍﻻﺳـﺘﺜﻤﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﻟﻌﻮﺍﻣﻞ ﺍﻟﻘﻮﻯ ﺍﳌﺎﺩﻳﺔ ﻭﺍﳌﻌﻨﻮﻳﺔ ﻣﻦ ﻟﺪﻥ ﺻﺎﻧﻊ ﺍﻟﻘﺮﺍﺭ ﻭﺍﻟـﺬﻱ ﳝﻠـﻚ ﺇﺭﺍﺩﺓ ﻗﻮﻳﺔ ﻭﻓﻜﺮﺍﹰ ﻭﻋﻘﻼﹰ ﺭﺍﺟﺤﺎﹰ ﻭﺇﳝﺎﻧﺎﹰ ﺭﺍﺳﺨﺎﹰ ﺑﺎﳌﺒﺎﺩﺉ ﺍﻟﱵ ﻳﺴﻌﻰ ﻟﺘﺤﻘﻴﻘﻬﺎ ﻭ ﲢﻮﻳﻠﻬﺎ ﺇﱃ ﻓﻌﻞ ﻣﺆﺛﺮ ﳝﻜﻦ ﻣﻦ ﺧﻼﻟﻪ ﲢﻘﻴﻖ ﻣﺼﺎﱀ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﻠﻴﺎ ( ).١١( ﻭ ﻳﺬﻫﺐ ) .ﺩ ﺳـﻌﺪ ﺣﻘـﻲ ﺗﻮﻓﻴﻖ ( ﺇﱃ ﺭﺑﻂ ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ﺑـ ) ﺍﻟﺘﺄﺛﲑ ( ﰲ ﺳﻠﻮﻙ ﺍﻟﻄﺮﻑ ﺍﳌﻘﺎﺑﻞ ﻟﻠﻘﻴـﺎﻡ ﺑﺄﻓﻌـﺎﻝ ﳏﺪﺩﺓ ﻻ ﻳﻘﻮﻡ ﺎ ﻣﻦ ﺗﻠﻘﺎﺀ ﻧﻔﺴﻪ ).٢١( ﻭﻳﺮﻯ ﺑﻌﺾ ﺍﻟﺒﺎﺣﺜﲔ ﺇﻥ ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ﻳﺘﻮﻟﺪ ﻋﻦ ﺛﻼﺛﻴﺔ ﳝﻜﻦ ﺍﻟﺘﻌﺒﲑ ﻋﻨـﻬﺎ ﺑﺪﻻﻟـﺔ ﺍﻟﻌﻼﻗﺔ ﺍﻟﺮﻳﺎﺿﻴﺔ: ) ﺍﻟﻘﺪﺭﺓ = ﺍﻹﺭﺍﺩﺓ ×ﺍﻟ ﻘﻮﺓ × ﺍﻹﺩﺭﺍﻙ ( ، ﻓﺎﻹﺭﺍﺩﺓ ﻫﻲ ﻣﺸﻴﺌﺔ ﺍﻷﻣﺔ ﺍﻟﺴﺎﻋﻴﺔ ﻟﻼﺭﺗﻘﺎﺀ ﺑﻘﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﻋﱪ ﺍﺳﺘﺜﻤﺎﺭ ﻋﻨﺎﺻﺮ ﺍﻟﻘﻮﺓ ﺍﳌﺎﺩﻳﺔ ﺑﺸـﻜﻞ ﺳـﻠﻴﻢ ﳜـﺪﻡ ﺍﻷﻫﺪﺍﻑ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺪﻭﻟﺔ ، ﻓﺎﻹﺭﺍﺩﺍﺓ ﺗﻮﻟﺪ ﺍﻟﺮﻏﺒﺔ ﰲ ﺍﻟﻌﻘﻞ ، ﻭﺗﻌﱪ ﻋـﻦ ﺍﳌﺼـﺎﱀ ﺍﻟﻮﻃﻨﻴﺔ ، ﻭﺗﺴﺒﻖ ﺍﻟﻘﺪﺭﺓ ﺍﻟﱵ ﲤﺜﻞ ﺍﻟﻌﻤﻞ ، ﻭﺗﻌﻜﺲ ﺍﻹﺳﺘﺮﺍﺗﻴﺠ ﻴﺔ ﺍﻟﱵ ﺗﻀﻤﻦ ﺍﻷﻫـﺪﺍﻑ ،ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟﺔ . ﺑﺬﻟﻚ ﺗﻜﻮﻥ ﺍﻹﺭﺍﺩﺓ ﺟﻮﻫﺮ ﺍﻟﻔﻌﻞ ﺍﳊﻘﻴﻘﻲ ﻓﻬﻲ ﻣﻜﻤﻦ ﺍﻟﻘﺪﺭﺓ ﻭﺗﺄﺛﲑﻫـﺎ ﺎ ﺇﻻ ﰲ ﻇﻞ ﺇﺭﺍﺩﺓ ﻭﺍﻋﻴﺔ ﳏﺮﻛﺔﺫﻟﻚ ﺃﻥ ﺍﻹﻣﻜﺎﻧﺎﺕ ﻭﺍﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ﻟﻠﺪﻭﻟﺔ ﻻ ﺗﺘﺄﻛﺪ ﻗﺪﺭ ﳍﺎ . ﺃﻣﺎ ﺍﻟﻘﻮﺓ ﻓﺘﻌﲏ ﺗﻌﺒﺌﺔ ﺍﻟﻄﺎﻗﺎﺕ ﻭﲢﺮﻳﻜﻬﺎ ﻭﺇﺑﺮﺍﺯﻫﺎ ﺇﱃ ﺣﻴﺰ ﺍﻟﻮﺟﻮﺩ ﻣﻦ ﺧﻼﻝ ﺍﻹﺭﺍﺩﺓ ﻭﺍﻟﻘﺮﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ . ﺃﻣﺎ ﺍﻹﺩﺭﺍﻙ ﻓﻴﻌﺘﱪ ﺣﺎﻓﺰ ﺍﻹﺭﺍﺩﺓ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺩﺍﻓﻌﺎﹰ ﻟﻠﺪﻭﻟـﺔ ﳓـﻮ ﺎ ، ﻓﺎﻹﺩﺭﺍﻙ ﻳﺮﺗﺒﻂ ﺑﺎﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﻻﺳﺘﻨﺒﺎﻁ ﻋﱪ ﺭﺑﻂ ﺍﳌﻌﺮﻓﺔ ﺑﻔﻜﺮﺓ ﺍﳌﻨﻔﻌـﺔﺗﻨﻤﻴﺔ ﻗﺪﺭﺍ ). ﺍﻟﻌﻤﻠﻴﺔ ، ﻭﻳﻌﺘﻤﺪ ﻋﻠﻰ ﺍﻹﺣﺴﺎﺱ ﻭﺍﻟﺘﺨﻴﻞ ٣١( ﻭﺍﻟﺮﺩﻉ ، ﻭﺍﻟﺘﻬﺪﻳﺪ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻌﻨﻒ ﰲ ﺣﺎﻟﺔ ﻋﺪﻡ ﺍﻻﻧﺼﻴﺎﻉ ﻹﺭﺍﺩﺓ ﻃﺮﻑ ﻣﻘﺘﺪﺭ ﻋﻠﻰ ﻃﺮﻑ ﺃﻗﻞ ﺍﻗﺘﺪﺍﺭﺍﹰ. ﻭﺇﱃ ﺟﺎﻧﺐ ﺍﻟﻘﺪﺭﺓ ﺍﳌﺎﺩﻳﺔ ﻫﻨﺎﻟﻚ ﺍﻟﻘﺪﺭﺓ ﺍﳌﻌﻨﻮﻳـﺔ ، ﺃﻱ ﻗـﺪﺭﺓ ، ﺍﻟﻔﺮﺩ ﻋﻠﻰ ﺍﻟﺘﺄﺛﲑ ﲝﻜﻢ ﺷﺨﺼﻴﺘﻪ ، ﻭﲤﻜﻨﻪ ﻣﻦ ﺇﻗﻨﺎﻉ ﺍﻵﺧـﺮﻳﻦ، ﺃﻭ ﲢﻴﻴـﺪ ﺃﻋﻤـﺎﳍﻢ ). ٣٧٣ ( ﻛﺮﺟﺎﻝ ﺍﻟﺪﻳﻦ ﻭﺍﻟﻘﺎﺩﺓ ﻭﺍﳊﻜﺎﻡ ٠١( ﻭﻫﻨﺎﻙ ﻣﻦ ﻳﻌﺮﻓﻬﺎ ﻋﻠﻰ ﺎﺍ : ) ﳑﺎﺭﺳ ﺔ ﻋﻤﻠﻴﺔ ﻣﻦ ﺧـﻼﻝ ﺍﻟﺘﻮﻇﻴـﻒ ﻭﺍﻻﺳـﺘﺜﻤﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﻟﻌﻮﺍﻣﻞ ﺍﻟﻘﻮﻯ ﺍﳌﺎﺩﻳﺔ ﻭﺍﳌﻌﻨﻮﻳﺔ ﻣﻦ ﻟﺪﻥ ﺻﺎﻧﻊ ﺍﻟﻘﺮﺍﺭ ﻭﺍﻟـﺬﻱ ﳝﻠـﻚ ﺇﺭﺍﺩﺓ ﻗﻮﻳﺔ ﻭﻓﻜﺮﺍﹰ ﻭﻋﻘﻼﹰ ﺭﺍﺟﺤﺎﹰ ﻭﺇﳝﺎﻧﺎﹰ ﺭﺍﺳﺨﺎﹰ ﺑﺎﳌﺒﺎﺩﺉ ﺍﻟﱵ ﻳﺴﻌﻰ ﻟﺘﺤﻘﻴﻘﻬﺎ ﻭ ﲢﻮﻳﻠﻬﺎ ﺇﱃ ﻓﻌﻞ ﻣﺆﺛﺮ ﳝﻜﻦ ﻣﻦ ﺧﻼﻟﻪ ﲢﻘﻴﻖ ﻣﺼﺎﱀ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﻠﻴﺎ ( ).١١( ﻭ ﻳﺬﻫﺐ ) .ﺩ ﺳـﻌﺪ ﺣﻘـﻲ ﺗﻮﻓﻴﻖ ( ﺇﱃ ﺭﺑﻂ ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ﺑـ ) ﺍﻟﺘﺄﺛﲑ ( ﰲ ﺳﻠﻮﻙ ﺍﻟﻄﺮﻑ ﺍﳌﻘﺎﺑﻞ ﻟﻠﻘﻴـﺎﻡ ﺑﺄﻓﻌـﺎﻝ ﺓﻻﻘ ﻠﻘ ﻔ ١ ﳏﺪﺩﺓ ﻻ ﻳﻘﻮﻡ ﺎ ﻣﻦ ﺗﻠﻘﺎﺀ ﻧﻔﺴﻪ ).٢١( ﻭﻳﺮﻯ ﺑﻌﺾ ﺍﻟﺒﺎﺣﺜﲔ ﺇﻥ ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ﻳﺘﻮﻟﺪ ﻋﻦ ﺛﻼﺛﻴﺔ ﳝﻜﻦ ﺍﻟﺘﻌﺒﲑ ﻋﻨـﻬﺎ ﺑﺪﻻﻟـﺔ ﺍﻟﻌﻼﻗﺔ ﺍﻟﺮﻳﺎﺿﻴﺔ: ) ﺍﻟﻘﺪﺭﺓ = ﺍﻹﺭﺍﺩﺓ ×ﺍﻟ ﻘﻮﺓ × ﺍﻹﺩﺭﺍﻙ ( ، ﻓﺎﻹﺭﺍﺩﺓ ﻫﻲ ﻣﺸﻴﺌﺔ ﺍﻷﻣﺔ ﺍﻟﺴﺎﻋﻴﺔ ﻟﻼﺭﺗﻘﺎﺀ ﺑﻘﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﻋﱪ ﺍﺳﺘﺜﻤﺎﺭ ﻋﻨﺎﺻﺮ ﺍﻟﻘﻮﺓ ﺍﳌﺎﺩﻳﺔ ﺑﺸـﻜﻞ ﺳـﻠﻴﻢ ﳜـﺪﻡ ﺍﻷﻫﺪﺍﻑ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺪﻭﻟﺔ ، ﻓﺎﻹﺭﺍﺩﺍﺓ ﺗﻮﻟﺪ ﺍﻟﺮﻏﺒﺔ ﰲ ﺍﻟﻌﻘﻞ ، ﻭﺗﻌﱪ ﻋـﻦ ﺍﳌﺼـﺎﱀ ﺍﻟﻮﻃﻨﻴﺔ ، ﻭﺗﺴﺒﻖ ﺍﻟﻘﺪﺭﺓ ﺍﻟﱵ ﲤﺜﻞ ﺍﻟﻌﻤﻞ ، ﻭﺗﻌﻜﺲ ﺍﻹﺳﺘﺮﺍﺗﻴﺠ ﻴﺔ ﺍﻟﱵ ﺗﻀﻤﻦ ﺍﻷﻫـﺪﺍﻑ ،ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟﺔ . ﺑﺬﻟﻚ ﺗﻜﻮﻥ ﺍﻹﺭﺍﺩﺓ ﺟﻮﻫﺮ ﺍﻟﻔﻌﻞ ﺍﳊﻘﻴﻘﻲ ﻓﻬﻲ ﻣﻜﻤﻦ ﺍﻟﻘﺪﺭﺓ ﻭﺗﺄﺛﲑﻫـﺎ ﺎ ﺇﻻ ﰲ ﻇﻞ ﺇﺭﺍﺩﺓ ﻭﺍﻋﻴﺔ ﳏﺮﻛﺔﺫﻟﻚ ﺃﻥ ﺍﻹﻣﻜﺎﻧﺎﺕ ﻭﺍﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ﻟﻠﺪﻭﻟﺔ ﻻ ﺗﺘﺄﻛﺪ ﻗﺪﺭ ﳍﺎ . ﺃﻣﺎ ﺍﻟﻘﻮﺓ ﻓﺘﻌﲏ ﺗﻌﺒﺌﺔ ﺍﻟﻄﺎﻗﺎﺕ ﻭﲢﺮﻳﻜﻬﺎ ﻭﺇﺑﺮﺍﺯﻫﺎ ﺇﱃ ﺣﻴﺰ ﺍﻟﻮﺟﻮﺩ ﻣﻦ ﺧﻼﻝ ﺍﻹﺭﺍﺩﺓ ﻭﺍﻟﻘﺮﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ . ﺃﻣﺎ ﺍﻹﺩﺭﺍﻙ ﻓﻴﻌﺘﱪ ﺣﺎﻓﺰ ﺍﻹﺭﺍﺩﺓ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺩﺍﻓﻌﺎﹰ ﻟﻠﺪﻭﻟـﺔ ﳓـﻮ ﺎ ، ﻓﺎﻹﺩﺭﺍﻙ ﻳﺮﺗﺒﻂ ﺑﺎﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﻻﺳﺘﻨﺒﺎﻁ ﻋﱪ ﺭﺑﻂ ﺍﳌﻌﺮﻓﺔ ﺑﻔﻜﺮﺓ ﺍﳌﻨﻔﻌـﺔﺗﻨﻤﻴﺔ ﻗﺪﺭﺍ ). ﺍﻟﻌﻤﻠﻴﺔ ، ﻭﻳﻌﺘﻤﺪ ﻋﻠﻰ ﺍﻹﺣﺴﺎﺱ ﻭﺍﻟﺘﺨﻴﻞ ٣١( ٦٧٣       ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ٧٧٣               ﺍﻷﻫﺪﺍﻑ ﻭﺍﳌﺼﺎﱀ ﺍﳊﻴﻮﻳﺔ ﺍﻟﱵ ﺗﺴﻌﻰ ﺇﻟﻴﻬﺎ ﺍﻟﺪﻭﻟﺔ ﻭﻳﻘﻊ ﺍﻷﻣﻦ ﻭﺍﻟﺴﻼﻡ ﻋﻠـﻰ . ﺭﺃﺱ ﻫﺬﻩ ﺍﻷﻫﺪﺍﻑ ، ﰒ ﺭﺳﻢ ﺳﻴﺎﺳﺔ ﻟﺘﺤﻘﻴﻘﻬﺎ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺴﻴﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﻟﺪﺍﺧﻠﻴﺔ ( : ﺑﺘﻌﺮﻳﻒ ﺑﺴﻴﻂ ﻟﻠﻘـﺪﺭﺓ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﺎ ﺍﻟﺘﻨﻔﻴﺬﻳـﺔﺩﺍﺧﻞ ﺣﺪﻭﺩ ﺍﻟﺪﻭﻟﺔ : ) ﻫﻮ ﺍﻟﻨﻬﺞ ﺍﻟﺬﻱ ﺗﺘﺒﻌﻪ ﺍﻟﺪﻭﻟﺔ ﺑﻜﺎﻓﺔ ﻫﻴﺌﺎ ﻭﺍﻟﺘﺸﺮﻳﻌﻴﺔ ﻭﺍﻟﻘﻀﺎﺋﻴﺔ ﻟﺘﻨﻈﻴﻢ ﺍ ﻟﻌﻼﻗﺔ ﺑﲔ ﺃﺑﻨﺎﺀ ﺍﻟﺸﻌﺐ ﻣﻦ ﺧﻼﻝ ﻧﻈﺎﻡ ﺳﻴﺎﺳﻲ ﺪﻑ ﺍﻟﻮﺻﻮﻝ ﺇﱃ ﺍﻟﻐﺎﻳﺔ ﺍﻟﻘﻮﻣﻴـﺔ ﰲ ﻣﺘﻮﺍﺯﻥ ﻣﺘﺤﺮﺭ ﻣﻦ ﺃﻱ ﺿﻐﻮﻁ ﺧﺎﺭﺟﻴﺔ . ( ﺍﻟﺒﻘﺎﺀ ﻭﺍﻟﺮﻓﺎﻫﻴﺔ ﻭﺍﻻﺯﺩﻫﺎﺭ ﻭﺍﳊﺮﻳﺔ  ﺍﻟﻘﺪﺭﺓ ﺍﳌﻌﻨﻮﻳﺔ : ﻫﻲ ﺣﺎﻟﺔ ﻧﻔﺴﻴﺔ ﻭﻋﻘﻠﻴﺔ ﻏﲑ ﳏﺴﻮﺳﺔ ﺗﻀﺎﻋﻒ ﺃﻭ ﲣﻔﺾ ﻣـﻦ ﺣﺠﻢ ﻭﻗﻴﻤﺔ ﺑﺎﻗﻲ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﻃﺒﻘﺎﹰ ﳌﺪﻯ ﻣﺎ ﺁ ﻟﺖ ﺇﻟﻴﻪ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﱵ ﺗﺘﺄﺛﺮ ﺘﻤﻊ ﻭﻣﺪﻯ ﺍﻧﻌﻜﺎﺳﻬﺎ ﻋﻠﻰ ﺃﳕـﺎﻁﺑﺎﻟﻘﻴﻢ ﻭﺍﻻﲡﺎﻫﺎﺕ ﻭﺍﳌﻌﺘﻘﺪﺍﺕ ﺍﻟﺴﺎﺋﺪﺓ ﰲ ﺍ ﺎ ﲣﺘﻠﻒ ﺑﺎﺧﺘﻼﻑ ﺍﻟﺒﻴﺌﺔ ﻭﺗﺒﻌـﺎﹰ ﻟﻠﻈـﺮﻭﻑ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔﺍﻟﺴﻠﻮﻙ ﻓﻴﻪ ، ﻛﻤﺎ ﺃ ﻭﺍﻻﺟﺘﻤﺎﻋﻴﺔ ﻭﺍﻟﺜﻘﺎﻓﻴﺔ ، ﻭﺗﻌﺘﱪ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺎﻣﻞ ﺍﳊﺎﺳﻢ ﰲ ﻛﻴﻔﻴﺔ ﺍﺳـﺘﺨﺪﺍﻡ ﻛﻞ ﻋﻨﺎﺻﺮ ﺍﻟﻘﻮ ﺓ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ، ﺇﺫ ﺗﻠﻘﻲ ﺑﻈﻼﳍﺎ ﺇﳚﺎﺑﺎﹰ ﻭﺳﻠﺒﺎﹰ ﻋﻠﻰ ﺍﺳﺘﺨﺪﺍﻡ . ﺑﺎﻗﻲ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ  ، ﻗﺪﺭﺍﺕ ﺃﺧﺮﻯ ﰲ ﺍﻟﺪﻭﻟﺔ : ) ﺍﻟﺘﻘﻨﻴﺔ ، ﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴﺔ ، ﺍﻟﺜﻘﺎﻓﻴـﺔ ، ﺍﻹﻋﻼﻣﻴـﺔ . ( ﻭﻏﲑﻫﺎ ﺍﶈﻮﺭ ﺍﻟﺜﺎﱐ / ﻋﻼﻗـﺔ ﺍﻟﻘـﺪﺭﺓ ﺑﺎﻹﺳﺘ ﺮﺍﺗﻴﺠﻴـﺔ ﻭﺃﺑﻌـﺎﺩﻫـﺎ ﺍﳌﻄﻠﺐ ﺍﻷﻭﻝ / ﻋﻼﻗـﺔ ﺍﻟﻘﺪﺭﺓ ﺑﺎﻹﺳﺘﺮﺍﺗﻴﺠﻴ. ﺔ ﺗﻌﺪ ﺍﻟﻘﺪﺭﺓ ﻇﺎﻫﺮﺓ ﻃﺒﻴﻌﻴﺔ ﻻﺯﻣﺖ ﺍﻟﺒﺸﺮﻳﺔ ﻣﻨﺬ ﺍﻟﻮﺟﻮﺩ ، ﺇﺫ ﺍﺳـﺘﺨﺪﻣﻬﺎ ﺍﻟﻔـﺮﺩ ﰲ . ﻣﺮﺍﺣﻞ ﺗﻄﻮﺭﻩ ﻟﻠﺒﺤﺚ ﻋﻦ ﻏﺎﻳﺎﺗﻪ ﻭﺍﳌﺘﻤﺜﻠﺔ ﺑﺎﻷﻣﻦ ﻭﺍﻟﺒﻘﺎﺀ ﻭﲢﺴﲔ ﺍﺣﻮﺍﻟـﻪ ﺍﳌﻌﻴﺸـﻴﺔ ﻭﺑﺘﻄﻮﺭ ﺍﻻﻧﺴﺎﻥ ﺍﺟﺘﻤﺎﻋﻴﺎﹰ ، ﻋﻤﻞ ﻋﻠﻰ ﺗﻨﻈﻴﻢ ﻧﻔﺴﻪ ﻭﺗﻨﻈﻴﻢ ﻋﻼﻗﺎﺗـﻪ ﻣـﻊ ﺍﳉﻤﺎﻋـﺎﺕ ﺍﳌﺨﺘﻠﻔﺔ ﺑﺸﻜﻞ ﻳﺆﻣﻦ ﻟﻪ ﲢﻘﻴﻖ ﻏﺎﻳﺎﺗﻪ ﺍﻋﻼﻩ ، ﻣﻌﺘﻤﺪﺍﹰ ﰲ ﺫﻟـﻚ ﻋﻠـﻰ ﻣـﺪﻯ ﺗـﻮﺍﻓﺮ ). ﺍﻻﻣﻜﺎﻧﺎﺕ ﻭﺍﻟﻘﺪﺭﺍﺕ ٥١( ﺍﻷﻫﺪﺍﻑ ﻭﺍﳌﺼﺎﱀ ﺍﳊﻴﻮﻳﺔ ﺍﻟﱵ ﺗﺴﻌﻰ ﺇﻟﻴﻬﺎ ﺍﻟﺪﻭﻟﺔ ﻭﻳﻘﻊ ﺍﻷﻣﻦ ﻭﺍﻟﺴﻼﻡ ﻋﻠـﻰ . ﺭﺃﺱ ﻫﺬﻩ ﺍﻷﻫﺪﺍﻑ ، ﰒ ﺭﺳﻢ ﺳﻴﺎﺳﺔ ﻟﺘﺤﻘﻴﻘﻬﺎ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺴﻴﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﻟﺪﺍﺧﻠﻴﺔ ( : ﺑﺘﻌﺮﻳﻒ ﺑﺴﻴﻂ ﻟﻠﻘـﺪﺭﺓ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﺎ ﺍﻟﺘﻨﻔﻴﺬﻳـﺔﺩﺍﺧﻞ ﺣﺪﻭﺩ ﺍﻟﺪﻭﻟﺔ : ) ﻫﻮ ﺍﻟﻨﻬﺞ ﺍﻟﺬﻱ ﺗﺘﺒﻌﻪ ﺍﻟﺪﻭﻟﺔ ﺑﻜﺎﻓﺔ ﻫﻴﺌﺎ ﻭﺍﻟﺘﺸﺮﻳﻌﻴﺔ ﻭﺍﻟﻘﻀﺎﺋﻴﺔ ﻟﺘﻨﻈﻴﻢ ﺍ ﻟﻌﻼﻗﺔ ﺑﲔ ﺃﺑﻨﺎﺀ ﺍﻟﺸﻌﺐ ﻣﻦ ﺧﻼﻝ ﻧﻈﺎﻡ ﺳﻴﺎﺳﻲ ﺪﻑ ﺍﻟﻮﺻﻮﻝ ﺇﱃ ﺍﻟﻐﺎﻳﺔ ﺍﻟﻘﻮﻣﻴـﺔ ﰲ ﻣﺘﻮﺍﺯﻥ ﻣﺘﺤﺮﺭ ﻣﻦ ﺃﻱ ﺿﻐﻮﻁ ﺧﺎﺭﺟﻴﺔ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ( ﺍﻟﺒﻘﺎﺀ ﻭﺍﻟﺮﻓﺎﻫﻴﺔ ﻭﺍﻻﺯﺩﻫﺎﺭ ﻭﺍﳊﺮﻳﺔ  ﺍﻟﻘﺪﺭﺓ ﺍﳌﻌﻨﻮﻳﺔ : ﻫﻲ ﺣﺎﻟﺔ ﻧﻔﺴﻴﺔ ﻭﻋﻘﻠﻴﺔ ﻏﲑ ﳏﺴﻮﺳﺔ ﺗﻀﺎﻋﻒ ﺃﻭ ﲣﻔﺾ ﻣـﻦ ﺣﺠﻢ ﻭﻗﻴﻤﺔ ﺑﺎﻗﻲ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﻃﺒﻘﺎﹰ ﳌﺪﻯ ﻣﺎ ﺁ ﻟﺖ ﺇﻟﻴﻪ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﱵ ﺗﺘﺄﺛﺮ ﺘﻤﻊ ﻭﻣﺪﻯ ﺍﻧﻌﻜﺎﺳﻬﺎ ﻋﻠﻰ ﺃﳕـﺎﻁﺑﺎﻟﻘﻴﻢ ﻭﺍﻻﲡﺎﻫﺎﺕ ﻭﺍﳌﻌﺘﻘﺪﺍﺕ ﺍﻟﺴﺎﺋﺪﺓ ﰲ ﺍ ﺎ ﲣﺘﻠﻒ ﺑﺎﺧﺘﻼﻑ ﺍﻟﺒﻴﺌﺔ ﻭﺗﺒﻌـﺎﹰ ﻟﻠﻈـﺮﻭﻑ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔﺍﻟﺴﻠﻮﻙ ﻓﻴﻪ ، ﻛﻤﺎ ﺃ ﻭﺍﻻﺟﺘﻤﺎﻋﻴﺔ ﻭﺍﻟﺜﻘﺎﻓﻴﺔ ، ﻭﺗﻌﺘﱪ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺎﻣﻞ ﺍﳊﺎﺳﻢ ﰲ ﻛﻴﻔﻴﺔ ﺍﺳـﺘﺨﺪﺍﻡ ﻛﻞ ﻋﻨﺎﺻﺮ ﺍﻟﻘﻮ ﺓ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ، ﺇﺫ ﺗﻠﻘﻲ ﺑﻈﻼﳍﺎ ﺇﳚﺎﺑﺎﹰ ﻭﺳﻠﺒﺎﹰ ﻋﻠﻰ ﺍﺳﺘﺨﺪﺍﻡ . ﺑﺎﻗﻲ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ  ﺍﻟﻘﺪﺭﺓ ﺍﳌﻌﻨﻮﻳﺔ : ﻫﻲ ﺣﺎﻟﺔ ﻧﻔﺴﻴﺔ ﻭﻋﻘﻠﻴﺔ ﻏﲑ ﳏﺴﻮﺳﺔ ﺗﻀﺎﻋﻒ ﺃﻭ ﲣﻔﺾ ﻣـﻦ ﺣﺠﻢ ﻭﻗﻴﻤﺔ ﺑﺎﻗﻲ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﻃﺒﻘﺎﹰ ﳌﺪﻯ ﻣﺎ ﺁ ﻟﺖ ﺇﻟﻴﻪ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﱵ ﺗﺘﺄﺛﺮ ﺘﻤﻊ ﻭﻣﺪﻯ ﺍﻧﻌﻜﺎﺳﻬﺎ ﻋﻠﻰ ﺃﳕـﺎﻁﺑﺎﻟﻘﻴﻢ ﻭﺍﻻﲡﺎﻫﺎﺕ ﻭﺍﳌﻌﺘﻘﺪﺍﺕ ﺍﻟﺴﺎﺋﺪﺓ ﰲ ﺍ ﺎ ﲣﺘﻠﻒ ﺑﺎﺧﺘﻼﻑ ﺍﻟﺒﻴﺌﺔ ﻭﺗﺒﻌـﺎﹰ ﻟﻠﻈـﺮﻭﻑ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔﺍﻟﺴﻠﻮﻙ ﻓﻴﻪ ، ﻛﻤﺎ ﺃ ﻭﺍﻻﺟﺘﻤﺎﻋﻴﺔ ﻭﺍﻟﺜﻘﺎﻓﻴﺔ ، ﻭﺗﻌﺘﱪ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺎﻣﻞ ﺍﳊﺎﺳﻢ ﰲ ﻛﻴﻔﻴﺔ ﺍﺳـﺘﺨﺪﺍﻡ ﻛﻞ ﻋﻨﺎﺻﺮ ﺍﻟﻘﻮ ﺓ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ، ﺇﺫ ﺗﻠﻘﻲ ﺑﻈﻼﳍﺎ ﺇﳚﺎﺑﺎﹰ ﻭﺳﻠﺒﺎﹰ ﻋﻠﻰ ﺍﺳﺘﺨﺪﺍﻡ . ﺑﺎﻗﻲ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ  ، ﻗﺪﺭﺍﺕ ﺃﺧﺮﻯ ﰲ ﺍﻟﺪﻭﻟﺔ : ) ﺍﻟﺘﻘﻨﻴﺔ ، ﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴﺔ ، ﺍﻟﺜﻘﺎﻓﻴـﺔ ، ﺍﻹﻋﻼﻣﻴـﺔ  ، ﻗﺪﺭﺍﺕ ﺃﺧﺮﻯ ﰲ ﺍﻟﺪﻭﻟﺔ : ) ﺍﻟﺘﻘﻨﻴﺔ ، ﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴﺔ ، ﺍﻟﺜﻘﺎﻓﻴـﺔ ، ﺍﻹﻋﻼﻣﻴـﺔ . ( ﻭﻏﲑﻫﺎ ﺗﻌﺪ ﺍﻟﻘﺪﺭﺓ ﻇﺎﻫﺮﺓ ﻃﺒﻴﻌﻴﺔ ﻻﺯﻣﺖ ﺍﻟﺒﺸﺮﻳﺔ ﻣﻨﺬ ﺍﻟﻮﺟﻮﺩ ، ﺇﺫ ﺍﺳـﺘﺨﺪﻣﻬﺎ ﺍﻟﻔـﺮﺩ ﰲ . ﻣﺮﺍﺣﻞ ﺗﻄﻮﺭﻩ ﻟﻠﺒﺤﺚ ﻋﻦ ﻏﺎﻳﺎﺗﻪ ﻭﺍﳌﺘﻤﺜﻠﺔ ﺑﺎﻷﻣﻦ ﻭﺍﻟﺒﻘﺎﺀ ﻭﲢﺴﲔ ﺍﺣﻮﺍﻟـﻪ ﺍﳌﻌﻴﺸـﻴﺔ ﻭﺑﺘﻄﻮﺭ ﺍﻻﻧﺴﺎﻥ ﺍﺟﺘﻤﺎﻋﻴﺎﹰ ، ﻋﻤﻞ ﻋﻠﻰ ﺗﻨﻈﻴﻢ ﻧﻔﺴﻪ ﻭﺗﻨﻈﻴﻢ ﻋﻼﻗﺎﺗـﻪ ﻣـﻊ ﺍﳉﻤﺎﻋـﺎﺕ ﺍﳌﺨﺘﻠﻔﺔ ﺑﺸﻜﻞ ﻳﺆﻣﻦ ﻟﻪ ﲢﻘﻴﻖ ﻏﺎﻳﺎﺗﻪ ﺍﻋﻼﻩ ، ﻣﻌﺘﻤﺪﺍﹰ ﰲ ﺫﻟـﻚ ﻋﻠـﻰ ﻣـﺪﻯ ﺗـﻮﺍﻓﺮ ). ﺍﻻﻣﻜﺎﻧﺎﺕ ﻭﺍﻟﻘﺪﺭﺍﺕ ٥١( ٨٧٣       ﺇﻥ ﺍﻟﻘﺪﺭﺓ ﲰﺔ ﻟﺼﻴﻘﺔ ﺑﺎﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻭﻣﻦ ﻏﲑ ﺍﳌﻤﻜﻦ ﻓﺼﻠﻬﺎ ﻋﻨـﻬﺎ ، ﻭﰲ ﺣﺎﻟـﺔ ﺎ . ﻭﻫـﻮﺍﻧﻔﺼﺎﳍﺎ ﻋﻨﻬﺎ ، ﺗﻜﻮﻥ ﺍﻷﺧﲑﺓ ﻏﲑ ﻗﺎﺩﺭﺓ ﻋﻠﻰ ﲢﻘﻴﻖ ﻣﺼﺎﳊﻬﺎ ﻭﺃﻫﺪﺍﻓﻬﺎ ﻭﻏﺎﻳﺎ ﻣﺎ ﺫﻫﺐ ﺍﻟﻴﻪ ﺍﳉ ﺎ : ) ﻋﻠـﻢ ﻭﻓـﻦﻨﺮﺍﻝ ) ﺃﻧﺪﺭﻳﻪ ﺑﻮﻓﺮ ( ﰲ ﺗﻌﺮﻳﻔﻪ ﻟﻺﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﻠﻰ ﺃ ﺍﺳﺘﺨﺪﺍﻡ ﺍﻟﻘﻮﺓ "ﺍﻟﻘﺪﺭﺓ" ﻟﺘﺤﻘﻴﻖ ﻫﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ( ﻭﺍﳌﻘﺼﻮﺩ ﻫﻨـﺎ "ﻗـﺪﺭﺍﺕ ﺍﻟﺪﻭﻟـﺔ ). "ﺍﻟﺸﺎﻣﻠﺔ ٦١( ﻓﺎﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﻤﻠﻴﺔ ﻓﻜﺮﻳﺔ ﻣﻨﻀﺒﻄﺔ ، ﺫﺍﺕ ﳐﺮﺟﺎﺕ ﻭﻃﺮﺍﺋﻖ ﻭﻭﺳﺎﺋﻞ ﳏﺪﺩﺓ ، ﻭﻫﻲ ﲣﺪﻡ ﺍﳍﺪﻑ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﻮﻃﲏ ، ﻭ ﲣﺪﻡ ﺍﻟﺴﻴﺎﺳﺔ ﰲ ﺇﻃﺎﺭ ﺍﻟﺘﻘﻠﺒﺎﺕ ﻭﺍﻟﺘﻌﻘﻴﺪﺍﺕ ﻭﺍﳍﻮﺍﺟﺲ ﻭﰲ ﻇﺮﻭﻑ ﻏﻤﻮﺽ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، . ﻭﻫﻲ ﲣﻀﻊ ﳌﻨﻄﻖ ﺍﻟﻨﻈﺮﻳـﺎﺕ ﺍﻹﺳـﺘﺮﺍﺗﻴﺠﻴﺔ . ﻭﻟﻌﻤﻠﻴﺔ ﻓﻜﺮﻳﺔ ﳏﺪﺩﺓ ﻭﻣﻨﻀﺒﻄﺔ ﺇﻥ ﺍﻟﻘﺪﺭﺓ ﲰﺔ ﻟﺼﻴﻘﺔ ﺑﺎﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻭﻣﻦ ﻏﲑ ﺍﳌﻤﻜﻦ ﻓﺼﻠﻬﺎ ﻋﻨـﻬﺎ ، ﻭﰲ ﺣﺎﻟـﺔ ﺎ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﻭﻫـﻮﺍﻧﻔﺼﺎﳍﺎ ﻋﻨﻬﺎ ، ﺗﻜﻮﻥ ﺍﻷﺧﲑﺓ ﻏﲑ ﻗﺎﺩﺭﺓ ﻋﻠﻰ ﲢﻘﻴﻖ ﻣﺼﺎﳊﻬﺎ ﻭﺃﻫﺪﺍﻓﻬﺎ ﻭﻏﺎﻳﺎ ﻣﺎ ﺫﻫﺐ ﺍﻟﻴﻪ ﺍﳉ ﺎ : ) ﻋﻠـﻢ ﻭﻓـﻦﻨﺮﺍﻝ ) ﺃﻧﺪﺭﻳﻪ ﺑﻮﻓﺮ ( ﰲ ﺗﻌﺮﻳﻔﻪ ﻟﻺﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﻠﻰ ﺃ ﺍﺳﺘﺨﺪﺍﻡ ﺍﻟﻘﻮﺓ "ﺍﻟﻘﺪﺭﺓ" ﻟﺘﺤﻘﻴﻖ ﻫﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ( ﻭﺍﳌﻘﺼﻮﺩ ﻫﻨـﺎ "ﻗـﺪﺭﺍﺕ ﺍﻟﺪﻭﻟـﺔ ). "ﺍﻟﺸﺎﻣﻠﺔ ٦١( ﻓﺎﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﻤﻠﻴﺔ ﻓﻜﺮﻳﺔ ﻣﻨﻀﺒﻄﺔ ، ﺫﺍﺕ ﳐﺮﺟﺎﺕ ﻭﻃﺮﺍﺋﻖ ﻭﻭﺳﺎﺋﻞ ﳏﺪﺩﺓ ، ﻭﻫﻲ ﲣﺪﻡ ﺍﳍﺪﻑ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﻮﻃﲏ ، ﻭ ﲣﺪﻡ ﺍﻟﺴﻴﺎﺳﺔ ﰲ ﺇﻃﺎﺭ ﺍﻟﺘﻘﻠﺒﺎﺕ ﻭﺍﻟﺘﻌﻘﻴﺪﺍﺕ ﻭﺍﳍﻮﺍﺟﺲ ﻭﰲ ﻇﺮﻭﻑ ﻏﻤﻮﺽ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، . ﻭﻫﻲ ﲣﻀﻊ ﳌﻨﻄﻖ ﺍﻟﻨﻈﺮﻳـﺎﺕ ﺍﻹﺳـﺘﺮﺍﺗﻴﺠﻴﺔ . ﻭﻟﻌﻤﻠﻴﺔ ﻓﻜﺮﻳﺔ ﳏﺪﺩﺓ ﻭﻣﻨﻀﺒﻄﺔ . ﻭﻟﻌﻤﻠﻴﺔ ﻓﻜﺮﻳﺔ ﳏﺪﺩﺓ ﻭﻣﻨﻀﺒﻄﺔ ﺇﻥ ﺍﻟﻌﻼﻗﺔ ﺑﲔ ﺍﻟﻘﺪﺭﺓ ﻭﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻻﺯﻣﺖ ﺍﻫﺘﻤﺎﻡ ﺍﳌﺨﺘﺼﲔ ﰲ ﺣﻘﻞ ﺍﻟﺪﺭﺍﺳـﺎﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﱪ ﺍﻟﻌﺼﻮﺭ ، ﻓﻬﻲ ﻋﻼﻗﺔ ﻣﺘﺼﻠﺔ ﻭﻣﺘﻼﺯﻣﺔ ، ﻓﻤﻦ ﺩﻭﻥ ﺍﻟﻘـﺪﺭﺓ ﻻ ﳝﻜـﻦ ﺍﳊﺪﻳﺚ ﻋﻦ ﻭﺟﻮﺩ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﻫﻲ ﻭﺳﻴﻠﺔ ﻭﻟﻴﺴﺖ ﻏﺎﻳﺔ . ﻓﻤـﻦ ﺧـﻼﻝ ، ﺍﻟﻘﺪﺭﺓ ﺗﺴﻌﻰ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻹﺣﺪﺍﺙ ﺗﺄﺛﲑﺍﺕ ﰲ ﺍﻟﺪﻭﻟﺔ ﻟﺘﺤﻘﻴﻖ ﻧﺘﺎﺋﺞ ﻣﺮﻏﻮﺏ ﻓﻴﻬـﺎ "ﲢﻘﻴﻖ ﺍﳌﺼﺎﱀ ﻭﺍﻷﻫﺪﺍﻑ ﺍﻟﻘﻮﻣﻴﺔ ﺍﻟﻌﻠﻴﺎ ﻭﻓﻖ ﺁﻃﺎﺭ ﺇﺳﺘﺮﺍﺗﻴﺠ ﻲ ﳏﺪﺩ" ﻭﺍﺳﺘﺒﻌﺎﺩ ﺍﻟﻨﺘـﺎﺋﺞ ). ﻏﲑ ﺍﳌﺮﻏﻮﺏ ﻓﻴﻬﺎ ٧١ ( ﲟﻌﲎ ﺍﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺗﻌﻜﺲ ﺗﻔﻀﻴﻼﹰ ﻣﻌﻴﻨﺎﹰ ﳊﺎﻟﺔ ﻣﺴﺘﻘﺒﻠﻴﺔ ﺃﻭ ﻟﻮﺿﻊ ﻣﺴﺘﻘﺒﻠﻲ ﰲ ﺑﻴﺌﺔ ﺍﻟﺪﻭﻟﺔ . ﻭﻫﻲ ﺗﻔﺘﺮﺽ ﺃﻧﻪ ﳝﻜﻦ ﺩﺭﺍﺳﺔ ﺑﻴﺌﺔ ﺍﻟﺪﻭﻟﺔ ﻭﺗﻘﻮﳝﻬﺎ ، ﻣﻊ ﺍﻷﺧﺬ ﰲ ﺍﻻﻋﺘﺒﺎﺭ ﺃﻧـﻪ ﻻ ﳝﻜﻦ ﺍﻟﺘﻨﺒﺆ ﺑﺎﳌﺴﺘﻘﺒﻞ . ﻭﻣﻦ ﺧﻼﻝ ﺍﺳﺘﺨﺪﺍﻡ ﺍﻟﺘﻘﻮﱘ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ، ﳝﻜـﻦ ﺍﻟﺘﻌـﺮﻑ ﻋﻠﻰ ﺍﻟﺘﻮﺟﻬﺎﺕ ، ﻭﺍﳌﺸﻜﻼﺕ ﻭﺍﻟﺘﻬﺪﻳﺪﺍﺕ ، ﻭﺍﳌﻤﺎﻧﻌﺎﺕ ، ﻭﺍﻟﻔﺮﺹ ، ﻭﺍﻟﻌﻮﺍﻣﻞ ﺍﻷﺧﺮﻯ ) ﺑﺎﺳﺘﺜﻨﺎﺀ ﺍﳌﺼﺎﺩﻓﺎﺕ ( ﺍﻟﱵ ﺗﺘﺄﺛﺮ ﻭﺗﺘﺸﻜﻞ ﺣﺴﺐ ﺍﻻﺧﺘﻴﺎﺭﺍﺕ ﺍﻟﱵ ﺗﺘﺒﻨﺎﻫﺎ ﺍﻟﺪﻭﻟﺔ ﻟﺘﻘﻮﻡ ﺑﻌﻤﻞ ﻣﺎ ﺃﻭ ﻻ ﺗﻘﻮﻡ . ﻭﻫﻜﺬﺍ ، ﻓﺈﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺗﺴﻌﻰ ﺇﱃ ﺍﻟﺘﺄﺛﲑ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﳌﺴـﺘﻘﺒﻠﻴﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺗﺸﻜﻴﻠﻬﺎ ، ﺑﺪﻻﹰ ﻣﻦ ﺍﻻﻛﺘﻔﺎﺀ ﺑﺒﺴﺎﻃﺔ ﺑﺈﺑﺪﺍﺀ . ﺭﺩﻭﺩ ﺃﻓﻌﺎﻝ ﻋﻠﻰ ﻣﺎ ﳛﺪﺙ ﻓﻴﻬـﺎ ﻭﳝﻜﻦ ﺗﻌﺮﻳﻒ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﻠﻰ ﻣﺴﺘﻮﻯ ﺍﻟﺪﻭﻟﺔ ﻭﺍﻟﻘﺪﺭﺍﺕ ﻛﻤﺎ ﻫﻮ ﺁﺕ : ) ﻓﻦ ﻭﻋﻠـﻢ ﺗﻄﻮﻳﺮ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ، ﺍﻻﺟﺘﻤﺎﻋﻴﺔ ﺍﻟﺴﻴﻜﻮﻟﻮﺟﻴﺔ ، ﻭﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﺳﺘﺨﺪﺍﻡ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺍﺕ ﳋﻠﻖ ﺗﺄﺛﲑﺍﺕ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﲢﻤﻲ ﺍﳌﺼﺎﱀ ﺍﻟﻮﻃﻨﻴـﺔ ﻭﺗﻌﺰﺯﻫـﺎ ﰲ ﺇﻥ ﺍﻟﻌﻼﻗﺔ ﺑﲔ ﺍﻟﻘﺪﺭﺓ ﻭﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻻﺯﻣﺖ ﺍﻫﺘﻤﺎﻡ ﺍﳌﺨﺘﺼﲔ ﰲ ﺣﻘﻞ ﺍﻟﺪﺭﺍﺳـﺎﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﱪ ﺍﻟﻌﺼﻮﺭ ، ﻓﻬﻲ ﻋﻼﻗﺔ ﻣﺘﺼﻠﺔ ﻭﻣﺘﻼﺯﻣﺔ ، ﻓﻤﻦ ﺩﻭﻥ ﺍﻟﻘـﺪﺭﺓ ﻻ ﳝﻜـﻦ ﺍﳊﺪﻳﺚ ﻋﻦ ﻭﺟﻮﺩ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﻫﻲ ﻭﺳﻴﻠﺔ ﻭﻟﻴﺴﺖ ﻏﺎﻳﺔ . ﻓﻤـﻦ ﺧـﻼﻝ ، ﺍﻟﻘﺪﺭﺓ ﺗﺴﻌﻰ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻹﺣﺪﺍﺙ ﺗﺄﺛﲑﺍﺕ ﰲ ﺍﻟﺪﻭﻟﺔ ﻟﺘﺤﻘﻴﻖ ﻧﺘﺎﺋﺞ ﻣﺮﻏﻮﺏ ﻓﻴﻬـﺎ "ﲢﻘﻴﻖ ﺍﳌﺼﺎﱀ ﻭﺍﻷﻫﺪﺍﻑ ﺍﻟﻘﻮﻣﻴﺔ ﺍﻟﻌﻠﻴﺎ ﻭﻓﻖ ﺁﻃﺎﺭ ﺇﺳﺘﺮﺍﺗﻴﺠ ﻲ ﳏﺪﺩ" ﻭﺍﺳﺘﺒﻌﺎﺩ ﺍﻟﻨﺘـﺎﺋﺞ ). ﻏﲑ ﺍﳌﺮﻏﻮﺏ ﻓﻴﻬﺎ ٧١ ( ﲟﻌﲎ ﺍﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺗﻌﻜﺲ ﺗﻔﻀﻴﻼﹰ ﻣﻌﻴﻨﺎﹰ ﳊﺎﻟﺔ ﻣﺴﺘﻘﺒﻠﻴﺔ ﺃﻭ ﻟﻮﺿﻊ ﻣﺴﺘﻘﺒﻠﻲ ﰲ ﺑﻴﺌﺔ ﺍﻟﺪﻭﻟﺔ . ﻭﻫﻲ ﺗﻔﺘﺮﺽ ﺃﻧﻪ ﳝﻜﻦ ﺩﺭﺍﺳﺔ ﺑﻴﺌﺔ ﺍﻟﺪﻭﻟﺔ ﻭﺗﻘﻮﳝﻬﺎ ، ﻣﻊ ﺍﻷﺧﺬ ﰲ ﺍﻻﻋﺘﺒﺎﺭ ﺃﻧـﻪ ﻻ ﳝﻜﻦ ﺍﻟﺘﻨﺒﺆ ﺑﺎﳌﺴﺘﻘﺒﻞ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﻭﻣﻦ ﺧﻼﻝ ﺍﺳﺘﺨﺪﺍﻡ ﺍﻟﺘﻘﻮﱘ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ، ﳝﻜـﻦ ﺍﻟﺘﻌـﺮﻑ ﻋﻠﻰ ﺍﻟﺘﻮﺟﻬﺎﺕ ، ﻭﺍﳌﺸﻜﻼﺕ ﻭﺍﻟﺘﻬﺪﻳﺪﺍﺕ ، ﻭﺍﳌﻤﺎﻧﻌﺎﺕ ، ﻭﺍﻟﻔﺮﺹ ، ﻭﺍﻟﻌﻮﺍﻣﻞ ﺍﻷﺧﺮﻯ ) ﺑﺎﺳﺘﺜﻨﺎﺀ ﺍﳌﺼﺎﺩﻓﺎﺕ ( ﺍﻟﱵ ﺗﺘﺄﺛﺮ ﻭﺗﺘﺸﻜﻞ ﺣﺴﺐ ﺍﻻﺧﺘﻴﺎﺭﺍﺕ ﺍﻟﱵ ﺗﺘﺒﻨﺎﻫﺎ ﺍﻟﺪﻭﻟﺔ ﻟﺘﻘﻮﻡ ﺑﻌﻤﻞ ﻣﺎ ﺃﻭ ﻻ ﺗﻘﻮﻡ . ﻭﻫﻜﺬﺍ ، ﻓﺈﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺗﺴﻌﻰ ﺇﱃ ﺍﻟﺘﺄﺛﲑ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﳌﺴـﺘﻘﺒﻠﻴﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺗﺸﻜﻴﻠﻬﺎ ، ﺑﺪﻻﹰ ﻣﻦ ﺍﻻﻛﺘﻔﺎﺀ ﺑﺒﺴﺎﻃﺔ ﺑﺈﺑﺪﺍﺀ . ﺭﺩﻭﺩ ﺃﻓﻌﺎﻝ ﻋﻠﻰ ﻣﺎ ﳛﺪﺙ ﻓﻴﻬـﺎ ﻭﳝﻜﻦ ﺗﻌﺮﻳﻒ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﻠﻰ ﻣﺴﺘﻮﻯ ﺍﻟﺪﻭﻟﺔ ﻭﺍﻟﻘﺪﺭﺍﺕ ﻛﻤﺎ ﻫﻮ ﺁﺕ : ) ﻓﻦ ﻭﻋﻠـﻢ ﺗﻄﻮﻳﺮ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ، ﺍﻻﺟﺘﻤﺎﻋﻴﺔ ﺍﻟﺴﻴﻜﻮﻟﻮﺟﻴﺔ ، ﻭﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﺳﺘﺨﺪﺍﻡ ﻫﺬﻩ ﺍﻟﻘﺪﺭﺍﺕ ﳋﻠﻖ ﺗﺄﺛﲑﺍﺕ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﲢﻤﻲ ﺍﳌﺼﺎﱀ ﺍﻟﻮﻃﻨﻴـﺔ ﻭﺗﻌﺰﺯﻫـﺎ ﰲ ٩٧٣       ﺍﻟﺒﻴﺌﺔ ﺍﳌﺴﺘﻬﺪﻓﺔ ﲟﺎ ﻳﺘﻮﺍﻓﻖ ﻭﺗﻮﺟﻴﻬﺎﺕ ﺍﻟﺴﻴﺎﺳﺔ . ﻭﲢﻘﻴﻖ ﺍﻟﺘﺂﺯﺭ ﻭﺍﻟﺘﻨﺎﺳﻖ ﺑﲔ ﺍﻷﻫـﺪﺍﻑ ﻭﺍﻟﻄﺮﺍﺋﻖ ﻭﺍﳌﻮﺍﺭﺩ ، ﻟﺰﻳﺎﺩﺓ ﺍﺣﺘﻤﺎﻻﺕ ﳒﺎﺡ ﺍﻟﺴﻴﺎﺳﺔ ﻭﲢﻘﻴﻖ ﺍﻟﻨﺘﺎﺋﺞ ﺍﳌﺮﺟﻮﺓ ، ﻭﻟﺘﻘﻠﻴﺺ ). ( ﻓﺮﺹ ﻓﺸﻞ ﺍﻟﺴﻴﺎﺳﺔ ٨١( ﺍﻟﺒﻴﺌﺔ ﺍﳌﺴﺘﻬﺪﻓﺔ ﲟﺎ ﻳﺘﻮﺍﻓﻖ ﻭﺗﻮﺟﻴﻬﺎﺕ ﺍﻟﺴﻴﺎﺳﺔ . ﻭﲢﻘﻴﻖ ﺍﻟﺘﺂﺯﺭ ﻭﺍﻟﺘﻨﺎﺳﻖ ﺑﲔ ﺍﻷﻫـﺪﺍﻑ ﻭﺍﻟﻄﺮﺍﺋﻖ ﻭﺍﳌﻮﺍﺭﺩ ، ﻟﺰﻳﺎﺩﺓ ﺍﺣﺘﻤﺎﻻﺕ ﳒﺎﺡ ﺍﻟﺴﻴﺎﺳﺔ ﻭﲢﻘﻴﻖ ﺍﻟﻨﺘﺎﺋﺞ ﺍﳌﺮﺟﻮﺓ ، ﻭﻟﺘﻘﻠﻴﺺ ). ( ﻓﺮﺹ ﻓﺸﻞ ﺍﻟﺴﻴﺎﺳﺔ ٨١( ﻛﻤﺎ ﻭﻳﺮﺗﺒﻂ ﻣﻔﻬﻮﻡ ﺍﻟﻘﺪﺭﺓ ، ﺑـ ) ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ( ، ﻟﻴﺲ ﻷ ﻥ ﺍﻟﻘﺪﺭﺓ ﺗﻨﺒـﺊ ﻋﻦ ﺇﻣﻜﺎﻧﻴﺔ ﲢﻘﻴﻖ ﺍﻷﻫﺪﺍﻑ ) ﺍﻟﻮﻃﻨﻴﺔ / ﺍﻟﻘﻮﻣﻴﺔ ( ﺑﻄﺮﻕ ﳐﺘﻠﻔﺔ ، ﺭﲟﺎ ﺃﻭﳍـﺎ ﺃﻭ ﺁﺧﺮﻫـﺎ ﺎ ﲤﺜﻞ ﻧﺘﺎﺝ ﺍﻟﺸـﻌﻮﺭ ﺑﺎﻻﻧﺴـﺠﺎﻡ ، ﻭﺍﻟﺘﻨـﺎﻏﻢ ﻣـﻊ ﺗﻄـﻮﺭﺍﺳﺘﺨﺪﺍﻡ ﺍﻟﻘﻮﺓ ، ﺑﻞ ﻷ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺭﲟﺎ ﺗﻜﻮﻥ ﺣﺎﻣﻠﺔ ﳍﺎ ﰲ ﻛﻞ ﻣﺎ ﻟﻪ ﺻﻠﺔ ﺑﺎﻟﺸﺆﻭﻥ ﺍﳋﺎﺭﺟﻴﺔ "ﺍﻟﺪﻭﻟﻴﺔ" ﻛﻤـﺎ ﻳﻘﻮﻝ ﺑﺬﻟ ﻚ "ﺑﻮﻝ ﻛﻴﻨﺪﻱ" . ﻋﻨﺪﻫﺎ ﺗﺒﺪﻭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺃﻭﺳﻊ ﻭﺃﺭﺣﺐ ﻟﻴﻤﺘﺪ ﺃﺛﺮﻫﺎ ﺇﱃ ﻣﺎ ﺑﻌﺪ ﺃﻭﻗﺎﺕ ﺍﳊﺮﺏ . ﺃﻭ ﺍﻷﺻﺢ ﺗﺒﺪﻭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺑﺴﺒﺐ ﺣﻀﻮﺭ ﺍﻟﻘﺪﺭﺓ ﻓﻨـﺎﹰ ﻹﺩﺍﺭﺓ ﺍﻟﺪﻭﻟﺔ ﰲ ﲨﻴﻊ ﺍﻷﻭﻗﺎﺕ ﻋﻠﻰ ﺣﺪ ) ﻗـﻮﻝ "ﺃﺩﻭﻳـﻦ ﻣﻴـﺪ ﺃﻳـﺮﻝ" ﰲ ﻛﺘﺎﺑـﻪ ﺻـﻨﺎﻉ ﺍﻹﺳ(ﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻃﺎﳌﺎ ﺑﺪﺕ ﺍﻟﻘﺪﺭﺓ ﺍﳉﻮﻫﺮ ﺍﻷﺻﻴﻞ .ﻟﻸﺩﺍﺀ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻟﺸﺎﻣﻞ ﻭﻫﻜﺬﺍ ﺗﺒﺪﻭ ﺍﻟﻘﺪﺭﺓ ﻛﻤﻌﻄﻰ ، ﻫﻲ ﺍﳌﺴﺆﻭﻟﺔ ﻋﻦ ﺍﻛﺘﺸـﺎﻑ ﺍﻷﻫـﺪﺍﻑ ﻭﺍﳌﺼـﺎﱀ ﺎ ﺍﳌﺴﺘﻮﺩﻉ ﺍﻟﺬﻱ ﻳﻔﺼـﺢ ﻋـﻦ ﺃﺳـﺮﺍﺭﻭﺗﻌﺮﻳﻔﻬﺎ ﻭﲢﺪﻳﺪ ﺃﳕﺎﻃﻬﺎ ﻭﺳﺒﻞ ﲢﻘﻴﻘﻬﺎ ، ﻛﻮ ﺎ ﺍﻟﺪﻭﻟﺔ ﻟﻘﻮﺍﻫﺎ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻟﺴﻴﻜﻮﻟﻮﺟﻴﺔ ، ﻓﻀﻼﹰ ﻋـﻦ ﺍﻟﺘﻮﻇﻴﻒ ﺍﻟﱵ ﺗﻘﻮﻡ ﺍﻟﻌﺴﻜ ﺎ ﺗﺸﻴﻴﺪ ﻟﻺﺳﺘﺮﺍﺗﻴﺠﻴﺔﺮﻳﺔ ﻭﻭﺿﻌﻬﺎ ﳏﻞ ﺍﻻﺳﺘﺨﺪﺍﻡ ، ﺣﱴ ﺗﺒﺪﻭ ﺗﻠﻚ ﺍﻟﻌﻤﻠﻴﺔ ﻭﻛﺄ ).ﺍﻟﻘﻮﻣﻴﺔ ٩١( ﻭﰲ ﺁﻃﺎﺭ ﺍﻟﻌﻼﻗﺔ ﺑﲔ ﺍﻟﻘﺪﺭﺓ ﻭﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﺍﺷﺎﺭ ﺍﻟﻌﺪﻳﺪ ﻣﻦ ﺍﻟﺒﺎﺣﺜﲔ ﺇﱃ ﻭﺟـﻮﺩ ﻋﻼﻗﺔ ﻃﺮﺩﻳﺔ ﻣﻮﺟﺒﺔ ﺑﲔ ﺣﺠﻢ ﺍﻟﻘﺪﺭﺓ ﺍﳌﺘﺎﺡ ﻟﺪﻭﻟﺔ ﻣﺎ ﻭﻓﺎﻋﻠﻴﺘﻬﺎ ﺍﻹﺳـﺘﺮﺍﺗﻴﺠﻴﺔ ، ﺇﺫ ﺃﻥ ﺍﻟﺪﻭﻝ ﺍ. ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﻟﱵ ﲤﺘﻠﻚ ﻗﻮﺓ ﻛﺎﻓﻴﺔ ﻟﺘﻮﻟﻴﺪ ﺍﻟﺘﺄﺛﲑ ﺗﻜﻮﻥ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺘﻬﺎ ﺍﻛﺜﺮ ﻧﺸﺎﻃﺎﹰ ﻭﻓﺎﻋﻠﻴﺔ ﻭﺃﺧﲑﺍﹰ ﳝﻜﻦ ﺍﻟﻘﻮﻝ ﺃﻥ ﺍﻟﻌﻼﻗﺔ ﺑﲔ ﺍﻟﻘـﺪﺭﺓ ﻭﺍﻹﺳـﺘ ﺮﺍﺗﻴﺠﻴﺔ ﺗﺘﻤﺤـﻮﺭ ﰲ ﺑﻌـﺪﻳﻦ ﺭﻭﻹ ﲔ ﻮ ﻦ ﻭﲑ ﻦ ﻮﺭﰲ ﺮ :ﺍﺳﺎﺳﻴﲔ)٠٢( - ، ﺍﻟﺒﻌﺪ ﺍﻷﻭﻝ / ﻳﺘﻤﺜﻞ ﰲ ﺩﻭﺭ ﺍﻟﻘـﺪﺭﺓ ﰲ ﺗـﺄﻣﲔ ﺧﺎﺻـﻴﱵ ) ﺍﻻﺳـﺘﻤﺮﺍﺭﻳﺔ ﻭﺍﻟﻔﺎﻋﻠﻴﺔ ( ﻟﻺﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍ ﻟﱵ ﲞﻼﻓﻬﺎ ﻻ ﳝﻜﻦ ﺍﳊﺪﻳﺚ ﻋﻦ ﻭﺟﻮﺩ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ . ﻋﻠﻰ ﺃﺭﺽ ﺍﻟﻮﺍﻗﻊ - ، ﺍﻟﺒﻌﺪ ﺍﻷﻭﻝ / ﻳﺘﻤﺜﻞ ﰲ ﺩﻭﺭ ﺍﻟﻘـﺪﺭﺓ ﰲ ﺗـﺄﻣﲔ ﺧﺎﺻـﻴﱵ ) ﺍﻻﺳـﺘﻤﺮﺍﺭﻳﺔ ﻭﺍﻟﻔﺎﻋﻠﻴﺔ ( ﻟﻺﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍ ﻟﱵ ﲞﻼﻓﻬﺎ ﻻ ﳝﻜﻦ ﺍﳊﺪﻳﺚ ﻋﻦ ﻭﺟﻮﺩ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ - ، ﺍﻟﺒﻌﺪ ﺍﻷﻭﻝ / ﻳﺘﻤﺜﻞ ﰲ ﺩﻭﺭ ﺍﻟﻘـﺪﺭﺓ ﰲ ﺗـﺄﻣﲔ ﺧﺎﺻـﻴﱵ ) ﺍﻻﺳـﺘﻤﺮﺍﺭﻳﺔ ﻭﺍﻟﻔﺎﻋﻠﻴﺔ ( ﻟﻺﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍ ﻟﱵ ﲞﻼﻓﻬﺎ ﻻ ﳝﻜﻦ ﺍﳊﺪﻳﺚ ﻋﻦ ﻭﺟﻮﺩ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ . ﻋﻠﻰ ﺃﺭﺽ ﺍﻟﻮﺍﻗﻊ ٠٨٣       - ﺍﻟﺒﻌﺪ ﺍﻟﺜﺎﱐ / ﺗﻘﺪﻡ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﻘﺪﺭﺓ ﺁﻃﺎﺭﺍﹰ ﻣﻦ ﺍﻟﱪﳎﺔ ﻭﺍﻟﺘﻨﻈـﻴﻢ ﻳـﺆﻣﻦ ﲢﺪﻳﺪ ﺍﳌﻬﺎﻡ ﻭﺍﻷﻫﺪﺍﻑ ﺍﳌﻄﻠﻮﺑﺔ ﻣـﻦ+ ﺍﻟﺘﻮﻇﻴﻒ ﺍﻻﻣﺜﻞ ﻟﻠﻤﻮﺍﺭﺩ ﻭﺍﻻﻣﻜﺎﻧﺎﺕ ﺎ ، ﻓـ ) ﺍﻟﻘﺪﺭﺓ ( ﻫﻨﺎ ﻣﺼـﺪﺭ ﻟﻼﺳـﺘﻤﺮﺍ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻘﻴﺎﻡ )ﺭﻳﺔ ﻭﺍﻟﻔﺎﻋﻠﻴـﺔ ﻭ . ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ( ﻣﺼﺪﺭ ﻟﻠﺘﻨﻈﻴﻢ ﻭﺍﻟﺘﻮﺟﻴﻪ ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺍﺑﻌـﺎﺩ ﺍﻟﻘـﺪﺭﺓ ﺍﻹﺳﺘـﺮﺍﺗﻴﺠﻴـﺔ ) : ﻭﻣﻦ ﺍﺑﻌﺎﺩ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺮﺋﻴﺴﺔ ﻫﻲ ١٢( ١- ﺍﻟﻘﺪﺭﺓ ﺍﻻﺩﺭﺍﻛﻴﺔ : ﺍﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﺩﺭﺍﻛﻴﺔ ﺗﺴﺎﻋﺪ ﺍﻟﺪﻭﻟﺔ ﻋﻠﻰ ﺍﻟﻜﺸـﻒ ﻋﻤـﺎ ﻣﻮﺟﻮﺩ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺘﺤ. ﺪﻳﺪ ﺮﻙ ﺍﺯﺍﺋﻬﺎ ﺳﻮﺍﺀ ﻛﺎﻧﺖ ﻓﺮﺻﺔ ﺃﻡ ٢- ﺍﻟﻘﺪﺭﺓ ﺍﻻﺳﺘﻴﻌﺎﺑﻴﺔ : ﻣﻦ ﺧﻼﻝ ﺍﻟﺘﻔﺎﻋﻞ ﻣﻊ ﺍﻟﺒﻴﺌﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺍﺳـﺘﻴﻌﺎﺏ . ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍﳉﺪﻳﺪﺓ ﻭﺍﺳﺘﺜﻤﺎﺭﻫﺎ ﻣﻦ ﻗﺒﻞ ﺻﺎﻧﻊ ﺍﻟﻘﺮﺍﺭ ٣- ﺍﻟﻘﺪﺭﺓ ﺍﻟﺘﺤﻮﻳﻠﻴﺔ : ﲤﺜﻞ ﺍﻟﻘﺪﺭﺓ ﺍﻟﺘﺤﻮﻳﻠﻴﺔ ﻣﺆﺷﺮﺍﹰ ﺗﺴﺘﺮﺷﺪ ﺑﻪ ﺍﻟﺪﻭﻟﺔ ﻟﺘﺤﺪﻳـﺪ ﻧﻮﻉ ﺍﻟﺘﺤﻮﻻﺕ ﺍﳌﻨﺎﺳﺒﺔ ﺍﻟﱵ ﺗﻘﺘﻀﻴﻬﺎ ﺍﻟﻈﺮﻭﻑ ﳉﻌﻞ ﺍﻟﺪﻭﻟﺔ ﲢﻘﻖ ﺍﻗﺼﻰ ﺩﺭﺟﺔ . ﻣﻦ ﺍﻻﺳﺘﺠﺎﺑﺔ ﻟﺒﻴﺌﺘﻬﺎ ٤- ﻓﺮﻳﻖ ﺍﻟﻌﻤﻞ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ : ﻋﺪﺩ ﻣﻦ ﺍﳋﱪﺍﺀ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﲔ ﻭﺍﻟﺬﻳﻦ ﳝﺘﻠﻜـﻮﻥ ﻣﺆﻫﻼﺕ ) ﺍﻟﻘﺎﺑﻠﻴﺔ ، ﺍﻟﻜﻔﺎﺀﺓ ، ﺍﳌﻌﺮﻓﺔ ، ﺍﻟﺘﻌﺎﻭﻥ ، ﺍﻟﻮﻗﺖ "ﻣﻦ ﺍﻟﻌﻮﺍﻣﻞ ﺍﳊﺎﲰﺔ ﰲ ﺻﻨﻊ ﺍﻟﻘﺮﺍﺭ" ( ﻭﻳﻜﻮﻧﻮﺍ ﻣﻠﺘﺰﻣﲔ ﺑﺎﺩﺍﺀ ﺃﻫﺪﺍﻑ ﻋﺎﻣـﺔ ، ﻭﻋﻼﻗـﺎﺕ ﻋﻤـﻞ . ﻣﺘﺒﺎﺩﻟﺔ ﺑﻴﻨﻬﻢ ﺍﶈـﻮﺭ ﺍﻟﺜﺎﻟﺚ: ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻮﺍﺟﻪ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﰲ ﺍﻟﺒﺪﺍﻳﺔ ﳝﻜﻦ ﺍﻟﻘﻮﻝ ، ﺍﻥ ﻣﺼﻄﻠﺢ ﺍﳌﻤﺎﻧﻌﺎﺕ ﰲ ﺍﻟﻠﻐﺔ ﺍﻟﻌﺮﺑﻴﺔ ، ﻛﻤﺼـﺪﺭ ، ﻋـﻦ ﺻﻴﻐﺔ ﻣﺒﺎﻟﻐﺔ ﺗﺸﺘﻖ ﻣﻦ ﺍﻟﻔﻌﻞ ) ﻣﻨﻊ ( ﺍﻟﺸﻲﺀ ﲟﻌﲎ ﻧﺎﺯﻋﻪ ﻭﻣﻨﻌﻪ ﺇﻳﺎﻩ ، ﻭﻛﺬﻟﻚ ﺟﻌﻠﻪ ﻏﲑ ). ﻗﺎﺩﺭ ﻋﻠﻰ ﲢﻘﻴﻖ ﺍﻷﻫﺪﺍﻑ ٢٢ ( ﻭﻗﺪ ﻭﺭﺩ ﻣﻔﻬﻮﻡ ﺍﳌﻨﻊ ﰲ ﺍﻟﻘـﺮﺁﻥ ﺍﻟﻜـﺮﱘ ﰲ ﻗﻮﻟـﻪ ﺗﻌـﺎﱃ : ﺑﺴـﻢ ﺍﷲ ﺍﻟـﺮ ﲪﻦ ﺍﻟﺮﺣﻴﻢ:)ﻴﻢﹴ ﺃﹶﺛﺪﺘﻌﺮﹺ ﻣﻴﺎﻉﹴ ﻟﱢﻠﹾﺨﻨﻣ( )،٣٢ (ﻭﻗﻮﻟﻪ ﺗﻌﺎﱃ: : ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ ) ـﺎﻉﹴﻨﻣ ﺮﹺﻳﺐﹴ ﻣﺪﺘﻌﺮﹺ ﻣﻴﻟﱢﻠﹾﺨ). ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ (٤٢( - ﺍﻟﺒﻌﺪ ﺍﻟﺜﺎﱐ / ﺗﻘﺪﻡ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﻘﺪﺭﺓ ﺁﻃﺎﺭﺍﹰ ﻣﻦ ﺍﻟﱪﳎﺔ ﻭﺍﻟﺘﻨﻈـﻴﻢ ﻳـﺆﻣﻦ ﲢﺪﻳﺪ ﺍﳌﻬﺎﻡ ﻭﺍﻷﻫﺪﺍﻑ ﺍﳌﻄﻠﻮﺑﺔ ﻣـﻦ+ ﺍﻟﺘﻮﻇﻴﻒ ﺍﻻﻣﺜﻞ ﻟﻠﻤﻮﺍﺭﺩ ﻭﺍﻻﻣﻜﺎﻧﺎﺕ ﺎ ، ﻓـ ) ﺍﻟﻘﺪﺭﺓ ( ﻫﻨﺎ ﻣﺼـﺪﺭ ﻟﻼﺳـﺘﻤﺮﺍ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻘﻴﺎﻡ )ﺭﻳﺔ ﻭﺍﻟﻔﺎﻋﻠﻴـﺔ ﻭ . ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ( ﻣﺼﺪﺭ ﻟﻠﺘﻨﻈﻴﻢ ﻭﺍﻟﺘﻮﺟﻴﻪ ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺍﺑﻌـﺎﺩ ﺍﻟﻘـﺪﺭﺓ ﺍﻹﺳﺘـﺮﺍﺗﻴﺠﻴـﺔ ) : ﻭﻣﻦ ﺍﺑﻌﺎﺩ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺮﺋﻴﺴﺔ ﻫﻲ ١٢( ١- ﺍﻟﻘﺪﺭﺓ ﺍﻻﺩﺭﺍﻛﻴﺔ : ﺍﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﺩﺭﺍﻛﻴﺔ ﺗﺴﺎﻋﺪ ﺍﻟﺪﻭﻟﺔ ﻋﻠﻰ ﺍﻟﻜﺸـﻒ ﻋﻤـﺎ ﻣﻮﺟﻮﺩ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﺘﺤ. ﺪﻳﺪ ﺮﻙ ﺍﺯﺍﺋﻬﺎ ﺳﻮﺍﺀ ﻛﺎﻧﺖ ﻓﺮﺻﺔ ﺃﻡ ٢- ﺍﻟﻘﺪﺭﺓ ﺍﻻﺳﺘﻴﻌﺎﺑﻴﺔ : ﻣﻦ ﺧﻼﻝ ﺍﻟﺘﻔﺎﻋﻞ ﻣﻊ ﺍﻟﺒﻴﺌﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺍﺳـﺘﻴﻌﺎﺏ . ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍﳉﺪﻳﺪﺓ ﻭﺍﺳﺘﺜﻤﺎﺭﻫﺎ ﻣﻦ ﻗﺒﻞ ﺻﺎﻧﻊ ﺍﻟﻘﺮﺍﺭ ٣- ﺍﻟﻘﺪﺭﺓ ﺍﻟﺘﺤﻮﻳﻠﻴﺔ : ﲤﺜﻞ ﺍﻟﻘﺪﺭﺓ ﺍﻟﺘﺤﻮﻳﻠﻴﺔ ﻣﺆﺷﺮﺍﹰ ﺗﺴﺘﺮﺷﺪ ﺑﻪ ﺍﻟﺪﻭﻟﺔ ﻟﺘﺤﺪﻳـﺪ ﻧﻮﻉ ﺍﻟﺘﺤﻮﻻﺕ ﺍﳌﻨﺎﺳﺒﺔ ﺍﻟﱵ ﺗﻘﺘﻀﻴﻬﺎ ﺍﻟﻈﺮﻭﻑ ﳉﻌﻞ ﺍﻟﺪﻭﻟﺔ ﲢﻘﻖ ﺍﻗﺼﻰ ﺩﺭﺟﺔ . ﻣﻦ ﺍﻻﺳﺘﺠﺎﺑﺔ ﻟﺒﻴﺌﺘﻬﺎ ٤- ﻓﺮﻳﻖ ﺍﻟﻌﻤﻞ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ : ﻋﺪﺩ ﻣﻦ ﺍﳋﱪﺍﺀ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﲔ ﻭﺍﻟﺬﻳﻦ ﳝﺘﻠﻜـﻮﻥ ﻣﺆﻫﻼﺕ ) ﺍﻟﻘﺎﺑﻠﻴﺔ ، ﺍﻟﻜﻔﺎﺀﺓ ، ﺍﳌﻌﺮﻓﺔ ، ﺍﻟﺘﻌﺎﻭﻥ ، ﺍﻟﻮﻗﺖ "ﻣﻦ ﺍﻟﻌﻮﺍﻣﻞ ﺍﳊﺎﲰﺔ ﰲ ﺻﻨﻊ ﺍﻟﻘﺮﺍﺭ" ( ﻭﻳﻜﻮﻧﻮﺍ ﻣﻠﺘﺰﻣﲔ ﺑﺎﺩﺍﺀ ﺃﻫﺪﺍﻑ ﻋﺎﻣـﺔ ، ﻭﻋﻼﻗـﺎﺕ ﻋﻤـﻞ . ﻣﺘﺒﺎﺩﻟﺔ ﺑﻴﻨﻬﻢ - ﺍﻟﺒﻌﺪ ﺍﻟﺜﺎﱐ / ﺗﻘﺪﻡ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﻘﺪﺭﺓ ﺁﻃﺎﺭﺍﹰ ﻣﻦ ﺍﻟﱪﳎﺔ ﻭﺍﻟﺘﻨﻈـﻴﻢ ﻳـﺆﻣﻦ ﲢﺪﻳﺪ ﺍﳌﻬﺎﻡ ﻭﺍﻷﻫﺪﺍﻑ ﺍﳌﻄﻠﻮﺑﺔ ﻣـﻦ+ ﺍﻟﺘﻮﻇﻴﻒ ﺍﻻﻣﺜﻞ ﻟﻠﻤﻮﺍﺭﺩ ﻭﺍﻻﻣﻜﺎﻧﺎﺕ ﺎ ، ﻓـ ) ﺍﻟﻘﺪﺭﺓ ( ﻫﻨﺎ ﻣﺼـﺪﺭ ﻟﻼﺳـﺘﻤﺮﺍ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻘﻴﺎﻡ )ﺭﻳﺔ ﻭﺍﻟﻔﺎﻋﻠﻴـﺔ ﻭ . ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ( ﻣﺼﺪﺭ ﻟﻠﺘﻨﻈﻴﻢ ﻭﺍﻟﺘﻮﺟﻴﻪ - ﺍﻟﺒﻌﺪ ﺍﻟﺜﺎﱐ / ﺗﻘﺪﻡ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻠﻘﺪﺭﺓ ﺁﻃﺎﺭﺍﹰ ﻣﻦ ﺍﻟﱪﳎﺔ ﻭﺍﻟﺘﻨﻈـﻴﻢ ﻳـﺆﻣﻦ ﲢﺪﻳﺪ ﺍﳌﻬﺎﻡ ﻭﺍﻷﻫﺪﺍﻑ ﺍﳌﻄﻠﻮﺑﺔ ﻣـﻦ+ ﺍﻟﺘﻮﻇﻴﻒ ﺍﻻﻣﺜﻞ ﻟﻠﻤﻮﺍﺭﺩ ﻭﺍﻻﻣﻜﺎﻧﺎﺕ ﺎ ، ﻓـ ) ﺍﻟﻘﺪﺭﺓ ( ﻫﻨﺎ ﻣﺼـﺪﺭ ﻟﻼﺳـﺘﻤﺮﺍ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻘﻴﺎﻡ )ﺭﻳﺔ ﻭﺍﻟﻔﺎﻋﻠﻴـﺔ ﻭ . ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ( ﻣﺼﺪﺭ ﻟﻠﺘﻨﻈﻴﻢ ﻭﺍﻟﺘﻮﺟﻴﻪ ﺍﶈـﻮﺭ ﺍﻟﺜﺎﻟﺚ: ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻮﺍﺟﻪ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﰲ ﺍﻟﺒﺪﺍﻳﺔ ﳝﻜﻦ ﺍﻟﻘﻮﻝ ، ﺍﻥ ﻣﺼﻄﻠﺢ ﺍﳌﻤﺎﻧﻌﺎﺕ ﰲ ﺍﻟﻠﻐﺔ ﺍﻟﻌﺮﺑﻴﺔ ، ﻛﻤﺼـﺪﺭ ، ﻋـﻦ ﺻﻴﻐﺔ ﻣﺒﺎﻟﻐﺔ ﺗﺸﺘﻖ ﻣﻦ ﺍﻟﻔﻌﻞ ) ﻣﻨﻊ ( ﺍﻟﺸﻲﺀ ﲟﻌﲎ ﻧﺎﺯﻋﻪ ﻭﻣﻨﻌﻪ ﺇﻳﺎﻩ ، ﻭﻛﺬﻟﻚ ﺟﻌﻠﻪ ﻏﲑ ). ﻗﺎﺩﺭ ﻋﻠﻰ ﲢﻘﻴﻖ ﺍﻷﻫﺪﺍﻑ ٢٢ ( ﺍﶈـﻮﺭ ﺍﻟﺜﺎﻟﺚ: ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻮﺍﺟﻪ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﰲ ﺍﻟﺒﺪﺍﻳﺔ ﳝﻜﻦ ﺍﻟﻘﻮﻝ ، ﺍﻥ ﻣﺼﻄﻠﺢ ﺍﳌﻤﺎﻧﻌﺎﺕ ﰲ ﺍﻟﻠﻐﺔ ﺍﻟﻌﺮﺑﻴﺔ ، ﻛﻤﺼـﺪﺭ ، ﻋـﻦ ﺻﻴﻐﺔ ﻣﺒﺎﻟﻐﺔ ﺗﺸﺘﻖ ﻣﻦ ﺍﻟﻔﻌﻞ ) ﻣﻨﻊ ( ﺍﻟﺸﻲﺀ ﲟﻌﲎ ﻧﺎﺯﻋﻪ ﻭﻣﻨﻌﻪ ﺇﻳﺎﻩ ، ﻭﻛﺬﻟﻚ ﺟﻌﻠﻪ ﻏﲑ ). ﻗﺎﺩﺭ ﻋﻠﻰ ﲢﻘﻴﻖ ﺍﻷﻫﺪﺍﻑ ٢٢ ( ﻭﻗﺪ ﻭﺭﺩ ﻣﻔﻬﻮﻡ ﺍﳌﻨﻊ ﰲ ﺍﻟﻘـﺮﺁﻥ ﺍﻟﻜـﺮﱘ ﰲ ﻗﻮﻟـﻪ ﺗﻌـﺎﱃ : ﺑﺴـﻢ ﺍﷲ ﺍﻟـﺮ ﲪﻦ ﺍﻟﺮﺣﻴﻢ:)ﻴﻢﹴ ﺃﹶﺛﺪﺘﻌﺮﹺ ﻣﻴﺎﻉﹴ ﻟﱢﻠﹾﺨﻨﻣ( )،٣٢ (ﻭﻗﻮﻟﻪ ﺗﻌﺎﱃ: : ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﲪﻦ ﺍﻟﺮﺣﻴﻢ ) ـﺎﻉﹴﻨﻣ ﺮﹺﻳﺐﹴ ﻣﺪﺘﻌﺮﹺ ﻣﻴﻟﱢﻠﹾﺨ). ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ (٤٢( ١٨٣       : ﺇﻥ ﺍﳌﻤﺎﻧﻌﺎﺕ ﻣﺜﻞ ﺃﻱ ﻇﺎﻫﺮﺓ ﻣﺜﲑﺓ ﻟﻼﻫﺘﻤﺎﻡ ﰲ ﻛﻞ ﺍﻟﻌﺼﻮﺭ ، ﺗﻌﲏ ﺑﺄﺑﺴﻂ ﺻﻮﺭﻫﺎ ) ﻛﻞ ﺍﻷﻭﺿﺎﻉ ﻭﺍﻟﻈﺮﻭﻑ ﺍﳌﻮﺟﻮﺩﺓ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﺍﺧﻠﻴﺔ ﻟﻠﺪﻭﻟﺔ ، ﻓﻀﻼﹰ ﻋـﻦ ﺍﳋﺎﺭﺟﻴـﺔ . ( ﻭﺍﻟﱵ ﺗﺸﻜﻞ ﻋﻘﺒﺔ ﺃﻣﺎﻡ ﲢﻘﻴﻖ ﺍﻟﻐﺎﻳﺎﺕ ﻭﺍﻷﻫﺪﺍﻑ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻘﺪ ﺑﺪﺕ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺑﻮﺍﺑﺔ ﻣﻦ ﺑﻮﺍﺑﺎﺕ ﺍﺳﺘﻘﺮﺍﺀ ﺍﻷﻭﺿﺎﻉ ، ﻭﺍﻟﺘﺤﺴﺐ ﺎﳌﺂﻻ ﻣﻦ ﺃﺟﻞ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﺍﳌﺴﺘﻘﺒﻞ ﺍﳌﻨﺸﻮﺩ ، ﻭﻫﻮ ﻣﺎ ﺃﻛﺪﻩ ) Porter ( ﰲ ﺗﻌﺮﻳﻔﻪ ﻟﻠﻘـﺪﺭﺓ ، ﺑــ ) ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﻋﻠﻰ ﲢﺪﻳﺪ ﺍﳌﻬﺪﺩﺍﺕ ﻭﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟﱵ ﺗﻮﺍﺟﻬﻬﺎ ﻭﺗﻌـﺎﱐ ﻣﻨـﻬﺎ ﻭﲡﺎﻭﺯﻫـﺎ ﻟﻼﻧﺘﻘﺎﻝ ﺇﱃ ﺍﻟﻨﺠﺎﺡ ﺍﻹ ﺳﺘﺮﺍﺗﻴﺠﻲ ﻭﺑﻠﻮﻍ ﺃﻫﺪﺍﻓﻬﺎ ( ).٥٢( ﻭﺍﳌﻤﺎﻧﻌﺔ ﻫﻲ : ) ﺇﻋﺘﻘﺎﺩ ﻣﻌﺎﻛﺲ ﻟﺮﻏﺒﺘﻨﺎ ( ، ﺃﻭ ﻫﻲ : ) ﻭﺿﻊ ﰲ ﺍﻟﻮﺍﻗﻊ ﺍﻟﺒﻴﺌﻲ ﻳﻌـﺎﻛﺲ ﲢﻘﻴﻖ ﺃﻫﺪﺍﻓﻨﺎ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ( . ﻭﻋﻠﻰ ﺍﻟﺮﻏﻢ ﻣﻦ ﺃﻥ ﺍﻟﺮﺅﻯ ﺍﻟﱵ ﺗﻨﺎﻭﻟﺖ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﳌﺆﺛﺮﺓ ﰲ ﺍﻟﻘـﺪﺭﺓ ﺍﻹﺳـﺘﺮﺍﺗﻴﺠﻴﺔ ﺗﺒﺎﻳﻨﺖ ﺑﻌﻀﻬﺎ ﻋﻦ ﺍﻟﺒﻌﺾ ﺍﻵﺧﺮ ، ﳒﺪ ﺃ ﻥ ﺑﻌﻀﻬﻢ ﺭﺑﻄﻬﺎ ﺑﺎﻟﻮﺍﻗﻊ ﺍﻟﺪﺍﺧﻠﻲ ﻟﻠﺪﻭﻟـﺔ ، ﰲ ﺣﲔ ﳒﺪ ﺃﻥ ﺍﻟﺒﻌﺾ ﺍﻵﺧﺮ ﻭﺟﺪﻫﺎ ﺗﻨﺒﻊ ﻣﻦ ﺧﺎﺭﺝ ﺣﺪﻭﺩ ﺍﻟﺪﻭﻟﺔ . ﻭﻣﻊ ﺍﻥ ﻫﺬﻩ ﺍﻟـﺮﺅﻯ ﻛﻼﹰ ﻋﻠﻰ ﺍﻧﻔﺮﺍﺩ ﺗﻌﱪ ﻋﻦ ﺟﺎﻧﺐ ﻣﻌﲔ ﻣﻦ ﻫﺬﻩ ﺍﻟﻌﻘﺒﺎﺕ . ﻧﺮﻯ ﺃﻥ ﺍﻟﻮﺍﻗﻊ ﺍﻟﻌﻤﻠﻲ ﻳﻘـﻮﻡ ﻋﻠﻰ ﺃﺳﺎﺱ ﺃﻥ ﺍﻟﻮﺍﻗﻊ ﺍﻟﺪﺍﺧﻠﻲ ﻟﻠﺪﻭﻟﺔ ﻣﺘﻔﺎﻋﻞ ﻣﻊ ﻣﺎ ﲢﻤﻠﻪ ﺍﻟﺒﻴﺌﺔ ﺍﳋﺎ. ﺭﺟﻴﺔ ﻟﻘﺪ ﺑﺎﺕ ﻭﺍﺿﺤﺎﹰ ﺇﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺗﻌﺪ ﺍﶈﺮﻙ ﺍﻻﺳﺎﺱ ﻭﺍﳌﺴﺎﻋﺪ ﻟﻠﺪﻭﻟﺔ ﻋﻠـﻰ ﳑﺎﺭﺳﺔ ﺍﻋﻤﺎﳍﺎ ﻭﺑﻠﻮﻍ ﺃﻫﺪﺍﻓﻬﺎ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ ﺑﺎﻻﻋﺘﻤﺎﺩ ﻋﻠﻰ ﳎﻤﻮﻋـﺔ ﻣـﻦ ﺎ . ﲟﻌﲎ ﻫﻲ ﻋﻤﻠﻴﺔ ﺗﺘﻌﺪﺩ ﻓﻴﻬـﺎ ﺍﳌﻘﺪﺭﺍﺕ ﺍﻟﺪﺍﺧﻠﻴﺔ ﻭﺍﻟﱵ ﺗﺘﻤﺜﻞ ﺑﻨﻘﺎﻁ ﺍﻟﻘﻮﺓ ﺍﻟﱵ ﺗﺘﻤﺘﻊ ﺍﳌ . ﺘﻐﲑﺍﺕ ﻭﻳﺆﺛﺮ ﺑﻌﻀﻬﺎ ﻋﻠﻰ ﺍﻟﺒﻌﺾ ﺍﻵﺧﺮ ﰲ ﺍﻃﺎﺭ ﴰﻮﱄ ﻣﺘﻜﺎﻣﻞ ﻭﺗﺸﲑ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ :ﺇﱃ )ﻗﺪﺭﺓ ﺍﻟﻌﺎﻣﻠﲔ ﰲ ﻣﺆﺳﺴﺎﺕ ﺍﻟﺪﻭﻟ ﺔ ﻋﻠﻰ ﺗﻮﻇﻴـﻒ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ "ﺳﻴﺎﺳﻴﺔ، ﺍﻗﺘﺼ،ﺎﺩﻳﺔ ،ﻋﺴﻜﺮﻳﺔ ،ﺃﻣﻨﻴﺔ ﺛﻘﺎﻓﻴﺔ، "ﺍﺟﺘﻤﺎﻋﻴﺔ ﻋـﺪﺓ ﺗﺴـﻤﺢ ﻟﻠﺪﻭﻟﺔ ﺑﺎﻟﺒﻘﺎﺀ ﻭﺯﻳﺎﺩﺓ ﻗﻴﻤﺘ ﻬﺎ ﻋﻠﻰ ﺍﳌﺴﺘﻮﻯ ﺍﻹ ﻗﻠﻴﻤﻲ (ﻭﺍﻟﺪﻭﱄ. ﻭﳝﻜـﻦ ﻟﻠﻌﺪﻳـﺪ ﻣـﻦ ﺍﻟﻌﻨﺎﺻﺮ ﺃﻥ ﺗﺴﻬﻢ ﰲ ﺗﻌﺰﻳﺰ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ . ﻛﻤﺎ ﺃﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﲢﺘﺎﺝ ﺇﱃ ﻋﻤﻠﻴﺔ ﺗﻘﻴﻴﻢ ﻋﻦ ﻃﺮﻳﻖ ﺍﻟﺒﻴﺎﻧﺎﺕ ﺍﳌﺴﺘﻤﺪﺓ ﻣﻦ ﺍﻟﺘﻘﺎﺭﻳﺮ ﻭﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻻﺳﺘﻘﺼﺎﺋﻴﺔ ﺍﻟﱵ ﻳﻘﻮﻡ ـﺎ ﺍﶈﻠﻠﻮﻥ ﰲ ﻣﺆﺳﺴﺎﺕ ﻭ ﺃﺟﻬﺰﺓ ﺍﻟﺪﻭﻟﺔ ﺍﳌﺨﺘﻠﻔﺔ ﻭﺑﺎﺳﺘﻤﺮﺍﺭ، ﺇﺫ ﻳﺘﻢ ﺒﻮﻳﺐﺗ ﺍﻷﻭﺿـﺎﻉ ﰲ ﺷﻜﻞ ﻧﻘﺎﻁ ﻗﻮﺓ ﻭﺿﻌﻒ ﻭﻣﻬﺪﺩﺍﺕ ﻭﻓﺮﺹ ، ﻣﻊ ﲢﺪﻳﺪ ﺍﻟﻘﻀﺎﻳﺎ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﳌﺨﺘﻠﻔﺔ . ٢٨٣       ﻭﻫﻜﺬﺍ ﳒﺪ ﺃﻥ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺗـﺆﺛﺮ ﺍﳚﺎﺑـﺎﹰ ﻋﻠـﻰ ﲢﻘﻴـﻖ ﺍﻷﻫـﺪﺍﻑ ﺎﻻﺕ ﻛﺎﻓﺔ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﻓﺎﻟﺪﻭﻟـﺔﺎ ﺗﺴﻬﻢ ﰲ ﲢﻘﻴﻖ ﺍﻻﺳﺘﺪﺍﻣﺔ ﻭﺍﻻﺳﺘﻤﺮﺍﺭﻳﺔ ﰲ ﺍﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻛﻮ ﺍﻟﱵ ﺗﻌﺎﱐ ﺎ ﻣﻦ ﻗﺼﻮﺭ ﰲ ﺍﻷﺩﺍﺀ ﲟﻌﲎ ﺿﻌﻒ ﺍﻟﻜﻔﺎﺀﺓ ﻋﻠﻰ ﺍﻟﺮﻏﻢ ﻣـﻦ ﺍﻣﺘﻼﻛﻬـﺎﺃﺟﻬﺰ ﺍﻻﻣﻜﺎﻧﻴﺎﺕ ﻭﺍﳌﻮﺍﺭﺩ ﺗﺒﻘﻰ ﺩﻭﻟﺔ ﳏﺒﻄﺔ ﳌﺎ ﺗﻌﺎﻧﻴﻪ ﻣﻦ ﻣﺸﻜﻼﺕ ﻣﺘﺰﺍﻳﺪﺓ ، ﻷﻥ ﺍﻷﺩﺍﺀ ﳛﻀﺮ ﻭﻳﻨﺸﻂ ﺑﺎﻟﺘﻮﺍﺯﻱ ﻣﻊ ﺍﻟﻘﻮﺓ ، ﻭﻣﱴ ﻣﺎ ﺣﻀﺮ ﻳﺘﺤﻘﻖ ﺍﻻﺳﺘﻘﺮﺍﺭ ﺍﻟﺴﻴﺎﺳـﻲ، ﻷﻥ ﺿـﻌﻔﻪ ﺳﻴﺆﺩﻱ ﺇﱃ ﺣﺎﻟﺔ ﻣﻦ ﺍﻟﻔﻮﺿﻰ ﻭﻋﺪﻡ ﺍﻻﺳ ﺘﻘﺮﺍﺭ ، ﻭﺍﻟﱵ ﻏﺎﻟﺒﺎﹰ ﻣﺎ ﺗـﺮﺩ ﺗﻠـﻚ ﺍﳊﺎﻟـﺔ ﺇﱃ ). ﺎ ﰲ ﺍﳌﺪﺭﻙ ﺍﻟﻮﻃﲏ ﻭﺍﳋﺎﺭﺟﻲﻓﻘﺪﺍﻥ ﺍﻟﺪﻭﻟﺔ ﻟﻠﻘﺪﺭﺓ ﻋﻠﻰ ﺗﺸﻜﻴﻞ ﺻﻮﺭ ٦٢( ﻭﺍﳊﻘﻴﻘﺔ ، ﺍﻧﻪ ﻛﻠﻤﺎ ﻛﺎﻧﺖ ﺍﻟﻘﺪﺭﺓ ﻣﻌﱪﺓ ﻋﻦ ﻓﻬﻢ ﻭﺃﺳـﺘﻴﻌﺎﺏ ﺟﺪﻳـﺪ ، ﺗﻌـﺪﺩﺕ ) ﺧﻴﺎﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﻋﻠﻰ ﻭﻓﻖ ﻧﻈﺮﻳﺔGESTALT ( ، ﺇﺫ ﻻ ﺑﺪ ﻟﻠﻘﺪﺭﺓ ﺃﻥ ﺗﻜﻮﻥ ﺫﺍﺕ ﻓﺎﻋﻠﻴ ﺔ ﻭﻣﺰﻭﺩﺓ ﲟﺠﺴﺎﺕ ﲢﺴﺲ ﻟﻠﺴﺒﺐ ﻭﺍﺳﺘﺸﺮﺍﻑ ﺍﻟﻨﺘﻴﺠﺔ ، ﻭﺍﻻ ﺳـﺘﻜﻮﻥ ﺍﻟﻘـﺪﺭﺓ ). ﺿﺤﻴﺔ ﻟﺘﻜﺎﻟﻴﻔﻬﺎ ، ﺑﺪﻻﹰ ﻣﻦ ﺍﳌﻜﻨﺔ ﰲ ﻣﻼﺣﻘﺔ ﺍﻟﺘﻬﺪﻳﺪﺍﺕ ٧٢ ( ﻟﺬﺍ ﻓﺈﻥ ﻋﻤﻠﻴﺔ ﺗﻘﻴـﻴﻢ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﻭﻇﺮﻭﻓﻬﺎ ﺍﻟﻌﺎﻣﺔ ﻳﻌﺪ ﺃﻣﺮﺍﹰ ﰲ ﻏﺎﻳﺔ ﺍﻷﳘﻴﺔ ، ﺫﻟﻚ ﻷﻥ ﺍﻟﺘﺤﺪﻳـﺪ ﺍﻟـﺪﻗﻴﻖ ﻟﺘﻠﻚ ﺍﻟﻘﺪﺭﺍﺕ ، ﻫﻮ ﺍﻟﺬﻱ ﺳﲑﺳﻢ ﺧﺎﺭﻃﺔ ﺃﻫﺪﺍﻑ ﺃﻣﻨﻬﺎ ﺍﻟﻘـﻮﻣﻲ ﻭﻣﺼـﺎﳊﻬﺎ ﺍﻟﻌﻠﻴـﺎ ﺍﻵﺧﺮﻯ . ﻭﻛﻤﺎ ﻳﻘﻮﻝ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻟﺼﻴﲏ "ﺳﺎﻥ ﺗﺰﻭ" : ) ﺇﺫﺍ ﻛﻨﺖ ﺗﻌﺮﻑ ﺍﻵﺧـﺮﻳﻦ ﻭﺗﻌﺮﻑ ﻧﻔﺴﻚ ، ﻓﺎﻧﻚ ﻟﻦ ﺗﻮﺍﺟﻪ ﺧﻄﺮﺍﹰ ﰲ ﻣﺎﺋﺔ ﻣﻌﺮﻛﺔ ، ﻭﺇﺫﺍ ﻛﻨﺖ ﻻ ﺗﻌﺮﻑ ﺍﻵﺧـﺮﻳﻦ ﻭﻟﻜﻨﻚ ﺗﻌﺮﻑ ﻧﻔﺴﻚ ، ﻓﺴﻮﻑ ﺗﻜﺴﺐ ﻣﻌﺮﻛﺔ ﻭﲣﺴﺮ ﺃﺧﺮﻯ ، ﻭﺇﺫﺍ ﻛﻨﺖ ﻻ ﺗﻌـﺮﻑ ﺍﻵﺧﺮﻳﻦ ﻭ). ( ﻻ ﺗﻌﺮﻑ ﻧﻔﺴﻚ ، ﻓﺴﻮﻑ ﺗﻠﻘﻰ ﺍﳌﺨﺎﻃﺮ ﰲ ﻛﻞ ﻣﻌﺮﻛﺔ ٨٢( ﻭﺑﻨﺎﺀﺍﹰ ﻋﻠﻰ ﺫﻟﻚ ﺗﻌﺪﺩﺕ ﺻﻮﺭ ﺍﻟﺘﻤﺎﺱ ﺍﳌﻤﺎﻧﻌﺔ ﺑﺎﻟﻘـﺪﺭﺓ ﻭﺍﻟـﱵ ﲢﺘـﺎﺝ ﺇﱃ ﺟﻬـﺪ ﺇﺳﺘﺮﺍﺗﻴﺠﻲ ﻣﻦ ﺃﺟﻞ ﺿﺒﻂ ﺇﻳﻘﺎﻉ ﺑﻨﺎﺅﻫﺎ "ﻟﻠﻘﺪﺭﺓ" ﻻ ﺍﻧﺪﺛﺎﺭﻫﺎ . ﻭﺫﻟﻚ ﻻ ﻳـﺘﻢ ﺇﻻ ﻣـﻦ ﺧﻼﻝ ﺃﻥ ﻳﺪﺭﻙ ﻭﳛﺪﺩ ﺻﺎﻧﻌﻮﺍ ﺍﻟﻘﺮﺍﺭ ﻭﺍﳌﺨﻄﻄﻮﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﻮﻥ ﺍﳌﻤﺎﻧﻌﺎﺕ ﻭﺍﳌﻬـﺪﺩﺍﺕ ﻭﺍﳌﺨﺎﻃﺮ ﺍﻟﱵ ﺗﻮﺍﺟﻪ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ، ﺳﻮﺍﺀ ﺃﻛﺎﻧﺖ ﺩﺍﺧﻠﻴﺔ ﺃﻡ ﺧﺎﺭﺟﻴﺔ ، ﺣـﱴ ﻳﺘﻤﻜﻨﻮﺍ ﻣﻦ ﻭﺿﻊ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ ﺍﻟﻔﺎﻋﻠﺔ ، ﺫﻟﻚ ﺍﻹﺩﺭﺍﻙ ﳚﺐ ﺃﻥ ﻳﻜﻮﻥ ﻗﺎﺋﻤـﺎﹰ ﻋﻠـﻰ ﺃﺳﺎﺱ ﻣﻦ ﺍﳌﻌﺮﻓﺔ ﺍﻟﻮﺛﻴﻘﺔ ﺑﻨﻘﺎﻁ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻀﻌﻒ ﰲ ﺍﻟﺪﻭﻟﺔ ، ﻭﺍﻻﺳﺘﻔﺎﺩﺓ ﻣﻦ ﻧﻘﺎﻁ ﺍﻟﻘـﻮﺓ ﻭﺗﻨﻤﻴﺘﻬﺎ ﻭﺗﻘﻠﻴﻞ ﻧﻘﺎﻁ ﺍﻟﻀﻌﻒ ﻭﺍﻟﺘﻐﻠﺐ ﻋﻠﻴﻬﺎ . ﻭﺑﺪﻭﻥ ﺫﻟﻚ ﺍﻟﺘﺤﺪﻳﺪ ﺍﻟـﺪﻗﻴﻖ ﻳﻜـﻮﻥ ﺻﺎﻧﻌﻮﺍ ﺍﻟﻘﺮﺍﺭ ﻭﺍﳌﺨﻄﻄﻮﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﻮﻥ ﻗﺪ ﺍﺭﺗﻜﺒﻮﺍ ﺃﺧﻄﺎﺀ ﲣـﺺ ﺍﳍـﺪﻑ ﺍﳌﻨﺸـﻮﺩ ٣٨٣       "ﻋﻤﻠﻴﺔ ﺍﻟﺒﻨﺎﺀ" ، ﻣﻦ ﺇﺟﺮﺍﺀ ﺗﻘﻮﱘ ﺇﺳﺘﺮﺍﺗﻴﺠﻲ ﺻـﺤﻴﺢ ، ﺇﱃ ﻋـﺪﻡ ﲢﺪﻳـﺪ ﺍﻟﻌﻮﺍﻣـﻞ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﱵ ﻳﺘﻮﻗﻒ ﻋﻠﻴ ﻬﺎ ﺍﳌﺴﺘﻘﺒﻞ ، ﻓﻀﻼﹰ ﻋﻦ ﻋﺪﻡ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﻻﺳـﺘﻔﺎﺩﺓ ﻣـﻦ ﺒﲎ ﻋﻠﻰﻢ ﺗﻢ ﻭﺇﺳﺘﺮﺍﺗﻴﺠﻴﺎﻫﺬﻩ ﺍﻟﻌﻮﺍﻣﻞ ﺑﺸﻜﻞ ﺻﺤﻴﺢ . ﻭﻧﺘﻴﺠﺔ ﻟﺬﻟﻚ ، ﻓﺈﻥ ﺳﻴﺎﺳﺎ ). ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﺃﺳﺲ ﺧﺎﻃﺌﺔ ﺃﻭ ﻏﲑ ﻣﻼﺋﻤﺔ ٩٢( ﻭﻋﻠﻰ ﻫﺬﺍ ﺍﻻﺳﺎﺱ ﰎ ﺗﻘﺴﻴﻢ ﺗﻠﻚ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺍﻟـﱵ ﺗﻮﺍﺟﻪ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﺑﻡ ﻨﻮﻋﲔ ﻣﻦ ﺍﳌﻤﺎﻧﻌﺎﺕ ﳘﺎ :  ﺍﳌﻄﻠـﺐ ﺍﻷﻭﻝ / ﳑـﺎﻧﻌـﺎﺕ ﺩﺍﺧﻠﻴـﺔ : ﻭﺍﻟﱵ ﺗﺸﻤﻞ : ﺃﻭﻻﹰ / ﺳﻴﺎﺳﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﰲ : )٠٣( ١- ﺍﻟﻘﺼﻮﺭ ﰲ ﻣﻮﺍﺩ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﺍﻟﺪﺍﺋﻢ ﻟﻌﺎﻡ ٥٠٠٢ ﻡ ، ﻓﺎﻟﺪﺳﺘﻮ ﺭ ﻻ ﻳﻀـﻊ ﳘﺎﹰ ﻟﻪ ﰲ ﺑﻨﺎﺀ ﺍﻟﺪﻭﻟﺔ ()، ﺃﻭ ﺃﻥ ﻳﻀﻊ ﻣﺸﺮﻭﻉ ﺍﻟﺪﻭﻟﺔ ﰲ ﺍﻭﻟﻮﻳﺎﺗﻪ . ٢- ﻓﻘﺪﺍﻥ ﺍﻟﻘﺮﺍﺭ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﻮﻃﲏ ، ﺟﻌﻞ ﻣﻦ ﺍﻟﻌﺮﺍﻕ ﺩﻭﻟـﺔ ﺑﺎﺋﺴـﺔ ، ﻻ ﻳﺘﺒـﻊ ﺳﻴﺎﺳﺎﺕ ﻭﺍﺿﺤﺔ ﻭﺛﺎﺑﺘﺔ ، ﻭﻭﺿﻌﻪ ﻏﲑ ﻣﺴﺘﻘﺮ ﻣﻨﺬ ﻋﺎﻡ ٣٠٠٢ ﻡ . ٣- ﻓﻘﺪﺍﻥ ﺍﻟﺜﻘﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺑﲔ ﺃﻃﺮﺍﻑ ﺍﻟﻌﻤﻠﻴﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻛﻜﻞ ﳑﻦ ﺍﺗﻔـﻖ ﺍﻟﺸـﻌﺐ ﻋﻠﻰ ﺍﺧﺘﻴﺎﺭﻫﻢ ﻟﺘﻤﺜﻴﻠﻪ ، ﻭﻫﺬﺍ ﻳﻌﺘﱪ ﻣﻦ ﺃﻭﺟﻪ ﺍﳌﻌﻀﻠﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺍﻟﱵ ﲢﻮﻝ ﺩﻭﻥ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻓﺎﻟﻌﺮﺍﻕ ﻛﻤﺎ ﻫﻮ ﻣﻌﺮﻭﻑ ﻣﻨﻘﺴـﻢ ﺇﱃ ﻋﺪﺓ ﻃﻮﺍﺋﻒ ﻭﺍﻗﻠﻴﺎﺕ ﺍﺛﻨﻴﺔ ﻭﺩﻳﻨﻴﺔ ، ﺇﺫ ﺗﻜﻤﻦ ﺍﳌﻌﻀﻠﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻣـﻦ ﻓﻘـﺪﺍﻥ ﺍﻟﺜﻘﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺑﲔ ﻛﻼﹰ ﻣﻦ ﺍﻟﻌﺮﺏ ﻣﻦ ﺟﻬﺔ ﻭﺍﻷﻛﺮﺍﺩ ﻣـﻦ ﺟﻬـﺔ ﺍﻟﺴـﺎﻋﲔ ﻟﺘﺤﻘﻴﻖ ﺃﻛﱪ ﻗﺪﺭ ﻣﻦ ﺍﳌﻜﺎﺳﺐ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻﺳﺘﻘﻼﻟﻴﺔ ﻣﻦ ﺍﻟﺴﻠﻄﺔ ﺍﻻﲢﺎﺩ ﻳـﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﺍﻻﻧﻘﺴﺎﻡ ﺍﻟﺪﻳﲏ ﺍﳌﺬﻫﱯ ﻣﻦ ﺟﻬﺔ ﺃﺧﺮﻯ ، ﻗﺪ ﺃﻟﻘﻰ ﺑﻀﻼﻟﻪ ﻫﻮ ﺍﻵﺧﺮ ﻋﻠﻰ ﺍﻟﻮﺍﻗﻊ ﺍﻟﺴﻴﺎﺳﻲ ﻭﻓﻖ ﻣﺒﺪﺃ "ﺍﶈﺎﺻﺼﺔ ."ﺍﻟﺴﻴﺎﺳﻴﺔ ٤- ﻋﺪﻡ ﻭﺟﻮﺩ ﺇﲨﺎﻉ ﻭﻃﲏ ﺣﻮﻝ ﺍﳌ ﺼﺎﱀ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟـﺔ، ﺑﺴـﺒﺐ ﺣﺮﺹ ﺍﻟﻘﻮﻯ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺍﳌﺨﺘﻠﻔﺔ ﻰﻋﻠ ﺗﻌﺰﻳـﺰ ﻭﲪﺎﻳـﺔ ﻣﻜﺎﺳـﺒﻬﺎ "ﺍﳊﺰﺑﻴـﺔ، ﻭﺍﻟ"ﺸﺨﺼﻴﺔ ﻋﻠﻰ ﺣﺴﺎﺏ ،ﺍﻵﺧﺮ ﲟﺎ ﻳﺴﻤﻰ ﺑـ)ﺍﻟﻔﺴﺎﺩ ﺍﻟﺴﻴﺎﺳﻲ( . ٤٨٣       ٥- ﺍﻟﻌﻨﻒ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺍﳌﺘﻤﺜﻞ ﺏ ) ﺍﳌﻠﻴﺸﻴﺎﺕ ﺍﳌﺴﻠﺤﺔ ( ﺍﳌﺮﺗﺒﻄﺔ ﺑـﺒﻌﺾ ﺍﻟﻘـﻮﻯ ﻭﺍﻷﺣﺰﺍﺏ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻟﺘﺤﻘﻴﻖ ﺃﻫﺪﺍﻑ ﺳﻴﺎﺳﻴﺔ ﻭﺍﻟﺼﺮﺍﻉ ﺣﻮﻝ ﺍﻟﻨﻔﻮﺫ ﻭﺍﻟﺴﻠﻄﺔ ﻭﺍﻟﺜﺮﻭﺓ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﺴﻴﻴﺲ ﺟﻬﺎﺯ ﺍﻟﺪﻭﻟﺔ ﻭﺗﻜﺮﻳﺴﻪ ﳋﺪﻣﺔ ﻣﺼﺎﳊﻬﺎ . ٦- ﻓﻘﺪﺍﻥ ) ﺍ ﻟﻮﺣﺪﺓ )ﺍﻟﻮﻃﻨﻴﺔ ١٣( ( ﻧﺘﻴﺠﺔ ﳑﺎﺭﺳﺎﺕ ) ﺍﻟﻘﻮﻯ ﻭﺍﻷﺣﺰﺍﺏ ﺍﻟﺴﻴﺎﺳﻴﺔ ( ﻣﻨﺬ ﻋﺎﻡ ٣٠٠٢ ﻡ ﻭﳊﺪ ﺍﻵﻥ ﻣﻦ ﺧﻼﻝ ﺍﳋﻄﺎﺑﺎﺕ ﺍﳌﻮﺟﻬـﺔ ﺇﱃ ﻗﻮﺍﻋـﺪﻫﻢ ﺍﳉﻬﺎﻫﲑﻳﺔ "ﺍﻟﻄﺎﺋﻔﺔ ﺍﻟﱵ "ﺎﳝﺜﻠﻮ ، ﺬﺍﻭ ﲢﻮﻝ ﺍﻟﺼﺮﺍﻉ ﻣـﻦ ) ﺳﻴﺎﺳـﻲ ( ﺇﱃ ﺻﺮﺍﻉ ) ﺍﺟﺘﻤﺎﻋﻲ ( ).٢٣( ٧- ﺍﳋﺪﻣﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺗﻘﻮﻡ ﻋﻠﻰ ﺍﻟﻘﺮﺍﺭ ﺍﻟﻔﺮﺩﻱ ﻭﺍﶈ ﺴﻮﺑﻴﺔ ﰲ ﻇـﻞ ﻋـﺪﻡ ﺳـﻴﺎﺩﺓ ﺍﻟﻘﺎﻧﻮﻥ. ٧- ﺍﳋﺪﻣﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺗﻘﻮﻡ ﻋﻠﻰ ﺍﻟﻘﺮﺍﺭ ﺍﻟﻔﺮﺩﻱ ﻭﺍﶈ ﺴﻮﺑﻴﺔ ﰲ ﻇـﻞ ﻋـﺪﻡ ﺳـﻴﺎﺩﺓ ﺍﻟﻘﺎﻧﻮﻥ. ٠١- ﺗﺸﺘﺖ ﻭﺿﻌﻒ ﺍﻹﺭﺍﺩﺓ ﺍﻟﻮﻃﻨﻴﺔ . ١١- ﺍﳊﻜﻢ ﻏﲑ ﺍﻟﺮﺍﺷﺪ ﺍﻟﺬﻱ ﻻ ﻳﺆﺳﺲ ﻟﺸﺮﺍﻛﺔ ﻭﻃﻨﻴﺔ ﻭﺗﺒﺎﺩﻝ ﺳﻠﻤﻲ ﻟﻠﺴـﻠﻄﺔ ﻭﺳﻴﺎﺩﺓ ﺍﻟﻘﺎﻧﻮﻥ ﻭﺇﺗﺎﺣﺔ ﻟﻠﺤﺮﻳﺎﺕ ﻭﺍﳊﻘﻮﻕ . ٢١- ﺍﻧﻌﺪﺍﻡ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﺣﺪﺍﺙ ﺍﻟﺘﻮﺍﺯﻥ ﺑﲔ ﻣﻘﺘﻀﻴﺎﺕ ﺍﳌﺼﺎﱀ ﺍﳊﺰﺑﻴﺔ ﻭﺍﻻﻟﺘـﺰﺍﻡ ﺍﻟﻮﻃﲏ . ﻓﻜﻞ ﻗﻮﺓ ﺗﻨﻈﺮ ﺇﱃ ﺍﻟﻌﺮﺍﻕ ﻣﻦ ﻣﻨﻈﻮﺭﻫﺎ ﻭﺭﺅﻳﺘﻬﺎ ﻭﻓﻬﻤﻬﺎ ﻭﻗﺪ ﺗﻜﻮﻥ ﻫـﺬﻩ ﺍﻟﺮﺅﻳﺔ ﻏﲑ ﻧﺎﺿﺠﺔ ﺃﻭ ﻣﺘﻜﺎﻣﻠﺔ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ٣١- ﺿﻌﻒ ﺍﳊﺲ ﺍﻟﻮﻃﲏ ﻭﺗﺒﺎﻳﻦ ﺍﳌﺸﺎﻋﺮ ﺍﻟﻮﻃﻨﻴﺔ . ٤١- ﺿﻌﻒ ﺍﻟﺸﺮﺍﻛﺎﺕ ﻭﺍﻟﺘﻘﺎﺳﻢ ﺍﻟﻮﻃﲏ ﻟﻸﺩﻭﺍﺭ . ٥١- ﻣﺴﺄﻟﺔ ﺍﻧﻔﺼﺎﻝ ""ﺍﻟﻜﻮﺭﺩ ﻭﻗﻴﺎﻡ ﺩﻭﻟﺔ ""ﻛﻮﺭﺩﻳﺔ ﻭﺗﺄﺛﲑﻫﺎ ﻋﻠﻰ ﺑﻨـﺎﺀ ﺍﻟﺪﻭﻟـﺔ ﺍﻟﻌ ﺮﺍﻗﻴﺔ ﻭﻋﻠﻰ ﺍﻟﺘﻮﺍﺯﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﰲ .ﺍﳌﻨﻄﻘﺔ ﺛﺎﻧﻴﺎﹰ / ﺍﺟﺘﻤﺎﻋﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﰲ : ١- ﺍﺷﻜﺎﻟﻴﺔ ﺍﳌﻮﺍﻃﻨﺔ ﻭﺃﺯﻣﺔ ﺍﳍﻮﻳﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺑﲔ ﺍﺑﻨﺎﺀ ﺍﻟﺸﻌﺐ ﺍﻟﻌﺮﺍﻗﻲ ﺑﺴﺒﺐ ﺿـﻌﻒ ﻟ ﻹ ﺔ ﻟ ﺔ ﻟ ﻓﻼﹰ ﻟﻞ ﳋ ﺔ ٠١- ﺗﺸﺘﺖ ﻭﺿﻌﻒ ﺍﻹﺭﺍﺩﺓ ﺍﻟﻮﻃﻨﻴﺔ . ١١- ﺍﳊﻜﻢ ﻏﲑ ﺍﻟﺮﺍﺷﺪ ﺍﻟﺬﻱ ﻻ ﻳﺆﺳﺲ ﻟﺸﺮﺍﻛﺔ ﻭﻃﻨﻴﺔ ﻭﺗﺒﺎﺩﻝ ﺳﻠﻤﻲ ﻟﻠﺴـﻠﻄﺔ ﻭﺳﻴﺎﺩﺓ ﺍﻟﻘﺎﻧﻮﻥ ﻭﺇﺗﺎﺣﺔ ﻟﻠﺤﺮﻳﺎﺕ ﻭﺍﳊﻘﻮﻕ . ٢١- ﺍﻧﻌﺪﺍﻡ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﺣﺪﺍﺙ ﺍﻟﺘﻮﺍﺯﻥ ﺑﲔ ﻣﻘﺘﻀﻴﺎﺕ ﺍﳌﺼﺎﱀ ﺍﳊﺰﺑﻴﺔ ﻭﺍﻻﻟﺘـﺰﺍﻡ ﺍﻟﻮﻃﲏ . ﻓﻜﻞ ﻗﻮﺓ ﺗﻨﻈﺮ ﺇﱃ ﺍﻟﻌﺮﺍﻕ ﻣﻦ ﻣﻨﻈﻮﺭﻫﺎ ﻭﺭﺅﻳﺘﻬﺎ ﻭﻓﻬﻤﻬﺎ ﻭﻗﺪ ﺗﻜﻮﻥ ﻫـﺬﻩ ﺍﻟﺮﺅﻳﺔ ﻏﲑ ﻧﺎﺿﺠﺔ ﺃﻭ ﻣﺘﻜﺎﻣﻠﺔ . ٠١- ﺗﺸﺘﺖ ﻭﺿﻌﻒ ﺍﻹﺭﺍﺩﺓ ﺍﻟﻮﻃﻨﻴﺔ . ١١- ﺍﳊﻜﻢ ﻏﲑ ﺍﻟﺮﺍﺷﺪ ﺍﻟﺬﻱ ﻻ ﻳﺆﺳﺲ ﻟﺸﺮﺍﻛﺔ ﻭﻃﻨﻴﺔ ﻭﺗﺒﺎﺩﻝ ﺳﻠﻤﻲ ﻟﻠﺴـﻠﻄﺔ ﻭﺳﻴﺎﺩﺓ ﺍﻟﻘﺎﻧﻮﻥ ﻭﺇﺗﺎﺣﺔ ﻟﻠﺤﺮﻳﺎﺕ ﻭﺍﳊﻘﻮﻕ . ٤١- ﺿﻌﻒ ﺍﻟﺸﺮﺍﻛﺎﺕ ﻭﺍﻟﺘﻘﺎﺳﻢ ﺍﻟﻮﻃﲏ ﻟﻸﺩﻭﺍﺭ . ٥١- ﻣﺴﺄﻟﺔ ﺍﻧﻔﺼﺎﻝ ""ﺍﻟﻜﻮﺭﺩ ﻭﻗﻴﺎﻡ ﺩﻭﻟﺔ ""ﻛﻮﺭﺩﻳﺔ ﻭﺗﺄﺛﲑﻫﺎ ﻋﻠﻰ ﺑﻨـﺎﺀ ﺍﻟﺪﻭﻟـﺔ ﺍﻟﻌ ﺮﺍﻗﻴﺔ ﻭﻋﻠﻰ ﺍﻟﺘﻮﺍﺯﻥ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﰲ .ﺍﳌﻨﻄﻘﺔ ﺛﺎﻧﻴﺎﹰ / ﺍﺟﺘﻤﺎﻋﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﰲ : ١- ﺍﺷﻜﺎﻟﻴﺔ ﺍﳌﻮﺍﻃﻨﺔ ﻭﺃﺯﻣﺔ ﺍﳍﻮﻳﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺑﲔ ﺍﺑﻨﺎﺀ ﺍﻟﺸﻌﺐ ﺍﻟﻌﺮﺍﻗﻲ ﺑﺴﺒﺐ ﺿـﻌﻒ ﺍﻟ ﻮﻋﻲ ﺍﻹﺟﺘﻤﺎﻋﻲ ﻭﺍﻟﺘﻨﺸﺌﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ، ﻓﻀﻼﹰ ﻋﻦ ﺩﻭﺭ ﺍﻟﻌﺎﻣﻞ ﺍﳋﺎﺭﺟﻲ ﻧﺘﻴﺠـﺔ ﺍﻻﺣﺘﻼﻝ ﺍﻟﱪﻳﻄﺎﱐ ﻭﺍﻻﺣﺘﻼﻝ ﺍﻷﻣﺮﻳﻜﻲ.       ٢- ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﻡ ﻭﰲ ﻇﻞ ﻏﻴﺎﺏ ﺍﻟﻘﺎﻧﻮﻥ ﻭﺍﻟﻔﺮﺍﻍ ﺍﻟﺴﻴﺎﺳﻲ ﲢﻮﻝ ﻭﻻﺀ ﺍﻟﻔﺮﺩ ﻣﻦ ﺍﻟﺪﻭﻟﺔ ﺇﱃ ﺍﻟﻄﺎﺋﻔﺔ ﻭﺍﻟﻌﺸﲑﺓ . ﻓﺎﻟﱰﺍﻋﺎﺕ ﺍﻟﻘﺒﻠﻴﺔ ""ﺍﻟﻌﺸـﺎﺋﺮﻳﺔ ، ﻭﺍﻧﺘﺸـﺎﺭ ﺍﻟﺴﻼﺡ )ﺍﳋﻔ،ﻴﻒ ،ﺍﳌﺘﻮﺳﻂ ﻭﺃﺣﻴﺎﻧﺎﹰ ﺍﻟﺜﻘﻴﻞ ( ﺑﺄﻳﺪﻱ ﳎﻤﻮﻋﺎﺕ ﻣـﻦ ﺍﻟﻌﺸـﺎ ﺋﺮ ﻭﺃﺑﻨﺎﺀ ﺍﻷﺭﻳﺎﻑ ﻭﺩﺍﺧﻞ ﺍﳌﺪﻳﻨﺔ، ﺑﺪﺃﺕ ﺗﻠﻌﺐ ﺍﻟﺪﻭﺭ ﺍﻷ ﻛﱪ ﰲ ﺍﻟﺘﺤﻜﻢ ﺑـﺎﻷﻣ ﻮﺭ ﺑﺪﻝ ﺍﻟﻘﺎﻧﻮﻥ ﻭﺍﳉﻬﺎﺕ ﺍﳊﻜﻮﻣﻴﺔ. ٣- ﺍﻟﺼﺮﺍﻋﺎﺕ ﺍﻟﺪﻳﻨﻴﺔ ﻭﺍﻟﻄﺎﺋﻔﻴﺔ ﻭﺍﻟﻘﺘﻞ ﻋﻠﻰ "ﺍﻷﺳﻢ "ﻭﺍﳍﻮﻳﺔ ﺧﺼﻮﺻﺎﹰ ﺑﻌﺪ ﺣﺎﺩﺛﺔ ﺗﻔﺠﲑ ﺿﺮﻳﺢ ﺍﻻﻣﺎﻣﻴﲔ ﺍﻟﻌﺴﻜﺮﻳﲔ ﰲ ﺳﺎﻣﺮﺍﺀ ﰲ ٢٢/٢/٦٠٠٢ ﻡ ، ﻭﺍﻟﺘﻬﺠﲑ ﰲ ﺍﻟﺪﺍﺧﻞ ﻭﺍﳋﺎﺭﺝ ﻭﻇﻬﻮﺭ ﺍﻟﺘﻐﻴﲑ ﺍﻟﺪﳝﻮﻏﺮﺍﰲ . ﻭﻣﻦ ﻫﻨﺎ ﺟﺮﻯ ﲤﺰﻳﻖ ﺍﻟﻨ ﺴﻴﺞ ﺍﻻﺟﺘﻤﺎﻋﻲ ﺑﻌﺪ ﻣﺎ ﺍﺿﺤﺖ ﻋﻤﻠﻴﺎﺕ ﺍﻟﻘﺘﻞ ﲡﺮﻱ ﻋﻠﻰ ﰲ ﺍﻟﻌﺮﺍﻕ ﻋﻠﻰ "ﺍﻷﺳـﻢ "ﻭﺍﳍﻮﻳﺔ . ٢- ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﻡ ﻭﰲ ﻇﻞ ﻏﻴﺎﺏ ﺍﻟﻘﺎﻧﻮﻥ ﻭﺍﻟﻔﺮﺍﻍ ﺍﻟﺴﻴﺎﺳﻲ ﲢﻮﻝ ﻭﻻﺀ ﺍﻟﻔﺮﺩ ﻣﻦ ﺍﻟﺪﻭﻟﺔ ﺇﱃ ﺍﻟﻄﺎﺋﻔﺔ ﻭﺍﻟﻌﺸﲑﺓ . ﻓﺎﻟﱰﺍﻋﺎﺕ ﺍﻟﻘﺒﻠﻴﺔ ""ﺍﻟﻌﺸـﺎﺋﺮﻳﺔ ، ﻭﺍﻧﺘﺸـﺎﺭ ﺍﻟﺴﻼﺡ )ﺍﳋﻔ،ﻴﻒ ،ﺍﳌﺘﻮﺳﻂ ﻭﺃﺣﻴﺎﻧﺎﹰ ﺍﻟﺜﻘﻴﻞ ( ﺑﺄﻳﺪﻱ ﳎﻤﻮﻋﺎﺕ ﻣـﻦ ﺍﻟﻌﺸـﺎ ﺋﺮ ﻭﺃﺑﻨﺎﺀ ﺍﻷﺭﻳﺎﻑ ﻭﺩﺍﺧﻞ ﺍﳌﺪﻳﻨﺔ، ﺑﺪﺃﺕ ﺗﻠﻌﺐ ﺍﻟﺪﻭﺭ ﺍﻷ ﻛﱪ ﰲ ﺍﻟﺘﺤﻜﻢ ﺑـﺎﻷﻣ ﻮﺭ ﺑﺪﻝ ﺍﻟﻘﺎﻧﻮﻥ ﻭﺍﳉﻬﺎﺕ ﺍﳊﻜﻮﻣﻴﺔ. ٣- ﺍﻟﺼﺮﺍﻋﺎﺕ ﺍﻟﺪﻳﻨﻴﺔ ﻭﺍﻟﻄﺎﺋﻔﻴﺔ ﻭﺍﻟﻘﺘﻞ ﻋﻠﻰ "ﺍﻷﺳﻢ "ﻭﺍﳍﻮﻳﺔ ﺧﺼﻮﺻﺎﹰ ﺑﻌﺪ ﺣﺎﺩﺛﺔ ﺗﻔﺠﲑ ﺿﺮﻳﺢ ﺍﻻﻣﺎﻣﻴﲔ ﺍﻟﻌﺴﻜﺮﻳﲔ ﰲ ﺳﺎﻣﺮﺍﺀ ﰲ ٢٢/٢/٦٠٠٢ ﻡ ، ﻭﺍﻟﺘﻬﺠﲑ ﰲ ﺍﻟﺪﺍﺧﻞ ﻭﺍﳋﺎﺭﺝ ﻭﻇﻬﻮﺭ ﺍﻟﺘﻐﻴﲑ ﺍﻟﺪﳝﻮﻏﺮﺍﰲ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﻭﻣﻦ ﻫﻨﺎ ﺟﺮﻯ ﲤﺰﻳﻖ ﺍﻟﻨ ﺴﻴﺞ ﺍﻻﺟﺘﻤﺎﻋﻲ ﺑﻌﺪ ﻣﺎ ﺍﺿﺤﺖ ﻋﻤﻠﻴﺎﺕ ﺍﻟﻘﺘﻞ ﲡﺮﻱ ﻋﻠﻰ ﰲ ﺍﻟﻌﺮﺍﻕ ﻋﻠﻰ "ﺍﻷﺳـﻢ "ﻭﺍﳍﻮﻳﺔ . ٤- ﺍﻟﱰﻭﺡ ﺍﻟﻜﺒﲑ ﺑﻌﺪ ﺳﻴﻄﺮﺓ ﺗﻨﻈﻴﻢ ""ﺩﺍﻋﺶ ﺍﻹﺭﻫﺎﰊ ﻋﻠﻰ ﻣﺪﻥ ﻋﺪﺓ ﰲ ﻣﻨـﺎﻃﻖ ﴰﺎﻝ ﻭﻏﺮﺏ ﺍﻟﻌﺮﺍﻕ . ﺇﺫ ﻭﺍﺟﻪ ﺍﻟﻨﺎﺯﺣﲔ ﻇﺮﻭﻓﺎﹰ ﺇﻧﺴﺎﻧﻴﺔ ﺑﺎﻟﻐﺔ ﺍﻟﺼﻌﻮﺑﺔ ﺗﺎﺭﻛﲔ ﻭﺭﺍﺅﻫﻢ ﻛﻞ ﺷﻲﺀ . ﻭﺗﻌﺪﺩﺕ ﻣﻈﺎﻫﺮ ﻣﻌﺎﻧﺎﺓ ﺍ ﻟﻨﺎﺯﺣﲔ ﻣﻦ ﻧﻘﺺ ﺣﺎﺩ ﰲ ﺍﻟﺼﺤﺔ ﻭﺍﻟﺘﻌﻠﻴﻢ ﻭﺍﳋﺪﻣﺎﺕ ﺍﻟﻀﺮﻭﺭﻳﺔ ﺧﺎﺻﺔ ﻣﺎﺀ ﺍﻟﺸﺮﺏ ﻭﺍﻟﺼﺮﻑ ﺍﻟﺼﺤﻲ ، ﻓﻀـﻼﹰ ﻋﻦ ﺗﻜﺪﺱ ﺍﻟﻌﻮﺍﺋﻞ ﺍﻟﻜﺒﲑﺓ ﺍﻟﻌﺪﺩ ﰲ ﳐﻴﻤﺎﺕ ﺻﻐﲑﺓ ﺩﻓﻌﺖ ﺑﻌـﺾ ﺃﻓﺮﺍﺩﻫـﺎ ﻟﻼﻧﺘﺤﺎﺭ ﻧﺘﻴﺠﺔ ﺍﻟﻈﺮﻭﻑ ﺍﻻﺟﺘﻤﺎﻋﻴﺔ ﻭﺍﻟﻨﻔﺴﻴﺔ ﺍﻟﺼﻌﺒﺔ ﻭﺍﻟﻈـﺮﻭﻑ ﺍﳌﻌﻴﺸـﻴﺔ ). ﺍﻟﻘﺎﺳﻴﺔ ﺩﺍﺧﻞ ﺍﳌﺨﻴﻤﺎﺕ ٣٣( ٥- ﺍﻟﺘﻔﻜﻚ ﺍ ﻻﺳﺮﻱ . ٦- ﺍﻟﺘﺠﺎﻭﺯ ﻋﻠﻰ ﺍﻟﻘﺎﻧﻮﻥ . ٧- ﺍﳉﺮﺍﺋﻢ ﺘﻤﻌﻴﺔﺍ ﻭﺍﻷﺧﻼﻗﻴﺔ ، ﻭﻇﺎﻫﺮﺓ ﺧﻄﻒ ﺍﻻﺷﺨﺎﺹ ﻣﻘﺎﺑﻞ ﺩﻓﻊ ﺍﻟﻔﺪﻳﺔ . ٨- ﻋﺪﻡ ﻗﺒﻮﻝ ﺍﻵﺧﺮ . ٩- ﺗﺪﱐ ﻣﺴﺘﻮﻯ ﺍﻻﻧﺘﻤﺎﺀ ﺍﻟﻮﻃﲏ ﻧﺘﻴﺠﺔ ﺍﻻﻧﻘﺴﺎﻡ ﻭﺍﻟﺘﻜﺘـﻞ ﺍﻟﻄـﺎﺋﻔﻲ ﺍﻟﺴﻴﺎﺳـﻲ ﻭﺍﻟﺬﻱ ﺍﻟﻘﻰ ﺑﻈﻼﻟﻪ ﻋﻠﻰ ﺍﻟﻨﺴﻴﺞ ﺍﻻﺟﺘﻤﺎﻋﻲ ﻟﻠﻤﺠﺘﻤﻊ ﺍﻟﻌﺮﺍﻗﻲ . ٠١- ﺍﻟﻜﺴﺐ ﻏﲑ ﺍﳌﺸﺮﻭﻉ ﻧﺘﻴﺠﺔ ﺍﻟﺮﺷﻮﺓ ﻭﺍﻟﻔﺴﺎﺩ ﺍﻹﺩﺍﺭﻱ . ٧- ﺍﳉﺮﺍﺋﻢ ﺘﻤﻌﻴﺔﺍ ﻭﺍﻷﺧﻼﻗﻴﺔ ، ﻭﻇﺎﻫﺮﺓ ﺧﻄﻒ ﺍﻻﺷﺨﺎﺹ ﻣﻘﺎﺑﻞ ﺩﻓﻊ ﺍﻟﻔﺪﻳﺔ . ٨- ﻋﺪﻡ ﻗﺒﻮﻝ ﺍﻵﺧﺮ .       ١١- ﺍﺭﺗﻔﺎﻉ ﻣﻌﺪﻻﺕ ﺍﻟﻨﻤﻮ ﺍﻟﺴﻜﺎﱐ ﻣﻘﺎﺑﻞ ﺗﺪﱐ ﺍﳋﺪﻣﺎﺕ ﻭﺍﻧﺘﺸﺎﺭ ﻇﺎﻫﺮﺓ ﺃﺯﻣـﺔ ﺍﻟﺴﻜﻦ . ٢١- ﺍﻻﻭﺿﺎﻉ ﺍﻟﺼﺤﻴﺔ "ﺗﺪﱐ ﻣﺴﺘﻮﻯ ﻛﻔﺎﺀﺓ ﺍﳌﺴﺘﺸﻔﻴﺎﺕ ﻭﺍﳌﺮﺍﻛﺰ "ﺍﻟﺘﺸﺨﻴﺼﻴﺔ . ٣١- ﺍﻧﺘﺸﺎﺭ ﺍﳌﺨﺪﺭﺍﺕ ﻭﺍﻟﻌﻘﺎﻗﲑ ﺍﳌﺜﺒﻄﺔ ﺫﻫﻨﻴﺎﹰ . ٤١- ﺍﳍﺠﺮﺓ ﳓﻮ ﺍﳋﺎﺭﺝ ﻭﻣﺎ ﺗﺸﻜﻠﻪ ﻣﻦ ﻧﺰﻑ ﻟﻠﻄﺎﻗﺎﺕ ﺍﻟﺒﺸﺮﻳﺔ ﻛﻮﻧﻪ ﺍﳊﻞ ﺍﻷﻣﺜﻞ ﻟﻠﺸﺎﺏ ﺍﻟﻌﺮﺍﻗﻲ . ﺛﺎﻟﺜﺎﹰ / ﻋﺴﻜﺮﻳﺔ ﻭﺃﻣﻨﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : )٤٣( ١- ﺿﻌﻒ ﺍﺩﺍﺀ ﺍﻟﻘﻴﺎﺩﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﻷﻣﻨﻴﺔ ﻋﻠﻰ ﻣﺴﺘﻮﻯ ﺍﳋﻄﻂ ﻭﺇﺩﺍﺭﺓ ﺍﻟﻘﺘـﺎﻝ ﻻﻓﺘﻘﺎﺭﻫﺎ ﻟﻠﻤﻬﻨﻴﺔ ﻭﺍﻟﻜﻔﺎﺀﺓ . ٢- ﻋﺪﻡ ﺍﻟﺪﻗﺔ ﰲ ﺇﻋﺎﺩﺓ ﺗﺸﻜﻴﻞ ﺍﳌﺆﺳﺴﺔ ﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﻋـﺪﻡ ﺍﺳـﺘﺨﺪﺍﻡ ﺍﳌﻌـﺎﻳﲑ ﺍﳌﻌﺮﻭﻓﺔ ﰲ ﺍﺧﺘﻴﺎﺭ ﺍﻟﻘﺎﺩﺓ ﻭﺃﻣﺮﻳﻬﺎ ، ﺃﺩﻯ ﺇﱃ ﳕﻮ ﺪﺩﻭﲤ ﺍﳉﻤﺎﻋـﺎﺕ ﺍﳌﺴـﻠﺤﺔ ""ﺍﻹﺭﻫﺎﺑﻴــﺔ، ﻭﻣــﻦ ﻧﺘﻴﺠﺘــﻪ ﻴــﺎﺭﺍﻻ ﺍﻟﻌﺴــﻜﺮﻱ ﻭﺍﻷﻣــﲏ ﻟﻠﺠــﻴﺶ ﰲ ٠١/٦/٤١٠٢ ﻡ ﰲ ﲪﺎﻳﺔ ﻋﺪﺩ ﻣﻦ ﺍﶈﺎﻓﻈﺎﺕ ﻭﻣﻨﺎﻃﻖ ﺃﺧﺮﻯ ﻣﻦ ﺍﻻ ﻴﺎﺭ ﺑﻴـﺪ ﺗﻨﻈﻴﻢ ﻣﺎ ﻳﺴﻤﻰ ""ﺩﺍﻋﺶ، ﻭﺗﺪﺍﻋﻴﺎﺗﻪ ﻋﻠﻰ ﺍﳌﺸﻬﺪﻳﻦ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺍﻻﻗﺘﺼـﺎﺩﻱ . ﺇﺫ ﲢﻮﻝ ﺍﳉﻴﺶ ﻣﻦ ﺣﺎﻡﹴ ﻟﻠﻌﺮﺍﻕ ، ﺇﱃ ﻣﻨﻔﺬﺍﹰ ﻟﻌﻮﺩﺓ ﺍﻟﻮﻻﻳﺎﺕ ﺍﳌﺘﺤﺪﺓ ﺍﻷﻣﺮﻳﻜﻴﺔ ﻣﻦ ﺟﺪﻳﺪ . ٣- ﺩﻣﺞ ""ﺍﳌﻠﻴﺸﻴﺎﺕ ﰲ ﺍﳉﻴﺶ ﳑﺎ ﺃﺩﻯ ﺇﱃ ﻓﻘﺪﺍﻥ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ﻣﻘﻮﻣﺎﺕ ﺑﻨﺎﺋـﻪ ﻭﺩﳝﻮﻣﺘﻪ ﺍﻻ ﻭﻫﻲ ) ﺍﻟﻀﺒﻂ ، ﻭﺍﻟﻨﻈﺎﻡ ، ﻭﺍﻟﻘﺪﻡ ﺍﻟﻌﺴـﻜﺮﻱ ، ﻭﺍﻟﺘـﺪﺭﺝ ﰲ ﺍﻟﺮﺗﺐ ، ﻭﺍﳌﻬﻨﻴﺔ ﻭﺍﻟﻜﻔﺎﺀﺓ ، ﻭﺍﻟﺪﺭﺍﺳﺔ ﺍﻻﻛﺎﺩﳝﻴﺔ ( . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ٤- ﺍﺑﻌﺎﺩ ﻛﻞ ﺍﳋﱪﺍﺕ ﺍﻟﻮﻃﻨﻴﺔ ﻭﺍﻟﻜﻔﺎﺀﺍﺕ ﺍﳌﻬﻨﻴﺔ ﻭﺍ ﻻﻛﺎﺩﳝﻴـﺔ ﲢـﺖ ﻣﺴـﻤﻴﺎﺕ ،""ﺍﻻﺟﺘﺜﺎﺙ ﻭﺍﺯﻻﻡ "ﺍﻟﻨﻈﺎﻡ "ﺍﻟﺴﺎﺑﻖ، ﻭ ﺟﻴﺶ ""ﺻﺪﺍﻡ ﻭﻏﲑﻫﺎ ﻣﻦ ﺍﳌـﱪﺭﺍﺕ ﺍﻟﻐﲑ ﻣﻨﻄﻘﻴﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻗﻴﺎﻡ ﺍﻷﺣﺰﺍﺏ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺑﺎﻟﺘﺪﺧﻞ ﰲ ﻋﻤﻞ ﺍﳌﺆﺳﺴـﺔ ﺍﻟﻌﺴﻜ ﺮﻳﺔ ﺑﻔﺮﺽ ﺍﻻﺷﺨﺎﺹ ﺍﳌﻨﺘﻤﲔ ﺍﻟﻴﻬﺎ).٥٣( ٣- ﺩﻣﺞ ""ﺍﳌﻠﻴﺸﻴﺎﺕ ﰲ ﺍﳉﻴﺶ ﳑﺎ ﺃﺩﻯ ﺇﱃ ﻓﻘﺪﺍﻥ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ﻣﻘﻮﻣﺎﺕ ﺑﻨﺎﺋـﻪ ﻭﺩﳝﻮﻣﺘﻪ ﺍﻻ ﻭﻫﻲ ) ﺍﻟﻀﺒﻂ ، ﻭﺍﻟﻨﻈﺎﻡ ، ﻭﺍﻟﻘﺪﻡ ﺍﻟﻌﺴـﻜﺮﻱ ، ﻭﺍﻟﺘـﺪﺭﺝ ﰲ ﺍﻟﺮﺗﺐ ، ﻭﺍﳌﻬﻨﻴﺔ ﻭﺍﻟﻜﻔﺎﺀﺓ ، ﻭﺍﻟﺪﺭﺍﺳﺔ ﺍﻻﻛﺎﺩﳝﻴﺔ ( . ٧٨٣              ٥- ﺿﻌﻒ ﺍﻟﺪﻭﻟﺔ ﰲ ﺍﻟﺘﻌﺎﻣﻞ ﻣﻊ ﻣﺴﺄﻟﺔ ﺗﻨﺎﻣﻲ ﻭﻻﺩﺓ "ﺍﳌﻠﻴﺸﻴﺎﺕ "ﺍﳌﺴﻠﺤﺔ ﻭﺣﻠـﻬﺎ ﰲ ﻇﻞ ﻏﻴﺎﺏ ﺍﲨﺎﻉ ﻭﻃﲏ ﻣﻦ ﺍﻟﻜﺘﻞ ﻭﺍﻻﺣﺰﺍﺏ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﻧﺘﻴﺠـﺔ ﺍﻻﺳـﻨﺎﺩ ﺍﻟﺬﻱ ﲢﻈﻰ ﺑﻪ "ﺍﳌﻠﻴﺸﻴﺎﺕ "ﺍﳌﺴﻠﺤﺔ ﻣﻦ ﺃﺟﻬﺰﺓ ﺣﺎﻛﻤـﺔ ﻭﻗﻮﻳـﺔ ، ﻭﺍﺭﺗﺒـﺎﻁ ﺑﻌﻀﻬﺎ ﺑﺪﻭﻝ ﺍﳉﻮﺍﺭ ﻭﻣﺎ ﺗﻘﺪ ﻣﻪ ﳍﺎ ﻣﻦ ﺍﻟﺴﻼﺡ ﻭﺍﻟﺪﻋﻢ ﺍﳌﺎﱄ . ﻭﻫـﺬﺍ ﻳﻌـﲏ ﲢﻮﻝ ﺍﻟﻌﺮﺍﻕ ﺇﱃ ﺃﻛﱪ ﻣﻴﺪﺍﻥ ﻟﻠﺘﺪﺭﻳﺐ ﺍﻟﻌﺴﻜﺮﻱ ﰲ ﺍﻟﻌﺎﱂ ﻭﺃﺿـﺨﻢ ﺗﺮﺳـﺎﻧﺔ ﻟﻠﺴﻼﺡ ﺍﳌﺘﺪﺍﻭﻝ ﺑﲔ ﺍﻷﻓﺮﺍﺩ ﻭﺍﳉﻤﺎﻋﺎﺕ ﺍﳌﺴﻠﺤﺔ . ٥- ﺿﻌﻒ ﺍﻟﺪﻭﻟﺔ ﰲ ﺍﻟﺘﻌﺎﻣﻞ ﻣﻊ ﻣﺴﺄﻟﺔ ﺗﻨﺎﻣﻲ ﻭﻻﺩﺓ "ﺍﳌﻠﻴﺸﻴﺎﺕ "ﺍﳌﺴﻠﺤﺔ ﻭﺣﻠـﻬﺎ ﰲ ﻇﻞ ﻏﻴﺎﺏ ﺍﲨﺎﻉ ﻭﻃﲏ ﻣﻦ ﺍﻟﻜﺘﻞ ﻭﺍﻻﺣﺰﺍﺏ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﻧﺘﻴﺠـﺔ ﺍﻻﺳـﻨﺎﺩ ﺍﻟﺬﻱ ﲢﻈﻰ ﺑﻪ "ﺍﳌﻠﻴﺸﻴﺎﺕ "ﺍﳌﺴﻠﺤﺔ ﻣﻦ ﺃﺟﻬﺰﺓ ﺣﺎﻛﻤـﺔ ﻭﻗﻮﻳـﺔ ، ﻭﺍﺭﺗﺒـﺎﻁ ﺑﻌﻀﻬﺎ ﺑﺪﻭﻝ ﺍﳉﻮﺍﺭ ﻭﻣﺎ ﺗﻘﺪ ﻣﻪ ﳍﺎ ﻣﻦ ﺍﻟﺴﻼﺡ ﻭﺍﻟﺪﻋﻢ ﺍﳌﺎﱄ . ﻭﻫـﺬﺍ ﻳﻌـﲏ ﲢﻮﻝ ﺍﻟﻌﺮﺍﻕ ﺇﱃ ﺃﻛﱪ ﻣﻴﺪﺍﻥ ﻟﻠﺘﺪﺭﻳﺐ ﺍﻟﻌﺴﻜﺮﻱ ﰲ ﺍﻟﻌﺎﱂ ﻭﺃﺿـﺨﻢ ﺗﺮﺳـﺎﻧﺔ ﻟﻠﺴﻼﺡ ﺍﳌﺘﺪﺍﻭﻝ ﺑﲔ ﺍﻷﻓﺮﺍﺩ ﻭﺍﳉﻤﺎﻋﺎﺕ ﺍﳌﺴﻠﺤﺔ . ٦- ﺗﺄﺳﻴﺲ ﻛﻠﻴﺎﺕ ﻋﺴﻜﺮﻳﺔ ()ﺧﺎﺻﺔ ﺗﻨﻘﺼﻬﺎ ﺃﻫـﻢ ﻣﻘﻮﻣـﺎﺕ ﻋﻤـﻞ ﺍﻟﻜﻠﻴـﺔ ﺍﻟﻌﺴﻜﺮﻳﺔ ﻣﻦ ﺍﳌﻌﻠﻤﲔ ﺍﻟﻀﺒﺎﻁ ﺍﳉﻴﺪﻳﻦ ﻭﺿﺒﺎﻁ ﺍﻟﺼﻒ ، ﻭﻣﻴﺎﺩﻳﻦ ﺍﻟﺘـﺪﺭﻳﺐ ﻭﺍﻟﻘﺘﺎﻝ ﻭﺳﺎﺣﺎﺕ ﺍﻟﺘﺪﺭﻳﺐ ﺍﳌﻼﺋﻤﺔ ﻭﻗﺎﻋﺎﺕ ﺍﻟﺪﺭﻭﺱ ﺍﻟﻨﻈﺮﻳﺔ ﻭﺍﻟﻌﻤﻠﻴﺔ ﻭﻏـﲑ ﺫﻟﻚ ﻣﻦ ﺍﻟﻘﺎﻋﺪﺓ ﺍﳌﺎﺩﻳﺔ ﻟﻠﺘﺪﺭﻳﺐ ﻭﺍﻟﺘﺪﺭﻳﺲ . ٧- ﻓﻴﻤﺎ ﳜﺺ "ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ "ﺍﻟﻌﻠﻴﺎ ، ﱂ ﻳﺘﻢ ﺗﺄﻫﻴﻞ ﻛﻠﻴﺔ ﺍﻷﺭﻛﺎﻥ ﺃﻭ ﺟﺎﻣﻌﺔ ﺍﻟﺒﻜﺮ ﻟﻠﺪﺭﺍﺳﺎﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺍﻟﻌﻠﻴﺎ ﺑﺎﻻﺳﺘﻔﺎﺩﺓ ﻣﻦ ﻛﻮﺍﺩ ﺭﻫﺎ ﺍﻟﺘﺪﺭﻳﺴﻴﺔ ﺍﻟﻜﻔﻮﺀﺓ ﻷﻥ ﺃﺣﺰﺍﺏ ﺍﻟﺴﻠﻄﺔ ﻻ ﺗﺮﻏﺐ ﺑﺬﻟﻚ . ٨- ﺃﺿﻌﺎﻑ ﺍﻟﺮﻭﺡ ﺍﻟﻮﻃﻨﻴﺔ ﻭﺣﺐ ﺍﻟﻮﻃﻦ "ﺍﻟﻌﻘﻴﺪﺓ "ﺍﻟﻌﺴﻜﺮﻳﺔ ﻟﺪﻯ ﻣﻨﺘﺴﱯ ﺍﳉﻴﺶ ﻭﺣﻞ ﳏﻠﻬﺎ ﺣﺐ ﺍﻟﻄﺎﺋﻔﺔ ﻭﺍﳌﺬﻫﺐ ﻭﺍﳊﺰﺏ . ٩- "ﺍﺳﺘﺸﺮﺍ ﺀ ﻇﺎﻫﺮﺓ ﺍﻟﻔﺴﺎﺩ ﺍﻹﺩﺍﺭﻱ "ﻭﺍﳌﺎﱄ، ﻛﻘﻴﺎﻡ ﺑﻌـﺾ ﺍﻟﺴﻴﺎﺳـﻴﲔ ﺑﺒﻴـﻊ ﻣﻨﺎﺻﺐ ﻗﻴﺎﺩﻳﺔ ﰲ ﺍﳉﻴﺶ ﻟﻘﺎﺩﺓ ﻏﲑ ﻛ ﻔﻮﺋﲔ ﳑﺎ ﺍﻧﻌﻜﺲ ﺳﻠﺒﺎﹰ ﻋﻠﻰ ﺃﺩﺍﺀ ﺍﳉﻴﺶ، ﻓﻀﻼﹰ ﻋﻦ ﺍﻋﺪﺍﺩ ﻭﳘﻴﺔ ﻳﺘﻘﺎﺿﻮﻥ ﺭﻭﺍﺗﺐ ، ﻭﺃﺧﲑﺍﹰ ﺍﻟﻔﺴﺎﺩ ﰲ ﺻﻔﻘﺎﺕ ﺍﻷﺳﻠﺤﺔ ﻭﺍﻟﻌﻘﻮﺩ ﻟﺘﺠﻬﻴﺰ ﺍﻟﺴﻼﺡ ﻭﺍﳌﻌﺪﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ . ٠١- ﺗﻔﺸﻲ ﻇﺎﻫﺮﺓ ﺍﻟﺘﺴﺮﺏ ﰲ ﺍﻷﺟﻬﺰﺓ ﺍﻷﻣﻨﻴﺔ . ١١- ﺿﻌﻒ ﺍﳉﺎﻧﺐ ﺍﻷﻣﲏ ﻭﺍﻹﺳﺘﺨﺒﺎﺭﺍﰐ ﺑﺴﺒﺐ ﻋﺪﻡ ﺍﻻﻋﺘﻤـﺎﺩ ﻋﻠـﻰ ﺍﳌﻌـﺎﻳ ﲑ ﺍﻷﺳﺎﺳﻴﺔ ﺍﻹﺳﺘﺨﺒﺎﺭﺍﺗﻴﺔ . ٢١- ﻋﺪﻡ ﺍﻟﺘﻌﺎﻭﻥ ﻣﻊ ﺩﻭﻝ ﺍﳉﻮﺍﺭ ﰲ ﺿﺒﻂ ﺍﳊﺪﻭﺩ ﳌﻨﻊ ﺗﺴﻠﻞ ﺍﻹﺭﻫﺎﺑﻴﲔ . ٣١-ﻋﺪﻡ ﺍﻣﺘﻼﻙ ﺃﺳﻠﺤﺔ ﺗﻮﺍﺯﻥ ﺇﺳﺘﺮﺍﺗﻴﺠﻲ(). ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ٢١- ﻋﺪﻡ ﺍﻟﺘﻌﺎﻭﻥ ﻣﻊ ﺩﻭﻝ ﺍﳉﻮﺍﺭ ﰲ ﺿﺒﻂ ﺍﳊﺪﻭﺩ ﳌﻨﻊ ﺗﺴﻠﻞ ﺍﻹﺭﻫﺎﺑﻴﲔ . ٣١- ﻋﺪﻡ ﺍﻣﺘﻼﻙ ﺃﺳﻠﺤﺔ ﺗﻮﺍﺯﻥ ﺇﺳﺘﺮﺍﺗﻴﺠﻲ ().       ﺭﺍﺑﻌﺎ / ﺍﻗﺘﺼﺎﺩﻳﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : )٦٣( ١- ﺍﻟﻔﺴﺎﺩ ﺍﻻﻗﺘﺼﺎﺩﻱ ﻭﺍﻻﺩﺍﺭﻱ ﻭﺍﳌﺸﺎﺭﻳﻊ ﺍﻟﻮﳘﻴﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﺍﻫﺪﺍﺭ ﺍﳌﺎﻝ ﺍﻟﻌـﺎﻡ ﻭﺍﻟﺬﻱ ﺪﺩﻳﻬ ﻋﻤﻠﻴﺔ ﺍﻻﺳﺘﻘﺮﺍﺭ ﺍﻻﻗﺘﺼﺎﺩﻱ . ﻭﻣﺎ ﺿﻴﺎﻉ ﻣﻠﻴﺎﺭﺍﺕ ﺍﻟـﺪﻭﻻﺭﺍﺕ ﺍﻟﱵ ﻛﺎﻧﺖ ﳐﺼﺼﺔ ﻟﺘﻨﻤﻴﺔ ﺍﶈﺎﻓﻈﺎﺕ ﻭﺍﺳﺘﺤﻮﺍﺫ ﺍﻟﻔﺎﺳﺪﻳﻦ ﻋﻠﻰ ﺗﻠﻚ ﺍﻻﻣـﻮﺍﻝ ﺩﻭﻥ ﺣﺪﻭﺙ ﺗﻨﻤﻴﺔ ﺍﻗﺘﺼﺎﺩﻳﺔ ﰲ ﺗﻠﻚ ﺍﶈﺎﻓﻈﺎﺕ ﺁﺛﺮ ﺳﻠﺒﺎﹰ ﺑﺪﻭﺭﻩ ﻋﻠﻰ ﺍﻻﻭﺿﺎﻉ ﺩﺍﺧﻞ ﺗﻠﻚ ﺍﶈﺎﻓﻈﺎﺕ ﻣﻦ ﺧﻼﻝ ﺍﻗﺘﺤﺎﻡ ﺍﳌﺘﻈﺎﻫﺮﻳﻦ ﰲ ) ﺍﻟﻨﺠﻒ ، ﻭﻣﻴﺴـ ﺎﻥ ، ﻭﺍﻟﺒﺼﺮﺓ ( ﳌﺒﺎﱐ ﺍﶈﺎﻓﻈﺎﺕ ﺑﺴﺒﺐ ﺍﻟﻔﺴﺎﺩ ﺍﻟﺬﻱ ﺍﺳﺘﺸﺮﻯ ﰲ ﺍﳉﺴﺪ ﺍﻟﻌﺮﺍﻗـﻲ ﻭﺗﺮﺩﻱ ﺍﳋﺪﻣﺎﺕ ﺍﳊﻜﻮﻣﻴﺔ ﺍﳌﻘﺪﻣﺔ ﻟﻠﻤﻮﺍﻃﻨﲔ ).٧٣( ٢- ﺍﻧﺘﺸﺎﺭ ﻭﺗﻔﺎﻗﻢ ﺍﻟﻔﻘﺮ ﻭﺍﻟﺘﺴﻮﻝ ، ﻭﺗﻌﻄﻴﻞ ﺍﳌﺼﺎﻧﻊ ﻭﺍﻟﺒﻄﺎﻟﺔ ، ﻭﺗﺮﺍﺟـﻊ ﻧﺴـﺐ ﻓﺮﺹ ﺍﻟﻌﻤﻞ ﻟﻠﻤﻮﺍﻃﻨﲔ ﲰﺢ ﺑﺎﺭﺗﻔﺎﻉ ﻧﺴﺒﺔ ﺍﳉﺮﳝﺔ . ٣- ﺗﺰﻭﻳﺮ ﺍﻟﻌﻤﻠﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻭﻣﺎ ﳍﺎ ﻣﻦ ﺁﺛﺎﺭ ﻋﻠﻰ ﺍﻻﻗﺘﺼﺎﺩ ﺍﻟﻌﺮﺍﻗﻲ . ٤- ﺇﺧﺮﺍﺝ ﺍﻟﻌﻤﻠﺔ ﺍﻟﺼﻌﺒﺔ ""ﺍﻟﺪﻭﻻﺭ ﻣﻦ ﺍﻟﻌﺮﺍﻕ ﺇﱃ ﺩﻭﻝ ﺍﳉﻮﺍﺭ ﻭﲢﺪﻳﺪﺍﹰ ""ﺇﻳﺮﺍﻥ ، ﳑﺎ ﺍﻧﻌﻜﺲ ﺳﻠﺒﺎﹰ ﻋﻠﻰ ﺳﻌﺮ ﺻﺮﻑ ﺍﻟﺪﻳﻨﺎﺭ ﺍﻟﻌﺮﺍﻗﻲ . ٥- ﻋﺪﻡ ﻗﺪﺭﺓ ﺍﻟﻔﺮﺩ ﺍﳌﺴﺆﻭﻝ ﰲ ﺍﻟﻌﺎﺋﻠﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻋﻠﻰ ﺗﺄﻣﲔ ﲨﻴﻊ ﺣﺎﺟﺎﺕ ﺍﺳـﺮﺗﻪ ﻧﺘﻴﺠﺔ ﻟﺘﺪﱐ ﺍﻟﺪﺧﻞ ﺍﻟﺸﻬﺮﻱ ﻭﺃ ﺍﻟﻴﻮﻣﻲ ﺍﳌﻨﺎﺳﺐ ﻟﺬﻟﻚ . ٦- ﺍﺭﺗﻔﺎﻉ ﻣﻌﺪﻻﺕ ﺍﻟﺘﻀﺨﻢ ﻭﺑﺮﻭﺯ ﻇﺎﻫﺮﺓ ﺍﻟﻜﺴﺎﺩ . ٧- ﺗﻠﻜﺆ ﺣﺮﻛﺎﺕ ﺍﻻﻋﻤﺎﺭ . ٨- ﻋﺪﻡ ﻭﺟﻮﺩ ﺭﺅﻳﺔ ﻭﻃﻨﻴﺔ ﺣﻮﻝ ﺍﳌﺼﺎﱀ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ . ٠١- ﺍﻣﺘﻼﻙ ﺍﳌﻮﺍﺭﺩ ﺍﻟﻄﺒﻴﻌﻴﺔ ﻭﺍﳌﺰﺍﻳﺎ ﺍﳉﻐﺮﺍﻓﻴﺔ ﰲ ﻇﻞ ﻋـﺪﻡ ﻭﺟـﻮﺩ ﺍﻟﺘﺮﺗﻴﺒـﺎﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻼﺯﻣﺔ ، ﳛﻮﳍﺎ ﺇﱃ ﻣﻬﺪﺩ ﺃﻣﲏ ، ﺇﺫ ﺃﻥ ﻣﻌﻈﻢ ﻣﻨﺎﻃﻖ ﺍﻟﺼﺮﺍﻉ ﰲ ﺍﻟﻌﺎﱂ ﻫﻲ ﻣﻨﺎﻃﻖ ﺛﺮﻭﺍﺕ ﻃﺒﻴﻌﻴﺔ ﻳﻔﺘﻘﺮ ﺃﻫﻠﻬﺎ ﻟﻠﺘﺮﺗﻴﺐ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻟﻼﺯﻡ . ١١- ﻋﺪﻡ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﻻﺳﺘﻔﺎﺩﺓ ﻣﻦ ﺍﳌﻮﺍﺭﺩ ﺍﻟﻄﺒﻴﻌﻴﺔ ﻭﺗﻨﻤﻴﺘﻬﺎ . ٢١- ﺗﻔﺎﻗﻢ ﻇﺎﻫﺮﺓ ﺗﺮﺍﺟﻊ ﺍﻟﺘﻨﻮﻉ ﺍﻻﺣﻴﺎﺋﻲ ﻭﺍﻧﺘﺸﺎﺭ ﺍﻻﻣﺮﺍﺽ ﺑﲔ ﺍﻟﺜﺮﻭﺓ ﺍﳊﻴﻮﺍﻧﻴـﺔ "ﻧﻔﻮﻕ "ﺍﻻﲰﺎﻙ ﻭﻣﺎﻟﻪ ﻣﻦ ﺁﺛﺮ ﻋﻠﻰ ﺗﺪﱐ ﺍﻟﻨﺸﺎﻃﺎﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ . ٩٨٣                  ٣١- ﺗﺪﱐ ﺍﻻﻧﺘﺎﺝ ﺍﻟﺰﺭﺍﻋﻲ ﻭﺍﻟﺼﻨﺎﻋﻲ ﻭﻣﺎ ﺭﺍﻓﻘﻪ ﻣﻦ ﺗﺮﺍﺟﻊ ﰲ ﺍﻻﻳﺪﻱ ﺍﻟﻌﺎﻣﻠﺔ . ٤١- ﺗﺬﺑﺬﺏ ﺍﺳﻌﺎﺭ ﺍﻟﻨﻔﻂ . ٥١- ﺗﺪﻫﻮﺭ "ﺍﻟﺒﲎ "ﺍﻟﺘﺤﺘﻴﺔ ﻧﺘﻴﺠﺔ ﻻﻋﻤﺎﻝ ﺍﻟﺴﻠﺐ ﻭﺍﻟﻨﻬﺐ ﻭﺍﻟﺘﺤﻄـﻴﻢ ﻭﻣﻨـﻬﺎ ﻣﻨﺸﺂﺕ "ﺍﻟﺘﺼﻨﻴﻊ "ﺍﻟﻌﺴﻜﺮﻱ . ٦١- ﻋﺪﻡ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﻘﻴﻖﲢ ﺍﻷﻣﻦ ﺍﻟﻐﺬﺍﺋﻲ ﻧﺘﻴﺠﺔ ﺍﻹﻟﻐـﺎﺀ ﺍﻟﺘـﺪﺭﳚﻲ ﻟﻠﺒﻄﺎﻗـﺔ ﺍﻟﺘﻤﻮﻳﻨﻴﺔ . ٧١- ﺍﻻﻭﺿﺎﻉ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺍﻟﺼﻌﺒﺔ ﰲ ﺇﻗﻠﻴﻢ "ﻛﻮﺭﺩﺳﺘﺎﻥ "ﺍﻟﻌﺮﺍﻕ ﻭﻋـﺪﻡ ﻗـﺪﺭﺓ ﺣﻜﻮﻣﺔ ﺍﻹﻗﻠﻴﻢ ﻋﻠﻰ ﺩﻓﻊ ﺭﻭﺍﺗﺐ ﻣﻮﻇﻔﻲ ﺍﻟﻘﻄﺎﻉ ﺍﻟﻌﺎﻡ ﺑﺸﻜﻞ ﻣﻨـﺘﻈﻢ . ﻭﱂ ﺗﻘـﻒ ﺍﻷﻭﺿﺎﻉ ﺇﱃ ﺫﻟﻚ ﺍﳊﺪ ﺑﻞ ﰎ ﺍﺗﺒﺎﻉ ﺇﺟﺮﺍﺀﺍﺕ ﺗﻘﺸﻔﻴﺔ ﻣﻨﻬﺎ ﺗﻘﻠﻴﺺ ﺍ ﻟﺮﻭﺍﺗﺐ ﺑﻨﺴـﺒﺔ ﺗﺘﺮﺍﻭﺡ ﺑﲔ ) ٥١% ٥٢% ( ﻭﺧﻔﺾ ﻧﺴﺒﺔ ) ٥٧% ( ﻣﻦ ﺭﻭﺍﺗﺐ ﺍﻟـﺪﺭﺟﺎﺕ ﺍﳋﺎﺻﺔ ﳋﻔﺾ ﻋﺐﺀ ﺍﻷﺟﻮﺭ ﺍﻟﺸﻬﺮﻳﺔ ﻋﻦ ﻛﺎﻫﻞ ﺣﻜﻮﻣﺔ ﺍﻹﻗﻠﻴﻢ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﻛﻞ ﺫﻟﻚ ﺟـﺎﺀ ﻧﺘﻴﺠﺔ ﺍﳋﻼﻓﺎﺕ ﺍﳌﺘﻜﺮﺭﺓ ﻣﻊ ﺣﻜﻮﻣﺔ ﺑﻐﺪﺍﺩ ﺣﻮﻝ ﻧﺴﺒﺔ ﻋﺎﺋﺪﺍﺕ ﺍﻟـﻨﻔﻂ ﻟﻺﻗﻠـﻴﻢ ، ﻧﺎﻫﻴﻚ ﻋﻦ ﺍﻟﻔﺴﺎﺩ ﺍﳌﺴﺘﺸﺮﻱ ﻛﻤﺎ ﻫﻮ ﺍﳊﺎﻝ ﰲ ﺑﺎﻗﻲ ﳏ ﺎﻓﻈﺎﺕ ﺍﻟﻌﺮﺍﻕ ).٨٣( ﺧﺎﻣﺴﺎﹰ / ﻋﻠﻤﻴﺔ ﻭﺛﻘﺎﻓﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : ١- ﺍﻓﺘﻘﺎﺭ ﻭﻋﺠﺰ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻋﻠﻰ ﺃﻥ ﻳﻜﻮﻥ ﳍﺎ ﺗﺎﺛﲑﻫﺎ ﻋﻠـﻰ ﺛﻘﺎﻓـﺔ ﺍﻟﻔـﺮﺩ ﺍﻟﻌﺮﺍﻗﻲ).٩٣( ٢- ﺍﻧﺘﺸﺎﺭ ﺍﻷﻣﻴﺔ "ﺃﻣﻴﺔ ﺍﻟﻌﻠﻢ "ﻭﺍﻟﺘﻘﻨﻴﺔ . ٣- ﺍﻧﺘﺸﺎﺭ ﺛﻘﺎﻓﺔ ﺍﻟﻼﻣﺒﺎﻻﺓ ﰲ ﺍﳊﻔﺎﻅ ﻋﻠﻰ ﳑﺘﻠﻜﺎﺕ ﺍﻟﺪﻭﻟﺔ . ٤- ﺍﻟﻐﺰﻭ ﺍﻟﻔﻜﺮﻱ . ٥- ﺿﻌﻒ ﺍﻟﻜﻮﺍﺩﺭ ﰲ ﳎﺎﻻﺕ ﻣﻌﻴﻨﺔ ﺍﻟﱵ ﺗﻨﺎﺳﺐ ﺍﻟﺘﻨﺎﻓﺲ ﺍﻟﻌﺎﳌﻲ . ٦- ﻋﺪﻡ ﻭﺟﻮﺩ ﺗﻌﻠﻴﻢ ﺇﺳﺘﺮﺍﺗﻴﺠﻲ ﻤﻜﻦﻳ ﻣﻦ ﲢﻘﻴﻖ ﻗﺪﺭﺍﺕ ﺗﻨﺎﻓﺴﻴﺔ ﻣﻦ ﻣﻨﻈﻮﺭ ﻋﺎﳌﻲ ﻟﻠﻤﺆﺳﺴﺎﺕ ﺍﻟﺘﻌﻠﻴﻤﻴﺔ ﺍﻟﻮﻃﻨﻴﺔ . ٧- ﺍﻟﻔﺴﺎﺩ ﺍﻟﺘﺮﺑﻮﻱ ﻭﻇﺎﻫﺮﺓ ﺗﺴﺮﻳﺐ ﺍﻷﺳﺌﻠﺔ ﺍﻻﻣﺘﺤﺎﻧﻴﺔ . ٣١- ﺗﺪﱐ ﺍﻻﻧﺘﺎﺝ ﺍﻟﺰﺭﺍﻋﻲ ﻭﺍﻟﺼﻨﺎﻋﻲ ﻭﻣﺎ ﺭﺍﻓﻘﻪ ﻣﻦ ﺗﺮﺍﺟﻊ ﰲ ﺍﻻﻳﺪﻱ ﺍﻟﻌﺎﻣﻠﺔ . ٤١- ﺗﺬﺑﺬﺏ ﺍﺳﻌﺎﺭ ﺍﻟﻨﻔﻂ . ٥١- ﺗﺪﻫﻮﺭ "ﺍﻟﺒﲎ "ﺍﻟﺘﺤﺘﻴﺔ ﻧﺘﻴﺠﺔ ﻻﻋﻤﺎﻝ ﺍﻟﺴﻠﺐ ﻭﺍﻟﻨﻬﺐ ﻭﺍﻟﺘﺤﻄـﻴﻢ ﻭﻣﻨـﻬﺎ ﻣﻨﺸﺂﺕ "ﺍﻟﺘﺼﻨﻴﻊ "ﺍﻟﻌﺴﻜﺮﻱ . ٦١- ﻋﺪﻡ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﻘﻴﻖﲢ ﺍﻷﻣﻦ ﺍﻟﻐﺬﺍﺋﻲ ﻧﺘﻴﺠﺔ ﺍﻹﻟﻐـﺎﺀ ﺍﻟﺘـﺪﺭﳚﻲ ﻟﻠﺒﻄﺎﻗـﺔ ﺍﻟﺘﻤﻮﻳﻨﻴﺔ . ٧١- ﺍﻻﻭﺿﺎﻉ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺍﻟﺼﻌﺒﺔ ﰲ ﺇﻗﻠﻴﻢ "ﻛﻮﺭﺩﺳﺘﺎﻥ "ﺍﻟﻌﺮﺍﻕ ﻭﻋـﺪﻡ ﻗـﺪﺭﺓ ﺣﻜﻮﻣﺔ ﺍﻹﻗﻠﻴﻢ ﻋﻠﻰ ﺩﻓﻊ ﺭﻭﺍﺗﺐ ﻣﻮﻇﻔﻲ ﺍﻟﻘﻄﺎﻉ ﺍﻟﻌﺎﻡ ﺑﺸﻜﻞ ﻣﻨـﺘﻈﻢ . ﻭﱂ ﺗﻘـﻒ ﺍﻷﻭﺿﺎﻉ ﺇﱃ ﺫﻟﻚ ﺍﳊﺪ ﺑﻞ ﰎ ﺍﺗﺒﺎﻉ ﺇﺟﺮﺍﺀﺍﺕ ﺗﻘﺸﻔﻴﺔ ﻣﻨﻬﺎ ﺗﻘﻠﻴﺺ ﺍ ﻟﺮﻭﺍﺗﺐ ﺑﻨﺴـﺒﺔ ﺗﺘﺮﺍﻭﺡ ﺑﲔ ) ٥١% ٥٢% ( ﻭﺧﻔﺾ ﻧﺴﺒﺔ ) ٥٧% ( ﻣﻦ ﺭﻭﺍﺗﺐ ﺍﻟـﺪﺭﺟﺎﺕ ﺍﳋﺎﺻﺔ ﳋﻔﺾ ﻋﺐﺀ ﺍﻷﺟﻮﺭ ﺍﻟﺸﻬﺮﻳﺔ ﻋﻦ ﻛﺎﻫﻞ ﺣﻜﻮﻣﺔ ﺍﻹﻗﻠﻴﻢ . ﻛﻞ ﺫﻟﻚ ﺟـﺎﺀ ﻧﺘﻴﺠﺔ ﺍﳋﻼﻓﺎﺕ ﺍﳌﺘﻜﺮﺭﺓ ﻣﻊ ﺣﻜﻮﻣﺔ ﺑﻐﺪﺍﺩ ﺣﻮﻝ ﻧﺴﺒﺔ ﻋﺎﺋﺪﺍﺕ ﺍﻟـﻨﻔﻂ ﻟﻺﻗﻠـﻴﻢ ، ﻧﺎﻫﻴﻚ ﻋﻦ ﺍﻟﻔﺴﺎﺩ ﺍﳌﺴﺘﺸﺮﻱ ﻛﻤﺎ ﻫﻮ ﺍﳊﺎﻝ ﰲ ﺑﺎﻗﻲ ﳏ ﺎﻓﻈﺎﺕ ﺍﻟﻌﺮﺍﻕ ).٨٣( ﺧﺎﻣﺴﺎﹰ / ﻋﻠﻤﻴﺔ ﻭﺛﻘﺎﻓﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : ١- ﺍﻓﺘﻘﺎﺭ ﻭﻋﺠﺰ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻋﻠﻰ ﺃﻥ ﻳﻜﻮﻥ ﳍﺎ ﺗﺎﺛﲑﻫﺎ ﻋﻠـﻰ ﺛﻘﺎﻓـﺔ ﺍﻟﻔـﺮﺩ ﺍﻟﻌﺮﺍﻗﻲ).٩٣( ٢- ﺍﻧﺘﺸﺎﺭ ﺍﻷﻣﻴﺔ "ﺃﻣﻴﺔ ﺍﻟﻌﻠﻢ "ﻭﺍﻟﺘﻘﻨﻴﺔ . ٣- ﺍﻧﺘﺸﺎﺭ ﺛﻘﺎﻓﺔ ﺍﻟﻼﻣﺒﺎﻻﺓ ﰲ ﺍﳊﻔﺎﻅ ﻋﻠﻰ ﳑﺘﻠﻜﺎﺕ ﺍﻟﺪﻭﻟﺔ . ٣- ﺍﻧﺘﺸﺎﺭ ﺛﻘﺎﻓﺔ ﺍﻟﻼﻣﺒﺎﻻﺓ ﰲ ﺍﳊﻔﺎﻅ ﻋﻠﻰ ﳑﺘﻠﻜﺎﺕ ﺍﻟﺪﻭﻟﺔ . ٤- ﺍﻟﻐﺰﻭ ﺍﻟﻔﻜﺮﻱ . ٥- ﺿﻌﻒ ﺍﻟﻜﻮﺍﺩﺭ ﰲ ﳎﺎﻻﺕ ﻣﻌﻴﻨﺔ ﺍﻟﱵ ﺗﻨﺎﺳﺐ ﺍﻟﺘﻨﺎﻓﺲ ﺍﻟﻌﺎﳌﻲ . ٦- ﻋﺪﻡ ﻭﺟﻮﺩ ﺗﻌﻠﻴﻢ ﺇﺳﺘﺮﺍﺗﻴﺠﻲ ﻤﻜﻦﻳ ﻣﻦ ﲢﻘﻴﻖ ﻗﺪﺭﺍﺕ ﺗﻨﺎﻓﺴﻴﺔ ﻣﻦ ﻣﻨﻈﻮﺭ ﻋﺎﳌﻲ ﻟﻠﻤﺆﺳﺴﺎﺕ ﺍﻟﺘﻌﻠﻴﻤﻴﺔ ﺍﻟﻮﻃﻨﻴﺔ . ٧- ﺍﻟﻔﺴﺎﺩ ﺍﻟﺘﺮﺑﻮﻱ ﻭﻇﺎﻫﺮﺓ ﺗﺴﺮﻳﺐ ﺍﻷﺳﺌﻠﺔ ﺍﻻﻣﺘﺤﺎﻧﻴﺔ . ٥- ﺿﻌﻒ ﺍﻟﻜﻮﺍﺩﺭ ﰲ ﳎﺎﻻﺕ ﻣﻌﻴﻨﺔ ﺍﻟﱵ ﺗﻨﺎﺳﺐ ﺍﻟﺘﻨﺎﻓﺲ ﺍﻟﻌﺎﳌﻲ . ٦- ﻋﺪﻡ ﻭﺟﻮﺩ ﺗﻌﻠﻴﻢ ﺇﺳﺘﺮﺍﺗﻴﺠﻲ ﻤﻜﻦﻳ ﻣﻦ ﲢﻘﻴﻖ ﻗﺪﺭﺍﺕ ﺗﻨﺎﻓﺴﻴﺔ ﻣﻦ ﻣﻨﻈﻮﺭ ﻋﺎﳌﻲ ﻟﻠﻤﺆﺳﺴﺎﺕ ﺍﻟﺘﻌﻠﻴﻤﻴﺔ ﺍﻟﻮﻃﻨﻴﺔ . ٧- ﺍﻟﻔﺴﺎﺩ ﺍﻟﺘﺮﺑﻮﻱ ﻭﻇﺎﻫﺮﺓ ﺗﺴﺮﻳﺐ ﺍﻷﺳﺌﻠﺔ ﺍﻻﻣﺘﺤﺎﻧﻴﺔ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ٠٩٣        ٨- ﻧﻘﺺ ﺍﳌﺪﺍﺭﺱ ﰲ ﺍﳌﻨﺎﻃﻖ ""ﺍﻟﻨﺎﺋﻴﺔ ، ﻳﻘﺎﺑﻠﻬﺎ ﺍﺭﺗﻔﺎﻉ ﻇﺎﻫﺮﺓ "ﺍﳌﺪﺍﺭﺱ "ﺍﻷﻫﻠﻴﺔ ﰲ ﺍﳌﺪﻥ ﻧﺘﻴﺠﺔ ﺿﻌﻒ ﺍﻟﺘﻌﻠﻴﻢ ﺍﳊﻜﻮﻣﻲ ﻭﺍﻧﻌﺪﺍﻡ ﺍﻻﺟﻮﺍﺀ ﺍﻟﺪﺭﺍﺳـﻴﺔ ﻭﺍﻟﺼـﺤﻴﺔ ﻭﺍﻟﺒﻴﺌﻴﺔ ﻭﺍﳋﺪﻣﻴﺔ ﺍﳌﻨﺎﺳﺒﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﻠﻜﺆ ﻭﺯﺍﺭﺓ ﺍﻟﺘﺮﺑﻴﺔ ﰲ ﺗﺰﻭﻳﺪ ﺍﳌـﺪﺍﺭﺱ ﺑﺎﻟﻘﺮﻃﺎﺳﻴﺔ ).٠٤( ٩- ﺍﻗﺼﺎﺀ ﲪﻠﺔ ﺍﻟﺸﻬﺎﺩﺍﺕ ﺍﻟﻌﻠﻴﺎ ﻣﻦ ﺍﻻﺳﺎﺗﺬﺓ ﺍﳉﺎﻣﻌﲔ "ﺍﳌﺴﺎﺀﻟﺔ "ﻭﺍﻟﻌﺪﺍﻟـﺔ ﳑـﺎ ﻳﻨﻌﻜﺲ ﻠﺒﺎﹰﺳ ﻋﻠﻰ ﺍﻟﻮﺍﻗﻊ ﺍﻻﺟﺘﻤﺎﻋﻲ ﻭﺍﻟﺴﻴﺎﺳﻲ ﻟﻠﻌﺮﺍﻕ . ٠١- ﻫﺠﺮﺓ ﺍﻟﻜﻮﺍﺩﺭ ﺍﻟﻌﻠﻤﻴﺔ ﻧﺘﻴﺠﺔ ﻻﺳﺘﻬﺪﺍﻑ ﻭﺍﻏﺘﻴﺎﻝ ﺍﻻﺳـﺎﺗﺬﺓ ﺍﳉـﺎﻣﻌﻴﲔ ، ﺍﻻﻣﺮ ﺍﻟﺬﻱ ﺍﻧﻌﻜﺲ ﺳﻠﺒﺎﹰ ﻋﻠﻰ ﺍﳌﺴﺘﻮﻯ .ﺍﻟﻌﻠﻤﻲ ١١- ﻋﺪﻡ ﺍﻻﺳﺘﻔﺎﺩﺓ ﻣﻦ ﺧﱪﺍﺕ ﻭﻛﻔﺎﺀﺍﺕ ﻣﻮﻇﻔﻲ "ﻫﻴﺌﺔ ﺍﻟﺘﺼـﻨﻴﻊ "ﺍﻟﻌﺴـﻜﺮﻱ ﺍﳌﻨﺤﻠﺔ ﰲ ﻭﺯﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﳌﻌﻨﻴﺔ . ﺳ ﺎﺩﺳﺎﹰ / ﺇﻋﻼﻣﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : )١٤( ١- ﲢﻮﻝ ﺍﻹﻋﻼﻡ ﺇﱃ ﺃﺩﻭﺍﺕ ﺳﻴﺎﺳﻴﺔ ﺑﻴﺪ ﺣﺰﺏ ﺃﻭ ﺳـﻠﻄﺔ ، ﺃﻱ ﻭﺍﻗـﻊ ﺇﻋﻼﻣـﻲ ""ﻣﺘﻬﺮﻱ ﺯﺍﺩ ﻣﻦ ﺍﳓﻄﺎﻃﻪ ﻧﻈﺎﻡ ﺍﶈﺎﺻﺼﺔ ﺍﻟﻄﺎﺋﻔﻴﺔ ، ﺍﻻﻣﺮ ﺍﻟﺬﻱ ﺟﻌﻠﻪ ﺟﻬـﺎﺯﺍﹰ ﻏﲑ ﻣﺴﺘﻘﻞ ﻭﻻ ﺗﻄﻠﻌﺎﺕ ﺭﻳﺎﺩﻳﺔ ﻟﻪ . ٢- ﺍﻻﻧﺎﻧﻴﺔ ﻭﻗﺼﺮ ﺍﻟﻨﻈﺮ ﻟﻠﺴﺎﺳﺔ ﻭﺻﻨﺎﻉ ﺍﻟﻘﺮﺍﺭ ﻳﻌﺘﱪ ﻣﻦ ﺍﻛﱪ ﺍﳌﻬـﺪﺩﺍﺕ ﺍﻣـﺎﻡ ﺍﻟﺘﻄﻮﺭ ﺍﳌﻮﺿﻮﻋﻲ ﻟﻺﻋﻼﻡ ﺍﻟﺴﻴﺎﺳﻲ ، ﺇﺫ ﻳﺴﻌﻰ ﺑﻌـﺾ ﺍﻟﺴﺎﺳـﺔ ﺍﺳـﺘﻐﻼﻝ ﺍﻹﻋﻼﻡ ﺍﻟﺴﻴﺎﺳﻲ ﻟﺒﻨﺎﺀ "ﺍﳎﺎﺩﻫﻢ "ﺍﻟﺸﺨﺼﻴﺔ . ٣- "ﺍﻟﻔﻮﺿﻰ "ﺍﻹﻋﻼﻣﻴﺔ ﺑﺴﺒﺐ ﺗﺼﺮﳛﺎﺕ ﻭﺗﺼﺮﻓﺎﺕ ﺍﳌﺴﺆﻭﻟﲔ ﺍﻟﺴﻴﺎﺳﻴﲔ ﺍﳉـﺪﺩ ، ﺍﳌﺘﻨﺎﻗﻀﺔ ﻭﺍﳌﺘﺨﺒﻄﺔ ﻭﺍﳌﺨﺘﻠﻔﺔ ﻭﺍﳌﺘﺸﺎﺑﻜﺔ ﻭﺍﳌﺘﺼﺎﺭﻋﺔ ﻭﺍﻟﱵ ﺍﻧﻌﻜﺴﺖ ﺳـﻠﺒﺎﹰ ﻋﻠﻰ ﺍﳌﺘﻠﻘﻲ ﺍﻟﻌﺮﺍﻗﻲ ﻭﺍﺳﻬﻤﺖ ﻛﺜﲑﺍﹰ ﰲ ﺍﻋﻤﺎﻝ ﺍﻟﻌﻨﻒ ﻭﺍﻹﺭﻫﺎﺏ . ٤- ﻋﺪﻡ ﺗﻮﻓﺮ ﺍﻟﻜﻔﺎﺀﺍﺕ ﺍﻹﻋﻼﻣﻴﺔ ﺫﺍﺕ ﺍﳋﱪﺓ ﺍﻟﻜﺎﻓﻴﺔ ﰲ ﺍﻟﻌﻤﻞ ﰲ ﳎﺎﻝ ﺍﻹﻋـﻼﻡ ﺍﻟﺴﻴﺎﺳﻲ ، ﻭﺍﻥ ﺍﻏﻠﺒﻬﺎ ﺗﻌﺎﱐ ﺍﻟﻀﻌﻒ ﰲ ﺍﻻﺩﺍﺀ ﻭﺍﻟﺘﻘﺪﱘ ﻭﻃﺮﺡ ﺍﳌﻮﺿـﻮﻋﺎﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﻭﻫﺬﺍ ﻳﺸﻜﻞ ﻋﻘﺒﺔ ﺍﻣﺎﻡ ﳒﺎﺡ ﺍﻹﻋﻼﻡ ﺍﻟﺴﻴﺎﺳﻲ ﻭﲢﻘﻴ ﻖ ﺃﻫﺪﺍﻓﻪ . ٥- ﻋﺪﻡ ﲢﻤﻞ ﺍﻹﻋﻼﻡ ﻣﺴﺆﻭﻟﻴﺘﻪ ﺍﺯﺍﺀ ﺃﺟﻬﺰﺓ ﺍﻷﻣﻦ ، ﻭﺍﳌﻄﺎﻟﺒﺔ ﲜﻌﻠﻬﺎ ﺃﺟﻬﺰﺓ ﻓﺎﻋﻠﺔ       ٨- ﻧﻘﺺ ﺍﳌﺪﺍﺭﺱ ﰲ ﺍﳌﻨﺎﻃﻖ ""ﺍﻟﻨﺎﺋﻴﺔ ، ﻳﻘﺎﺑﻠﻬﺎ ﺍﺭﺗﻔﺎﻉ ﻇﺎﻫﺮﺓ "ﺍﳌﺪﺍﺭﺱ "ﺍﻷﻫﻠﻴﺔ ﰲ ﺍﳌﺪﻥ ﻧﺘﻴﺠﺔ ﺿﻌﻒ ﺍﻟﺘﻌﻠﻴﻢ ﺍﳊﻜﻮﻣﻲ ﻭﺍﻧﻌﺪﺍﻡ ﺍﻻﺟﻮﺍﺀ ﺍﻟﺪﺭﺍﺳـﻴﺔ ﻭﺍﻟﺼـﺤﻴﺔ ﻭﺍﻟﺒﻴﺌﻴﺔ ﻭﺍﳋﺪﻣﻴﺔ ﺍﳌﻨﺎﺳﺒﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﻠﻜﺆ ﻭﺯﺍﺭﺓ ﺍﻟﺘﺮﺑﻴﺔ ﰲ ﺗﺰﻭﻳﺪ ﺍﳌـﺪﺍﺭﺱ ﺑﺎﻟﻘﺮﻃﺎﺳﻴﺔ ).٠٤( ٩- ﺍﻗﺼﺎﺀ ﲪﻠﺔ ﺍﻟﺸﻬﺎﺩﺍﺕ ﺍﻟﻌﻠﻴﺎ ﻣﻦ ﺍﻻﺳﺎﺗﺬﺓ ﺍﳉﺎﻣﻌﲔ "ﺍﳌﺴﺎﺀﻟﺔ "ﻭﺍﻟﻌﺪﺍﻟـﺔ ﳑـﺎ ﻳﻨﻌﻜﺲ ﻠﺒﺎﹰﺳ ﻋﻠﻰ ﺍﻟﻮﺍﻗﻊ ﺍﻻﺟﺘﻤﺎﻋﻲ ﻭﺍﻟﺴﻴﺎﺳﻲ ﻟﻠﻌﺮﺍﻕ . ٠١- ﻫﺠﺮﺓ ﺍﻟﻜﻮﺍﺩﺭ ﺍﻟﻌﻠﻤﻴﺔ ﻧﺘﻴﺠﺔ ﻻﺳﺘﻬﺪﺍﻑ ﻭﺍﻏﺘﻴﺎﻝ ﺍﻻﺳـﺎﺗﺬﺓ ﺍﳉـﺎﻣﻌﻴﲔ ، ﺍﻻﻣﺮ ﺍﻟﺬﻱ ﺍﻧﻌﻜﺲ ﺳﻠﺒﺎﹰ ﻋﻠﻰ ﺍﳌﺴﺘﻮﻯ .ﺍﻟﻌﻠﻤﻲ ١١- ﻋﺪﻡ ﺍﻻﺳﺘﻔﺎﺩﺓ ﻣﻦ ﺧﱪﺍﺕ ﻭﻛﻔﺎﺀﺍﺕ ﻣﻮﻇﻔﻲ "ﻫﻴﺌﺔ ﺍﻟﺘﺼـﻨﻴﻊ "ﺍﻟﻌﺴـﻜﺮﻱ ﺍﳌﻨﺤﻠﺔ ﰲ ﻭﺯﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﳌﻌﻨﻴﺔ . ﺳ ﺎﺩﺳﺎﹰ / ﺇﻋﻼﻣﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : )١٤( ١- ﲢﻮﻝ ﺍﻹﻋﻼﻡ ﺇﱃ ﺃﺩﻭﺍﺕ ﺳﻴﺎﺳﻴﺔ ﺑﻴﺪ ﺣﺰﺏ ﺃﻭ ﺳـﻠﻄﺔ ، ﺃﻱ ﻭﺍﻗـﻊ ﺇﻋﻼﻣـﻲ ""ﻣﺘﻬﺮﻱ ﺯﺍﺩ ﻣﻦ ﺍﳓﻄﺎﻃﻪ ﻧﻈﺎﻡ ﺍﶈﺎﺻﺼﺔ ﺍﻟﻄﺎﺋﻔﻴﺔ ، ﺍﻻﻣﺮ ﺍﻟﺬﻱ ﺟﻌﻠﻪ ﺟﻬـﺎﺯﺍﹰ ﻏﲑ ﻣﺴﺘﻘﻞ ﻭﻻ ﺗﻄﻠﻌﺎﺕ ﺭﻳﺎﺩﻳﺔ ﻟﻪ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ٢ ﺍﻻﻧﺎﻧﺔ ﻭﻗﺼ ﺍﻟﻨﻈ ﻟﻠﺎﺳﺔ ﻭﺻﻨﺎﻉ ﺍﻟﻘﺍ ﻳﻌﺘ ﻣﻦ ﺍﻛ ﺍﳌﻬﺪﺩﺍﺕ ﺍﻣﺎﻡ ٨- ﻧﻘﺺ ﺍﳌﺪﺍﺭﺱ ﰲ ﺍﳌﻨﺎﻃﻖ ""ﺍﻟﻨﺎﺋﻴﺔ ، ﻳﻘﺎﺑﻠﻬﺎ ﺍﺭﺗﻔﺎﻉ ﻇﺎﻫﺮﺓ "ﺍﳌﺪﺍﺭﺱ "ﺍﻷﻫﻠﻴﺔ ﰲ ﺍﳌﺪﻥ ﻧﺘﻴﺠﺔ ﺿﻌﻒ ﺍﻟﺘﻌﻠﻴﻢ ﺍﳊﻜﻮﻣﻲ ﻭﺍﻧﻌﺪﺍﻡ ﺍﻻﺟﻮﺍﺀ ﺍﻟﺪﺭﺍﺳـﻴﺔ ﻭﺍﻟﺼـﺤﻴﺔ ﻭﺍﻟﺒﻴﺌﻴﺔ ﻭﺍﳋﺪﻣﻴﺔ ﺍﳌﻨﺎﺳﺒﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﻠﻜﺆ ﻭﺯﺍﺭﺓ ﺍﻟﺘﺮﺑﻴﺔ ﰲ ﺗﺰﻭﻳﺪ ﺍﳌـﺪﺍﺭﺱ ١- ﲢﻮﻝ ﺍﻹﻋﻼﻡ ﺇﱃ ﺃﺩﻭﺍﺕ ﺳﻴﺎﺳﻴﺔ ﺑﻴﺪ ﺣﺰﺏ ﺃﻭ ﺳـﻠﻄﺔ ، ﺃﻱ ﻭﺍﻗـﻊ ﺇﻋﻼﻣـﻲ ""ﻣﺘﻬﺮﻱ ﺯﺍﺩ ﻣﻦ ﺍﳓﻄﺎﻃﻪ ﻧﻈﺎﻡ ﺍﶈﺎﺻﺼﺔ ﺍﻟﻄﺎﺋﻔﻴﺔ ، ﺍﻻﻣﺮ ﺍﻟﺬﻱ ﺟﻌﻠﻪ ﺟﻬـﺎﺯﺍﹰ ﻏﲑ ﻣﺴﺘﻘﻞ ﻭﻻ ﺗﻄﻠﻌﺎﺕ ﺭﻳﺎﺩﻳﺔ ﻟﻪ . ٢- ﺍﻻﻧﺎﻧﻴﺔ ﻭﻗﺼﺮ ﺍﻟﻨﻈﺮ ﻟﻠﺴﺎﺳﺔ ﻭﺻﻨﺎﻉ ﺍﻟﻘﺮﺍﺭ ﻳﻌﺘﱪ ﻣﻦ ﺍﻛﱪ ﺍﳌﻬـﺪﺩﺍﺕ ﺍﻣـﺎﻡ ﺍﻟﺘﻄﻮﺭ ﺍﳌﻮﺿﻮﻋﻲ ﻟﻺﻋﻼﻡ ﺍﻟﺴﻴﺎﺳﻲ ، ﺇﺫ ﻳﺴﻌﻰ ﺑﻌـﺾ ﺍﻟﺴﺎﺳـﺔ ﺍﺳـﺘﻐﻼﻝ ﺍﻹﻋﻼﻡ ﺍﻟﺴﻴﺎﺳﻲ ﻟﺒﻨﺎﺀ "ﺍﳎﺎﺩﻫﻢ "ﺍﻟﺸﺨﺼﻴﺔ . ١٩٣              ﺳﺎﺑﻌﺎﹰ / ﺗﻘﻨﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : ﺳﺎﺑﻌﺎ / ﺗﻘﻨﻴﺔ : ﻭﺗﺘﻤﺜﻞ ﺑـ : ١- ﻧﻘﻞ ﺍﳌﻌﻠﻮﻣﺎﺕ ﻏﲑ ﺍﳍﺎﺩﻓﺔ ﻭﺍﳌﻐﻠﻮﻃﺔ ﻣﻦ ﺃﺟﻞ ﺑﺚ ﺍﻻﻛﺎﺫﻳﺐ ﺍﻟﱵ ﺗﺆﺛﺮ ﻋﻠـﻰ ﺍﻟﺮﺃﻱ ﺍﻟﻌﺎﻡ . ٢- ﺻﻌﻮﺑﺔ ﺍﻟﺘﺤﻘﻖ ﻣﻦ ﺍﳌﻌﻠﻮﻣﺎﺕ ﻭﺍﻻﺧﺒﺎﺭ ﻧﺘﻴﺠﺔ ﺍﻟﺴﺮﻋﺔ ﰲ ﺍﻻﻧﺘﺸـﺎﺭ ﻭﺗﻌـﺪﺩ ﺍﳌﺼﺎﺩﺭ . ٣- ﻧﺸﺮ ﺃﻓﻜﺎﺭ ﻭﻋﻘﺎﺋﺪ ﻣﻀﻠﻠﺔ ﻟﻠﻌﻘﻴﺪﺓ . ٤- ﺍﻻﺳﺎﺀﺓ ﻟﻶﺧﺮﻳﻦ ﳑﻦ ﳛﻤﻠﻮﻥ ﻣﺬﺍﻫﺐ ﺁﺧﺮﻯ .  ﺍﳌﻄﻠﺐ /ﺍﻟﺜـﺎﱐ ﳑﺎﻧﻌﺎﺕ ﺧﺎﺭﺟﻴﺔ: ﻭﺗﺘﻤﺜﻞ ﰲ : )٢٤( ﺃﻭﻻﹰ : ﻭﺟﻮﺩ ﺍﻹﺭﻫﺎﺏ ﰲ ﺍﻟﻌﺮﺍﻕ ، ﻭﻟﻪ ﻧﺼﻴﺒﻪ ﺍﻟﻜﺒﲑ ﰲ ﻋﺮﻗﻠﺔ ﺍﻟﺒﻨﺎﺀ ، ﺇﺫ ﺃﻥ ﺍﻻﺳﺘﻘﺮﺍﺭ ﺍﻷﻣﲏ ﻳﻨﻌﻜﺲ ﻋﻠﻰ ﺍﻻﺳﺘﻘﺮﺍﺭ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺍﻻﻗﺘﺼﺎﺩﻱ ﻭﺍﻻﺟﺘﻤﺎﻋﻲ ، ﻭﺍﳌﺸﻜﻠﺔ ﺍﻷﻣﻨﻴـﺔ ﺗﺸﻜﻞ ﻋﺎﺋﻘﺎﹰ ﻛﺒﲑﺍﹰ ﰲ ﻭﺟﻪ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ، ﻓﺤﻞ ﺍﳌﺆﺳﺴﺎﺕ ﺍﻷﻣﻨﻴﺔ ﻣﻦ ﻗﺒﻞ ﺳـﻠﻄﺎﺕ ﺍﻻﺣﺘﻼﻝ ﻗﺪ ﻓﺘﺢ ﺍﻟﺒﺎﺏ ﻋﻠﻰ ﻣﺼﺮﺍﻋﻴﻪ ﺃﻣﺎﻡ ﺗﻮﺍﻓﺪ ﺍﻟﻘﻮﻯ ﺍﻹﺭﻫﺎﺑﻴﺔ ، ﻓﻀﻼﹰ ﻋـﻦ ﺧﻠـﻖ ﺧﻼﻳﺎ ﻧﺎﺋﻤﺔ ﻟﺘﻨﻔﻴﺬ ﺗﻠﻚ ﺍﻻ ﻋﻤﺎﻝ ﻙ ) ﺍﻟﺴﻴﺎﺭﺍﺕ ﺍﳌﻔﺨﺨﺔ ، ﻭﺍﺳﺘﺨﺪﺍﻡ ﺍﻻﺣﺰﻣﺔ ﺍﻟﻨﺎﺳـﻔﺔ ﻭﺍﻟﺘﻔﺠﲑﺍﺕ ﺍﻻﻧﺘﺤﺎﺭﻳﺔ ، ﻭﺗﻌﺮﺽ ﺍﻟﻌﻠﻤﺎﺀ ﻭﺍﻻﻛﺎﺩﳝﻴﲔ ﻭﺍﻻﻃﺒـﺎﺀ ﻟﻌﻤﻠﻴـﺎﺕ ﺍﳋﻄـﻒ ﻭﺍﻟﻘﺘﻞ ﻭﺍﻻﻏﺘﻴﺎﻻﺕ ﺍﳌﻨﻈﻤﺔ ، ﻭﺗﺰﺍﻳﺪ ﺍﻟﻘﺘﻞ ﺍﳉﻤﺎﻋﻲ ، ﻭﻇﺎﻫﺮﺓ ﺍﳉﺜﺚ ﻬﻮﻟﺔﺍ ﺍﳍﻮﻳﺔ ( . ﺛﺎﻧﻴﺎﹰ : ﺗﺪﺧﻞ ﺩﻭﻝ ﺍﳉﻮﺍﺭ ) ﺇﻳﺮﺍﻥ ، ﺳﻮﺭﻳﺎ ، ﺍﳌﻤﻠﻜﺔ ﺍﻟﻌﺮﺑﻴﺔ ﺍﻟﺴﻌﻮﺩﻳﺔ ، ﺗﺮﻛﻴﺎ ( ﺍﻟﺴﺎﻓﺮ ﰲ ﺍﻟﺸﺄﻥ ﺍﻟﻌﺮﺍﻗﻲ . ﺛﺎﻟﺜﺎﹰ: ﺍﻟﻄﻤﻮﺣﺎﺕ ﺍﻹﻳﺮﺍﻧﻴﺔ ﻭﺍﻃﻤﺎﻋﻬﺎ ﺍﻟﺘﻮﺳﻌﻴﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺑﺸﻜﻞ ﻋﺎﻡ ﻭﺍﻟﻌـﺮﺍﻕ ﺑﺸـﻜﻞ ﺧﺎﺹ ، ﻣﻦ ﺧﻼﻝ ﺳﻠﺴﻠﺔ ﺭﺑﻂ ﺟﻐﺮﺍﻓﻴﺔ ﲤﺘﺪ ﻣﻦ ﻏﺮﺏ ﺇﻳﺮﺍﻥ ﻣﺮﻭﺭﺍﹰ ﺑﺎﻟﻌﺮﺍﻕ ﻭﺳـﻮﺭﻳﺎ، ﻭﺻﻮﻻﹰ ﺇﱃ ﺒﻨﺎﻥﻟ ﻭﺍﻷﺭﺍﺿﻲ ﺍﻟﻔﻠﺴﻄﻴﻨﺔ ﺍﶈﺘﻠﺔ، ﻭﺍﻧﺘـﻬﺎﺀﺍﹰ ﺑـﺎﻟﻴﻤﻦ ﰲ ﺍﳉـﺰﺀ ﺍﳉﻨـﻮﰊ ).ﺍﻟﻐﺮﰊ ٣٤( ﺭﺍﺑﻌﺎﹰ: ﺍﻻﺿﻄﺮﺍﺑﺎﺕ ﻭ ﺍﻟﺜﻮﺭﺍﺕ ﺍﻟﻌﺮﺑﻴﺔ "ﺍﻟﺮﺑﻴﻊ "ﺍﻟﻌﺮﰊ، ﺇﺫ ﺃﺣﺪﺛﺖ ﻫﺬﻩ ﺍﻟﺜﻮﺭﺍﺕ ﺗﻐﻴﲑﺍﺕ ﻛﱪﻯ ، ﻋﻠﻰ ﺍﻟﺼﻌﻴﺪﻳﻦ ﺍﻹﻗﻠﻴﻤﻲ ﻭﺍﻟﺪﻭﱄ ﻭﺗﺄﺛﲑﻫﺎ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻺﻗﻠﻴﻢ ، ﻛﻤﺎ ١- ﻧﻘﻞ ﺍﳌﻌﻠﻮﻣﺎﺕ ﻏﲑ ﺍﳍﺎﺩﻓﺔ ﻭﺍﳌﻐﻠﻮﻃﺔ ﻣﻦ ﺃﺟﻞ ﺑﺚ ﺍﻻﻛﺎﺫﻳﺐ ﺍﻟﱵ ﺗﺆﺛﺮ ﻋﻠـﻰ ﺍﻟﺮﺃﻱ ﺍﻟﻌﺎﻡ . ١- ﻧﻘﻞ ﺍﳌﻌﻠﻮﻣﺎﺕ ﻏﲑ ﺍﳍﺎﺩﻓﺔ ﻭﺍﳌﻐﻠﻮﻃﺔ ﻣﻦ ﺃﺟﻞ ﺑﺚ ﺍﻻﻛﺎﺫﻳﺐ ﺍﻟﱵ ﺗﺆﺛﺮ ﻋﻠـﻰ ﺍﻟﺮﺃﻱ ﺍﻟﻌﺎﻡ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ٢- ﺻﻌﻮﺑﺔ ﺍﻟﺘﺤﻘﻖ ﻣﻦ ﺍﳌﻌﻠﻮﻣﺎﺕ ﻭﺍﻻﺧﺒﺎﺭ ﻧﺘﻴﺠﺔ ﺍﻟﺴﺮﻋﺔ ﰲ ﺍﻻﻧﺘﺸـﺎﺭ ﻭﺗﻌـﺪﺩ ﺍﳌﺼﺎﺩﺭ . ٢- ﺻﻌﻮﺑﺔ ﺍﻟﺘﺤﻘﻖ ﻣﻦ ﺍﳌﻌﻠﻮﻣﺎﺕ ﻭﺍﻻﺧﺒﺎﺭ ﻧﺘﻴﺠﺔ ﺍﻟﺴﺮﻋﺔ ﰲ ﺍﻻﻧﺘﺸـﺎﺭ ﻭﺗﻌـﺪﺩ ﺍﳌﺼﺎﺩﺭ .  ﺍﳌﻄﻠﺐ /ﺍﻟﺜـﺎﱐ ﳑﺎﻧﻌﺎﺕ ﺧﺎﺭﺟﻴﺔ: ﻭﺗﺘﻤﺜﻞ ﰲ : )٢٤( ﺃﻭﻻﹰ : ﻭﺟﻮﺩ ﺍﻹﺭﻫﺎﺏ ﰲ ﺍﻟﻌﺮﺍﻕ ، ﻭﻟﻪ ﻧﺼﻴﺒﻪ ﺍﻟﻜﺒﲑ ﰲ ﻋﺮﻗﻠﺔ ﺍﻟﺒﻨﺎﺀ ، ﺇﺫ ﺃﻥ ﺍﻻﺳﺘﻘﺮﺍﺭ ﺍﻷﻣﲏ ﻳﻨﻌﻜﺲ ﻋﻠﻰ ﺍﻻﺳﺘﻘﺮﺍﺭ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺍﻻﻗﺘﺼﺎﺩﻱ ﻭﺍﻻﺟﺘﻤﺎﻋﻲ ، ﻭﺍﳌﺸﻜﻠﺔ ﺍﻷﻣﻨﻴـﺔ ﺗﺸﻜﻞ ﻋﺎﺋﻘﺎﹰ ﻛﺒﲑﺍﹰ ﰲ ﻭﺟﻪ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ، ﻓﺤﻞ ﺍﳌﺆﺳﺴﺎﺕ ﺍﻷﻣﻨﻴﺔ ﻣﻦ ﻗﺒﻞ ﺳـﻠﻄﺎﺕ ﺍﻻﺣﺘﻼﻝ ﻗﺪ ﻓﺘﺢ ﺍﻟﺒﺎﺏ ﻋﻠﻰ ﻣﺼﺮﺍﻋﻴﻪ ﺃﻣﺎﻡ ﺗﻮﺍﻓﺪ ﺍﻟﻘﻮﻯ ﺍﻹﺭﻫﺎﺑﻴﺔ ، ﻓﻀﻼﹰ ﻋـﻦ ﺧﻠـﻖ ﺧﻼﻳﺎ ﻧﺎﺋﻤﺔ ﻟﺘﻨﻔﻴﺬ ﺗﻠﻚ ﺍﻻ ﻋﻤﺎﻝ ﻙ ) ﺍﻟﺴﻴﺎﺭﺍﺕ ﺍﳌﻔﺨﺨﺔ ، ﻭﺍﺳﺘﺨﺪﺍﻡ ﺍﻻﺣﺰﻣﺔ ﺍﻟﻨﺎﺳـﻔﺔ ﻭﺍﻟﺘﻔﺠﲑﺍﺕ ﺍﻻﻧﺘﺤﺎﺭﻳﺔ ، ﻭﺗﻌﺮﺽ ﺍﻟﻌﻠﻤﺎﺀ ﻭﺍﻻﻛﺎﺩﳝﻴﲔ ﻭﺍﻻﻃﺒـﺎﺀ ﻟﻌﻤﻠﻴـﺎﺕ ﺍﳋﻄـﻒ ﻭﺍﻟﻘﺘﻞ ﻭﺍﻻﻏﺘﻴﺎﻻﺕ ﺍﳌﻨﻈﻤﺔ ، ﻭﺗﺰﺍﻳﺪ ﺍﻟﻘﺘﻞ ﺍﳉﻤﺎﻋﻲ ، ﻭﻇﺎﻫﺮﺓ ﺍﳉﺜﺚ ﻬﻮﻟﺔﺍ ﺍﳍﻮﻳﺔ ( . ﺛﺎﻧﻴﺎﹰ : ﺗﺪﺧﻞ ﺩﻭﻝ ﺍﳉﻮﺍﺭ ) ﺇﻳﺮﺍﻥ ، ﺳﻮﺭﻳﺎ ، ﺍﳌﻤﻠﻜﺔ ﺍﻟﻌﺮﺑﻴﺔ ﺍﻟﺴﻌﻮﺩﻳﺔ ، ﺗﺮﻛﻴﺎ ( ﺍﻟﺴﺎﻓﺮ ﺛﺎﻟﺜﺎﹰ: ﺍﻟﻄﻤﻮﺣﺎﺕ ﺍﻹﻳﺮﺍﻧﻴﺔ ﻭﺍﻃﻤﺎﻋﻬﺎ ﺍﻟﺘﻮﺳﻌﻴﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺑﺸﻜﻞ ﻋﺎﻡ ﻭﺍﻟﻌـﺮﺍﻕ ﺑﺸـﻜﻞ ﺧﺎﺹ ، ﻣﻦ ﺧﻼﻝ ﺳﻠﺴﻠﺔ ﺭﺑﻂ ﺟﻐﺮﺍﻓﻴﺔ ﲤﺘﺪ ﻣﻦ ﻏﺮﺏ ﺇﻳﺮﺍﻥ ﻣﺮﻭﺭﺍﹰ ﺑﺎﻟﻌﺮﺍﻕ ﻭﺳـﻮﺭﻳﺎ، ﻭﺻﻮﻻﹰ ﺇﱃ ﺒﻨﺎﻥﻟ ﻭﺍﻷﺭﺍﺿﻲ ﺍﻟﻔﻠﺴﻄﻴﻨﺔ ﺍﶈﺘﻠﺔ، ﻭﺍﻧﺘـﻬﺎﺀﺍﹰ ﺑـﺎﻟﻴﻤﻦ ﰲ ﺍﳉـﺰﺀ ﺍﳉﻨـﻮﰊ ).ﺍﻟﻐﺮﰊ ٣٤( ﺭﺍﺑﻌﺎﹰ: ﺍﻻﺿﻄﺮﺍﺑﺎﺕ ﻭ ﺍﻟﺜﻮﺭﺍﺕ ﺍﻟﻌﺮﺑﻴﺔ "ﺍﻟﺮﺑﻴﻊ "ﺍﻟﻌﺮﰊ، ﺇﺫ ﺃﺣﺪﺛﺖ ﻫﺬﻩ ﺍﻟﺜﻮﺭﺍﺕ ﺗﻐﻴﲑﺍﺕ ﻛﱪﻯ ، ﻋﻠﻰ ﺍﻟﺼﻌﻴﺪﻳﻦ ﺍﻹﻗﻠﻴﻤﻲ ﻭﺍﻟﺪﻭﱄ ﻭﺗﺄﺛﲑﻫﺎ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻺﻗﻠﻴﻢ ، ﻛﻤﺎ ٢٩٣              ﺎﺃ ﺃﺣﺪﺛﺖ ﺗﻐﻴﲑﺍﺕ ﻫﺎﺋﻠﺔ ﰲ ﺧﺮﻳﻄﺔ ﺍﻟﻔﺎﻋﻠﻴﲔ ﺍﻹﻗﻠﻴﻤﻴﲔ ﺑـﲔ ﺍﳍﺒـﻮﻁ ﻭﺍﻟﺼـﻌﻮﺩ ، ﻭﺍﻟﺘﺮﺩﺩ ﻭﺍﻻﻧﻜﻤﺎﺵ ، ﻭﺃﺧﲑﺍﹰ ﺍﻟﺘﻔﺎﻋﻼﺕ ﺍﳌﺨﺘﻠﻔﺔ ﺑﲔ ﺍﻟﻘﻮﻯ ﺍﻹﻗﻠﻴﻤﻴـﺔ ﺍﻟﻔﺎﻋﻠـﺔ ﺍﻟـﱵ ﺃﻋﺎﺩﺕ ﺳﺮﻳﻌﺎﹰ ﻗﺮﺍﺀﺓ ﺍﻷﺣﺪﺍﺙ ﻣﻦ ﻣﻨﻈﻮﺭ ﻭﺍﻗﻊ ﳐﺘﻠﻒ ﻭﺣﻘﺎﺋﻖ ﺟﺪﻳﺪﺓ ، ﱂ ﻳﻜﻦ ﺍﻟﻌﺮﺍﻕ ﺑﻌﻴﺪﺍﹰ ﻋﻨﻬﺎ ).٤٤( ﺧﺎﻣﺴﺎﹰ : ﺇﻋﺎﺩﺓ ﺍﳍﻴﻤﻨﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺍﳌﻌﺮﻭﻓﺔ ﺃﻫﺪﺍﻓﻬ ﺎ ﺎﻭﻏﺎﻳﺎ ﻟﻜـﻦ ﺑﻮﺳـﺎﺋﻞ ﻭﺃﺳـﺎﻟﻴﺐ ﻣﻌﺎﺻﺮﺓ ، ﻣﻦ ﺧﻼﻝ : - ﺇﻧﻀﺎﺝ ﻃﺒﻘﺔ ﺳﻴﺎﺳﻴﺔ ، ﻭﺃﺣﺰﺍﺏ ﺑﺪﻋﻢ ﻭﺗﺼﻨﻴﻊ ﺃﻣﺮﻳﻜﻲ ، ﲢﺖ ﻋﻨﻮﺍﻥ ) ﺣﻜﻮﻣـﺎﺕ ﻣﻨﺘﺨﺒﺔ ()( ، ﺫﺍﺕ ﺗﻮﺟﻬﺎﺕ ﻟﻴﱪﺍﻟﻴﺔ ، ﲣﺪﻡ ﻣﺼﺎﱀ ﺍﻟﻮﻻﻳـﺎﺕ ﺍﳌﺘﺤـﺪﺓ ﺍﻷﻣﺮﻳﻜﻴـﺔ ﻭﺣﻠﻴﻔﺘﻬﺎ ""ﺇﺳﺮﺍﺋﻴﻞ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ - ﺗﻮﻗﻴﻊ ﺍﻟﻮﻻﻳﺎﺕ ﺍﳌﺘﺤﺪﺓ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺍﺗﻔﺎﻗﻴﺔ ﺍﻹﻃﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﻣﻊ ﺍﻟﻌـﺮﺍﻕ ﺃﻛﺴـﺒﻬﺎ ﺃﻣﻮﺭ ﻋﺪﺓ ، ﻣﻨﻬﺎ : )٥٤( ﺎﺃ ﺃﺣﺪﺛﺖ ﺗﻐﻴﲑﺍﺕ ﻫﺎﺋﻠﺔ ﰲ ﺧﺮﻳﻄﺔ ﺍﻟﻔﺎﻋﻠﻴﲔ ﺍﻹﻗﻠﻴﻤﻴﲔ ﺑـﲔ ﺍﳍﺒـﻮﻁ ﻭﺍﻟﺼـﻌﻮﺩ ، ﻭﺍﻟﺘﺮﺩﺩ ﻭﺍﻻﻧﻜﻤﺎﺵ ، ﻭﺃﺧﲑﺍﹰ ﺍﻟﺘﻔﺎﻋﻼﺕ ﺍﳌﺨﺘﻠﻔﺔ ﺑﲔ ﺍﻟﻘﻮﻯ ﺍﻹﻗﻠﻴﻤﻴـﺔ ﺍﻟﻔﺎﻋﻠـﺔ ﺍﻟـﱵ ﺃﻋﺎﺩﺕ ﺳﺮﻳﻌﺎﹰ ﻗﺮﺍﺀﺓ ﺍﻷﺣﺪﺍﺙ ﻣﻦ ﻣﻨﻈﻮﺭ ﻭﺍﻗﻊ ﳐﺘﻠﻒ ﻭﺣﻘﺎﺋﻖ ﺟﺪﻳﺪﺓ ، ﱂ ﻳﻜﻦ ﺍﻟﻌﺮﺍﻕ ﺑﻌﻴﺪﺍﹰ ﻋﻨﻬﺎ ).٤٤( ﺧﺎﻣﺴﺎﹰ : ﺇﻋﺎﺩﺓ ﺍﳍﻴﻤﻨﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺍﳌﻌﺮﻭﻓﺔ ﺃﻫﺪﺍﻓﻬ ﺎ ﺎﻭﻏﺎﻳﺎ ﻟﻜـﻦ ﺑﻮﺳـﺎﺋﻞ ﻭﺃﺳـﺎﻟﻴﺐ ﻣﻌﺎﺻﺮﺓ ، ﻣﻦ ﺧﻼﻝ : - ﺇﻧﻀﺎﺝ ﻃﺒﻘﺔ ﺳﻴﺎﺳﻴﺔ ، ﻭﺃﺣﺰﺍﺏ ﺑﺪﻋﻢ ﻭﺗﺼﻨﻴﻊ ﺃﻣﺮﻳﻜﻲ ، ﲢﺖ ﻋﻨﻮﺍﻥ ) ﺣﻜﻮﻣـﺎﺕ ﻣﻨﺘﺨﺒﺔ ()( ، ﺫﺍﺕ ﺗﻮﺟﻬﺎﺕ ﻟﻴﱪﺍﻟﻴﺔ ، ﲣﺪﻡ ﻣﺼﺎﱀ ﺍﻟﻮﻻﻳـﺎﺕ ﺍﳌﺘﺤـﺪﺓ ﺍﻷﻣﺮﻳﻜﻴـﺔ ﻭﺣﻠﻴﻔﺘﻬﺎ ""ﺇﺳﺮﺍﺋﻴﻞ . - ﺇﻧﻀﺎﺝ ﻃﺒﻘﺔ ﺳﻴﺎﺳﻴﺔ ، ﻭﺃﺣﺰﺍﺏ ﺑﺪﻋﻢ ﻭﺗﺼﻨﻴﻊ ﺃﻣﺮﻳﻜﻲ ، ﲢﺖ ﻋﻨﻮﺍﻥ ) ﺣﻜﻮﻣـﺎﺕ ﻣﻨﺘﺨﺒﺔ ()( ، ﺫﺍﺕ ﺗﻮﺟﻬﺎﺕ ﻟﻴﱪﺍﻟﻴﺔ ، ﲣﺪﻡ ﻣﺼﺎﱀ ﺍﻟﻮﻻﻳـﺎﺕ ﺍﳌﺘﺤـﺪﺓ ﺍﻷﻣﺮﻳﻜﻴـﺔ ﻭﺣﻠﻴﻔﺘﻬﺎ ""ﺇﺳﺮﺍﺋﻴﻞ . - ﺗﻮﻗﻴﻊ ﺍﻟﻮﻻﻳﺎﺕ ﺍﳌﺘﺤﺪﺓ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺍﺗﻔﺎﻗﻴﺔ ﺍﻹﻃﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﻣﻊ ﺍﻟﻌـﺮﺍﻕ ﺃﻛﺴـﺒﻬﺎ ﺃﻣﻮﺭ ﻋﺪﺓ ، ﻣﻨﻬﺎ : )٥٤( ١. ﺣﻖ ﺇﻗﺎﻣﺔ ﻗﻮﺍﻋﺪ ﻋﺴﻜﺮﻳﺔ ﺃﻣﻨﻴﺔ ﺃﻣﺮﻳﻜﻴﺔ ، ﺗـﺆﻣﻦ ﺗـﺮﺍﺑﻂ ﻭﺭﺑـﻂ ﻭﺍﺗﺼﺎﻝ ﺍﻟﻘﻮﺍﺕ ﺍﻷﻣﺮﻳﻜﻴﺔ ﻣﻦ ﻏﺮﺏ ﺃﻭﺭﻭﺑﺎ ﺣﱴ ﺑﺎﻛﺴﺘﺎﻥ ﻭﺍﻟﻔﻠـﺒﲔ ، ﳌﻮﺍﺟﻬﺔ ﺃﻳﺔ ﺪﻳﺪﺍﺕ ﳏﺘﻤﻠﺔ ﻟﻸﻣﻦ ﺍﻟﻘﻮﻣﻲ ﺍﻷﻣﺮﻳﻜﻲ . ١. ﺣﻖ ﺇﻗﺎﻣﺔ ﻗﻮﺍﻋﺪ ﻋﺴﻜﺮﻳﺔ ﺃﻣﻨﻴﺔ ﺃﻣﺮﻳﻜﻴﺔ ، ﺗـﺆﻣﻦ ﺗـﺮﺍﺑﻂ ﻭﺭﺑـﻂ ﻭﺍﺗﺼﺎﻝ ﺍﻟﻘﻮﺍﺕ ﺍﻷﻣﺮﻳﻜﻴﺔ ﻣﻦ ﻏﺮﺏ ﺃﻭﺭﻭﺑﺎ ﺣﱴ ﺑﺎﻛﺴﺘﺎﻥ ﻭﺍﻟﻔﻠـﺒﲔ ، ﳌﻮﺍﺟﻬﺔ ﺃﻳﺔ ﺪﻳﺪﺍﺕ ﳏﺘﻤﻠﺔ ﻟﻸﻣﻦ ﺍﻟﻘﻮﻣﻲ ﺍﻷﻣﺮﻳﻜﻲ . ٢. ﺣﺮﻳﺔ ﺍﻟﻌﺒﺚ ﺑﺄﻣﻦ ﺍﻟﻌﺮﺍﻕ ﻭﺳﻠﻄﺎﺗﻪ ، ﻭﺣﱴ ﺍﻹﻃﺎﺣـﺔ ﺑﺎﳊﻜﻮﻣـﺎﺕ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﺎﻷﺳﻠﻮﺏ ﻧﻔﺴﻪ ﺍﻟﺬﻱ ﻛﺎﻧﺖ ﲤﺎﺭﺳﻪ ﺍﻹﺩﺍﺭﺍﺕ ﺍﻷﻣﺮﻳﻜﻴـﺔ ()""ﺍﻟﺴﺎﺑﻘﺔ . ﻛﻤﺎ ﺣﺼﻞ ﰲ ﺣﺰﻳـﺮﺍﻥ ﻋـﺎﻡ ٤١٠٢ ﻡ ، ﻋﻨـﺪﻣﺎ ﺃﺳﺘﻐﻠﺖ ﻫﺠﻤﺎﺕ ﺗﻨﻈﻴﻢ ""ﺩﺍﻋﺶ ﻟﺘﻐﻴﲑ ﻣﻮﺍﺯﻳﻦ ﺍﻟﻘﻮﻯ ﺍﻟﺴﻴﺎﺳﻴﺔ ﰲ ﺍﻟﻌﺮﺍﻕ . ٢. ﺣﺮﻳﺔ ﺍﻟﻌﺒﺚ ﺑﺄﻣﻦ ﺍﻟﻌﺮﺍﻕ ﻭﺳﻠﻄﺎﺗﻪ ، ﻭﺣﱴ ﺍﻹﻃﺎﺣـﺔ ﺑﺎﳊﻜﻮﻣـﺎﺕ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﺎﻷﺳﻠﻮﺏ ﻧﻔﺴﻪ ﺍﻟﺬﻱ ﻛﺎﻧﺖ ﲤﺎﺭﺳﻪ ﺍﻹﺩﺍﺭﺍﺕ ﺍﻷﻣﺮﻳﻜﻴـﺔ ()""ﺍﻟﺴﺎﺑﻘﺔ . ﻛﻤﺎ ﺣﺼﻞ ﰲ ﺣﺰﻳـﺮﺍﻥ ﻋـﺎﻡ ٤١٠٢ ﻡ ، ﻋﻨـﺪﻣﺎ ﺃﺳﺘﻐﻠﺖ ﻫﺠﻤﺎﺕ ﺗﻨﻈﻴﻢ ""ﺩﺍﻋﺶ ﻟﺘﻐﻴﲑ ﻣﻮﺍﺯﻳﻦ ﺍﻟﻘﻮﻯ ﺍﻟﺴﻴﺎﺳﻴﺔ ﰲ ٣. ﺗﺄﻣﲔ ﻣﺴﺘﻘﺒﻞ ﺍﻟﻌﺮﺍﻕ ﺑﺎﻟﺼﻮﺭﺓ ﺍﻟـﱵ ﺗﺮﻳـﺪﻫﺎ ﺍﻟﻮﻻﻳـﺎﺕ ﺍﳌﺘ ﺤـﺪﺓ ﺍﻷﻣﺮﻳﻜﻴﺔ ﻟﻪ ﻟﻀﻤﺎﻥ ﻋﺪﻡ ﺧﺮﻭﺟﻪ ﻣﻦ ﻗﻮﺍﻋـﺪ ﺍﻟﻠﻌﺒـﺔ ﺍﻹﻗﻠﻴﻤﻴـﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ ، ﺣﺴﺐ ﺍﻟﺮﺅﻳﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ).٦٤( ﺳﺎﺩﺳﺎﹰ : ﺗﻘﻴﻴﺪ ﺍﻟﺘﺴﻠﻴﺢ ﻭﻓﻘﺎﹰ ﳌﻌﺎﻳﲑ ﺍﻧﺘﻘﺎﺋﻴﺔ ﲣﺪﻡ ﺍﳌﺼﺎﱀ ﺍﻟﻐﺮﺑﻴﺔ ، ﻭﺫﻟﻚ ﺑﺴﺒﺐ : )٧٤( - ﺍﳋﻮﻑ ﻣﻦ ﺍﺳﺘﺤﻮﺍﺫ ﺍﳌﻠﻴﺸﻴﺎﺕ ﻋﻠﻰ ﺍﻟﺴﻼﺡ ﺍﻷﻣﺮﻳﻜـﻲ ، ﻭﺍﻟـﱵ ﺗﺘﻬﻤﻬﺎ ﺍﻟﻮﻻﻳﺎﺕ ﺍﳌﺘﺤﺪﺓ ﺎﺑﺄ ﻣﺪﻋﻮﻣﺔ ﻣﻦ ﻃﻬﺮﺍﻥ . ٣٩٣                     - ﺳﻴﻄﺮﺓ ﺗﻨﻈﻴﻢ ""ﺩﺍﻋﺶ ﻋﻠﻰ ﺗﺮﺳﺎﻧﺔ ﺍﻷﺳﻠﺤﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺍﻟﱵ ﻛﺎﻧـﺖ ﻟﺪﻯ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ﻣﻊ ﺳﻘﻮﻁ ﺍﳌﻮﺻﻞ ﰲ ﺣﺰﻳﺮﺍﻥ ﻋـﺎﻡ ٤١٠٢ ﻡ ﺑﻴﺪ ﺗﻨﻈﻴﻢ ""ﺩﺍﻋﺶ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ - ﺳﻴﻄﺮﺓ ﺗﻨﻈﻴﻢ ""ﺩﺍﻋﺶ ﻋﻠﻰ ﺗﺮﺳﺎﻧﺔ ﺍﻷﺳﻠﺤﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺍﻟﱵ ﻛﺎﻧـﺖ ﻟﺪﻯ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ﻣﻊ ﺳﻘﻮﻁ ﺍﳌﻮﺻﻞ ﰲ ﺣﺰﻳﺮﺍﻥ ﻋـﺎﻡ ٤١٠٢ ﻡ ﺑﻴﺪ ﺗﻨﻈﻴﻢ ""ﺩﺍﻋﺶ . - ﺍﺳﺘﻤﺮﺍﺭ ﺍﻟﻌﺮﺍﻕ "ﻣﻀﻄﺮﺍﹰ" ﺇﱃ ﺍﻻﻋﺘﻤﺎﺩ ﻋﻠـﻰ ﺍﳌﻈﻠـﺔ ﺍﻟﻌﺴـﻜﺮﻳﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ، ﻭﺍﻻﺳﺘﻤﺮﺍﺭ ﺍﻷﻣﺮﻳﻜﻲ ﰲ ﺍﻟﺘﺤﻜﻢ ﻣﺴﺘﻘﺒﻼﹰ ﰲ ﺍﳌﺆﺳﺴـﺔ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ).٨٤( ﺳﺎﺑﻌﺎﹰ : ﺳﺒﺎﻕ ﺍﻟﺘﺴﻠﺢ ﰲ ﺍﳌﻨﻄﻘﺔ ، ﻛﺎﻟﺴﻌﻲ ﺍﻹﻳﺮﺍﱐ ﻟﺒﻨﺎﺀ ﻗﻮﺓ ﻋﺴﻜﺮﻳﺔ ﺭﺍﺩﻋﺔ ﻟﻠـﺪﻓﺎﻉ ﻭﺍﳍﺠﻮﻡ ﻋﻦ ﻃﺮﻳﻖ ﺗﻄﻮﻳﺮ ﺎﻗﺪﺭﺍ ﺍﻟﻨﻮﻭﻳﺔ ﻭﻭﺳﺎﺋﻞ ﺇﻳﺼﺎﳍﺎ ﺍﻟ ﺼﺎﺭﻭﺧﻴﺔ ﺍﻟﺒﺎﻟﺴﺘﻴﺔ . ﺳﺎﺑﻌﺎﹰ : ﺳﺒﺎﻕ ﺍﻟﺘﺴﻠﺢ ﰲ ﺍﳌﻨﻄﻘﺔ ، ﻛﺎﻟﺴﻌﻲ ﺍﻹﻳﺮﺍﱐ ﻟﺒﻨﺎﺀ ﻗﻮﺓ ﻋﺴﻜﺮﻳﺔ ﺭﺍﺩﻋﺔ ﻟﻠـﺪﻓﺎﻉ ﻭﺍﳍﺠﻮﻡ ﻋﻦ ﻃﺮﻳﻖ ﺗﻄﻮﻳﺮ ﺎﻗﺪﺭﺍ ﺍﻟﻨﻮﻭﻳﺔ ﻭﻭﺳﺎﺋﻞ ﺇﻳﺼﺎﳍﺎ ﺍﻟ ﺼﺎﺭﻭﺧﻴﺔ ﺍﻟﺒﺎﻟﺴﺘﻴﺔ . ﺧـﺎﲤـﺔ ﻭﺍﺳﺘﻨﺘـﺎﺟـﺎﺕ ﰲ ﺧﺘﺎﻡ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﺍﻟﱵ ﺍﺟﺮﻳﻨﺎﻫﺎ ﻋﻦ ﺿﺮﻭﺭﺓ ﺇﻋﺎﺩﺓ ﺑﻨـﺎﺀ ﺍﻟﻘـﺪﺭﺓ ﺍﻹﺳـﺘﺮﺍﺗﻴﺠﻴﺔ : ﺍﻟﻌﺮﺍﻗﻴﺔ ﳝﻜﻨﻨﺎ ﺍﻻﺷﺎﺭﺓ ﺇﱃ ﺑﻌﺾ ﺍﳌﻼﺣﻈﺎﺕ ١- ﻭﺍﺟﻬﺖ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﻡ ، ﳑﺎﻧﻌـﺎﺕ ﻭﲢـﺪﻳﺎﺕ ﻭﺍﺳﻌﺔ ﺷﻜﻠﺖ ﺣﺠﺮ ﺍﻟﺰﺍﻭﻳﺔ ﰲ ﺍﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ، "ﻗﺪﺭﺍﺕ" ﻛﺎﻧﺖ ﻭﻣﺎ ﺯﺍﻟـﺖ ﲝﺎﺟـﺔ ﺇﱃ ﺍﻟﻜﺜﲑ ﻣﻦ ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﻟﻠﺘﺼﺤﻴﺢ ، ﻭﻟﺘﻌﺰﻳﺰ ﻋﻮﺩﺓ ﺍﻟﻌﺮﺍﻕ ﻗﻮﺓ ﻣـﺆﺛﺮﺓ ﻋﻠـﻰ . ﺍﻟﺴﺎﺣﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ : ﻻﺭ ﺇﱃ ﺑﺾ ﳌﻼ ﺮﻴ ﳝ ١- ﻭﺍﺟﻬﺖ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﻡ ، ﳑﺎﻧﻌـﺎﺕ ﻭﲢـﺪﻳﺎﺕ ﻭﺍﺳﻌﺔ ﺷﻜﻠﺖ ﺣﺠﺮ ﺍﻟﺰﺍﻭﻳﺔ ﰲ ﺍﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ، "ﻗﺪﺭﺍﺕ" ﻛﺎﻧﺖ ﻭﻣﺎ ﺯﺍﻟـﺖ ﲝﺎﺟـﺔ ﺇﱃ ﺍﻟﻜﺜﲑ ﻣﻦ ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﻟﻠﺘﺼﺤﻴﺢ ، ﻭﻟﺘﻌﺰﻳﺰ ﻋﻮﺩﺓ ﺍﻟﻌﺮﺍﻕ ﻗﻮﺓ ﻣـﺆﺛﺮﺓ ﻋﻠـﻰ . ﺍﻟﺴﺎﺣﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ ٢- ﺿﻌﻒ ﺃﻭ ﻋﺪﻡ ﻭﺿﻮﺡ ﺭﺅﻳﺎ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺒﻨﺎ "ﺀ ﺍﻟﻘﺪﺭﺍﺕ ، ﻓﺈﻣﺎ ﺃﻥ ﺗﺴﻮﺩ "ﺍﻻﻧﺘﻘﺎﺋﻴﺔ ﻭﻳﺘﻢ ﺇﻗﺼﺎﺀ ﺍﻟﻄﺮﻑ ﺍﻵﺧﺮ ﺍﻟﺬﻱ ﳝﻜﻦ ﺃﻥ ﻳﻜﻮﻥ ﻟﻪ ﺍﻟـﺪﻭﺭ ﺍﻟﻔﻌـﺎﻝ ﰲ ﺑﻨـﺎﺀ ﺍﻟﺮﺅﻳـﺎ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﺃﻭ ﺃﻥ ﻳﺴﻮﺩ ﺍﻟﺘﺸﺘﺖ ﻭﺍﻟﺘﻮﺟﻪ ﳓﻮ ﺍﺧﺘﻴﺎﺭ ﺃﻱ ﺭﺅﻳﺎ ﺇﺳـﺘﺮﺍﺗﻴﺠﻴﺔ ﺑﻐـﺾ ﺍﻟﻨﻈﺮ ﻋﻦ ﻣﺪﻯ ﺍﺧﻔﺎﻗﻬﺎ ﺃﻭ ﳒﺎﺣﻬﺎ ، ﻭﺑﻐﺾ ﺍﻟﻨﻈﺮ ﻋﻦ ﻣﺪﺍﻫﺎ ﺍﻟﺰﻣﲏ ﻭﺍﳌﻜﺎﱐ ﺍﻟ ﺬﻱ ﺭﲟـﺎ . ﻻ ﻳﺘﻼﺋﻢ ﻭﺍﻟﻮﺿﻊ ﺍﻵﱐ ٣- ﺗﺘﺠﻪ ﻋﻤﻠﻴﺔ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﻠﻰ ﺗﻔﻌﻴﻞ ﺍﺩﺍﺓ ﺩﻭﻥ ﺃﺧﺮﻯ ، ﻓﻤﺜﻼﹰ ﺗﺮﻛـﺰ ﻔﻌﻞ ﺍﻟﻘﺪﺭﺓ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺩﻭﻥ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻟﺜﻘﺎﻓﻴﺔ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴﺔ ﻫﺬﺍ ﻣﺎ ﳚﻌـﻞ ﺍﻟﻘـﺪﺭﺓﻭﺗ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻏﲑ ﻣﺘﻜﺎﻣﻠﺔ ، ﻓﻘﺪ ﻳﻄﻐﻰ ﺍﳉﺎﻧﺐ ﺍﻟﺴﻴﺎﺳﻲ ﻋﻠﻰ ﺑﺎﻗﻲ ﺍﳉﻮﺍ ﻧﺐ ﺍﻷﺧﺮﻯ ﳑﺎ . ﻮﻝ ﺩﻭﻥ ﲢﻘﻴﻖ ﻗﺪﺭﺓ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻓﻌﺎﻟﺔﳛ ٤٩٣       ٤- ﳏﺪﻭﺩﻳﺔ ﺍﻟﺪﻭﺭ ﻛﺄﺣﺪﻯ ﻣﻌﻮﻗﺎﺕ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻓﺎﻟﺪﻭﺭ ﺍﻟـﺬﻱ ﺗﻠﻌﺒـﻪ ﺑﻌﺾ ﺍﳌﺆﺳﺴﺎﺕ ﺍﻟﺮﲰﻴﺔ ﻻ ﳝﻜﻦ ﺃﻥ ﲤﺎﺭﺳﻪ ﺍﳌﺆﺳﺴﺎﺕ ﺍﻷﺧﺮﻯ ﳑﺎ ﳚﻌﻠﻬﺎ ﺣﻜﺮﺍﹰ ﻋﻠـﻰ ﺑﻌﺾ ﺍﳌﺆﺳﺴﺎﺕ ﺩﻭﻥ ﺍﻷﺧﺮﻯ ﻭﺑﺎﻟﺘﺎﱄ ﻳﺴﻮﺩ ﻣﺒﺪﺃ ﻋﺪ ﺍﻟﺸﺮﺍ ﻛﺔ ﻭﺗﻮﺯﻳﻊ ﺍﻻﺩﻭﺍﺭ ﻓﺘﺼﺪﺭ ﺎ ﺃﻥ ﺗﻜﻮﻥ ﻋﺎﺋﻘﺎﹰ ﺍﻣﺎﻡ ﺑﻨﺎﺀﺍﻟﻘﺮﺍﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﱵ ﺗﺴﻤﻰ ) ﺍﳌﺼﻠﺤﻴﺔ ( ﺍﻟﱵ ﻣﻦ ﺷﺄ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﺍﻟﺘﻮﺟﻬﺎﺕ ﺍﻷﻣﻨﻴﺔ ﺍﳌﺴﺎﻋﺪﺓ ﰲ ﺣﺎﻟﺔ ﻭﺟﻮﺩ ﺗﻘﺒﻞ ﻟﻺﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺍﻷﻣﻨﻴﺔ ٥٩٣ ٥٩٣              ٤- ـﺎ ﰲ ﻋﻠﻰ ﺍﳉﺎﻣﻌﺎﺕ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺃﻥ ﺗﺆﺩﻱ ﺩﻭﺭﻫﺎ ﺍﻟﻮﻃﲏ ﻭﺍﻻﻛﺎﺩﳝﻲ ﺍﻟﺘﻌﻠﻴﻤﻲ ﺍﳌﻨﻮﻁ ﺍﻧﺸﺎﺀ ﻛﻠﻴﺎﺕ ﻟﻠﻌﻠﻮﻡ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﻛﺬﻟﻚ ﺍﻧﺸـﺎﺀ ﻣﺮﺍﻛـﺰ ﻟﻠ ﺪﺭﺍﺳـﺎﺕ ﻭﺍﻟﺒﺤـﻮﺙ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺘﺴﺎﻋﺪ ﲨﻴﻊ ﺍﳌﺆﺳﺴﺎﺕ ﰲ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺳـﻮﺍﺀ ﰲ ﺍﻟﻘﻄـﺎﻉ ﺍﻟﻌـﺎﻡ ﺃﻭ ﺍﳋﺎﺹ ﻋﻠﻰ ﻛﻴﻔﻴﺔ ﺑﻨﺎﺀ ﻭﺍﻋﺪﺍﺩ ﻭﲣﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ ﻭﺗﻨﻔﻴﺬﻫﺎ ﻭﺗﻄﻮﻳﺮﻫـﺎ ﺳـﻮﺍﺀ ﰲ ﺎﻝ ﺍﻟﺴﻴﺎﺳﻲ ﺃﻭ ﺍﻻﻗﺘﺼﺎﺩﻱ ﺃﻭ ﺍﻟﻌﺴﻜﺮﻱ ﺃﻭ ﺍﳌﻌﻠﻮﻣﺎﰐ ﻓﻬﻮ ﻣﻄﻠﺐ ﺣﻀﺎﺭﻱ ﻭﺇﻧﺴـﺎﱐﺍ ﻭﻋﻠﻤﻲ ﻭﺃﻣﲏ ﺿ. ﺮﻭﺭﻱ ٥- ﺍﻻﳘﻴﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻮﺟﻮﺩ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﺮﺍﻗﻴﺔ ﺷﺎﻣﻠﺔ ﻣﺮﻫﻮﻧﺔ ﺑﺘﻮﻓﲑ ﺍﺭﺍﺩﺓ ﻭﻃﻨﻴـﺔ ﻋﺮﺍﻗﻴﺔ ﻓﺎﻋﻠﺔ ﻳﺘﺤﺘﻢ ﻋﻠﻴﻬﺎ ﺇﻋﺎﺩﺓ ﺍﻟﻨﻈﺮ ﰲ ﺍﳌﺴﺎﺭ ﺍﳊﺎﱄ ﻭﺑﻨﺎﺀ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﻃﻨﻴﺔ ﺗﻨﺒﻊ ﻣﻨﻬﺎ ) : ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ ﴰﻮﻟﻴﺔ ﻭﺇﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ ﻓﺮﻋﻴﺔ ، ﺃﳘﻬﺎ ٩٤( : ﺳﻴﺎﺳﻴﺎﹰ / ﻣﻦ ﺧﻼﻝ ٤- ـﺎ ﰲ ﻋﻠﻰ ﺍﳉﺎﻣﻌﺎﺕ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺃﻥ ﺗﺆﺩﻱ ﺩﻭﺭﻫﺎ ﺍﻟﻮﻃﲏ ﻭﺍﻻﻛﺎﺩﳝﻲ ﺍﻟﺘﻌﻠﻴﻤﻲ ﺍﳌﻨﻮﻁ ﺍﻧﺸﺎﺀ ﻛﻠﻴﺎﺕ ﻟﻠﻌﻠﻮﻡ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﻛﺬﻟﻚ ﺍﻧﺸـﺎﺀ ﻣﺮﺍﻛـﺰ ﻟﻠ ﺪﺭﺍﺳـﺎﺕ ﻭﺍﻟﺒﺤـﻮﺙ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺘﺴﺎﻋﺪ ﲨﻴﻊ ﺍﳌﺆﺳﺴﺎﺕ ﰲ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺳـﻮﺍﺀ ﰲ ﺍﻟﻘﻄـﺎﻉ ﺍﻟﻌـﺎﻡ ﺃﻭ ﺍﳋﺎﺹ ﻋﻠﻰ ﻛﻴﻔﻴﺔ ﺑﻨﺎﺀ ﻭﺍﻋﺪﺍﺩ ﻭﲣﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ ﻭﺗﻨﻔﻴﺬﻫﺎ ﻭﺗﻄﻮﻳﺮﻫـﺎ ﺳـﻮﺍﺀ ﰲ ﺎﻝ ﺍﻟﺴﻴﺎﺳﻲ ﺃﻭ ﺍﻻﻗﺘﺼﺎﺩﻱ ﺃﻭ ﺍﻟﻌﺴﻜﺮﻱ ﺃﻭ ﺍﳌﻌﻠﻮﻣﺎﰐ ﻓﻬﻮ ﻣﻄﻠﺐ ﺣﻀﺎﺭﻱ ﻭﺇﻧﺴـﺎﱐﺍ ﻭﻋﻠﻤﻲ ﻭﺃﻣﲏ ﺿ. ﺮﻭﺭﻱ ٥- ﺍﻻﳘﻴﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﻮﺟﻮﺩ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻋﺮﺍﻗﻴﺔ ﺷﺎﻣﻠﺔ ﻣﺮﻫﻮﻧﺔ ﺑﺘﻮﻓﲑ ﺍﺭﺍﺩﺓ ﻭﻃﻨﻴـﺔ ﻋﺮﺍﻗﻴﺔ ﻓﺎﻋﻠﺔ ﻳﺘﺤﺘﻢ ﻋﻠﻴﻬﺎ ﺇﻋﺎﺩﺓ ﺍﻟﻨﻈﺮ ﰲ ﺍﳌﺴﺎﺭ ﺍﳊﺎﱄ ﻭﺑﻨﺎﺀ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﻃﻨﻴﺔ ﺗﻨﺒﻊ ﻣﻨﻬﺎ ) : ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ ﴰﻮﻟﻴﺔ ﻭﺇﺳﺘﺮﺍﺗﻴﺠﻴﺎﺕ ﻓﺮﻋﻴﺔ ، ﺃﳘﻬﺎ ٩٤( : ﺳﻴﺎﺳﻴﺎﹰ / ﻣﻦ ﺧﻼﻝ ﺑﻨﺎﺀ ﺍ ﺎ ﻭﺍﻟﻌﻤﻞ ﻋﻠﻰ ﺗﺮﺳﻴﺦ ﻣﺒﺎﺩﺋﻬﺎ ﻟﺘﻨﺴـﺠﻢ ﻭﻣﺘﻄﻠﺒـﺎﺕﻟﺪﻭﻟﺔ ﻭﺍﶈﺎﻓﻈﺔ ﻋﻠﻰ ﻛﻴﺎ . ﺍﻟﺪﳝﻮﻗﺮﺍﻃﻴﺔ * ﺿﺮﻭﺭﺓ ﺍﻻﲡﺎﻩ ﳓﻮ ﺍﳌﺼﺎﳊﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺍﻟﱵ ﻫﻲ ﺍﻻﺳﺎﺱ ﻭﺍﳌﻨﻄﻠﻖ ﻟﺘﺮﺳﻴﺦ ﻋﻤﻠﻴﺔ ﺍﻟﺒﻨـﺎﺀ ﺎ ﺍﻵﻃﺎﺭ ﺍﻟﺬﻱ ﳚﻤﻊ ﻛﻞ ﺍﳌﻮﺍﻃﻨﲔ ﻋﻠﻰ ﺍﺧﺘﻼﻑﻭﺇﻋﺎﺩﺓ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻛﻮ ﻢﺍﻧﺘﻤﺎﺀﺍ . ﺍﻟﻔﺮﻋﻴﺔ * ﻭﺿﻊ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻓﻌﺎﻟﺔ ﻣﻦ ﻗﺒﻞ ﺍﻟﺪﻭﻟﺔ ﻟﻠﻘﻀﺎﺀ ﻋﻠﻰ ﻇﺎﻫﺮﺓ ﺍﻟﻔﺴﺎﺩ ﺍﻟـﺬﻱ ﻳﺴـﺮﻱ ﰲ ، ﲨﻴﻊ ﻣﻔﺎﺻﻞ ﺍﻟﺪﻭﻟﺔ ﺳﻮﺍﺀ ﺍﻛﺎﻥ ﻓﺴﺎﺩ ) ﺳﻴﺎﺳﻲ ، ﺍﺟﺘﻤـﺎﻋﻲ ، ﺍﻗﺘﺼـﺎﺩﻱ ، ﺇﺩﺍﺭﻱ . ( ﻗﺎﻧﻮﱐ * ﺿﺮﻭﺭﺓ ﺍﻻﲡﺎﻩ ﳓﻮ ﺍﳌﺼﺎﳊﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺍﻟﱵ ﻫﻲ ﺍﻻﺳﺎﺱ ﻭﺍﳌﻨﻄﻠﻖ ﻟﺘﺮﺳﻴﺦ ﻋﻤﻠﻴﺔ ﺍﻟﺒﻨـﺎﺀ ﺎ ﺍﻵﻃﺎﺭ ﺍﻟﺬﻱ ﳚﻤﻊ ﻛﻞ ﺍﳌﻮﺍﻃﻨﲔ ﻋﻠﻰ ﺍﺧﺘﻼﻑﻭﺇﻋﺎﺩﺓ ﻗﺪﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻛﻮ ﻢﺍﻧﺘﻤﺎﺀﺍ . ﺍﻟﻔﺮﻋﻴﺔ * ﻭﺿﻊ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻓﻌﺎﻟﺔ ﻣﻦ ﻗﺒﻞ ﺍﻟﺪﻭﻟﺔ ﻟﻠﻘﻀﺎﺀ ﻋﻠﻰ ﻇﺎﻫﺮﺓ ﺍﻟﻔﺴﺎﺩ ﺍﻟـﺬﻱ ﻳﺴـﺮﻱ ﰲ ، ﲨﻴﻊ ﻣﻔﺎﺻﻞ ﺍﻟﺪﻭﻟﺔ ﺳﻮﺍﺀ ﺍﻛﺎﻥ ﻓﺴﺎﺩ ) ﺳﻴﺎﺳﻲ ، ﺍﺟﺘﻤـﺎﻋﻲ ، ﺍﻗﺘﺼـﺎﺩﻱ ، ﺇﺩﺍﺭﻱ . ( ﻗﺎﻧﻮﱐ * ﺍﻻﺭﺗﻘﺎﺀ ﺑﺎﻟﻌﻤﻠﻴﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻟﺘﻜﻮﻥ ﻗﺎﺩﺭﺓ ﻋﻠﻰ ﻣﻮﺍﺟﻬﺔ ﺍﻟﺘﺤﺪﻳﺎﺕ ﻭﺍﳌﺘﻐﲑﺍﺕ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴ ﺔ ﺑﺎﻻﻋﺘﻤﺎﺩ ﻋﻠﻰ ﻧﻔﺴﻬﺎ ، ﺩﻭﻥ ﺣﺎﺟﺔ ﻟﺮﻋﺎﻳﺔ ﺩﻭﻟﻴﺔ ﺃﻭ ﺇﻗﻠﻴﻤﻴﺔ ، ﺑﻮﺻﻠﺘﻬﺎ ﰲ ﺫﻟﻚ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺍﻟﺘـﻮﺻﻴـﺎﺕ ﻧﺘﻴﺠﺔ ﻟﻄﺒﻴﻌﺔ ﺍﻟﺘﺤﺪﻳﺎﺕ ﻭﺃﳕﺎﻃﻬﺎ ﺍﳌﺨﺘﻠﻔﺔ ﻭﺣﺎﺟﺔ ﺍﻟﺒﻠـﺪ ﺇﱃ ﺍﻟﺒﺤـﺚ ﻭﺍﻟﺪﺭﺍﺳـﺔ ﻭﺍﻟﺘﺤﻠﻴﻞ ﰲ ﺑﻌﺾ ﺍﳌﻌﻄﻴﺎﺕ ﻭﺍﳌﻘ ﻮﻣﺎﺕ ﻭﺍﳌﺆﺛﺮﺍﺕ ﻭﺍﳌﻜﻮﻧﺎﺕ ﻭﺍﳌﻬﺪﺩﺍﺕ ﻭﺍﻟﺘﺤﺪﻳﺎﺕ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺪﺍﺧﻠﻴﺔ ﻭﺍﳋﺎﺭﺟﻴﺔ ، ﻭﺑﻌﺪ ﺍﻟﻮﻗﻮﻑ ﻋﻠﻰ ﻣﺴﺘﻮﻯ ﺍﻟﺘﺨﻄﻴﻂ ﰲ ﺑﻌﺾ ، ﻭﺯﺍﺭﺍﺕ ﻭﻣﺆﺳﺴﺎﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ، ﻭﺍﻟﻨﻈﺮ ﰲ ﺍﻻﺑﻌﺎﺩ ) ﺍﻟﺴﻴﺎﺳـﻴﺔ ، ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﺍﻻﺟﺘﻤﺎﻋﻴﺔ ، ﺍﻟﺜﻘﺎﻓﻴﺔ ( ﻭﻣﺪﻯ ﺃﳘﻴﺔ ﻫﺬﺍ ﺍﻻ ﺑﻌﺎﺩ ﻟﻠﻤﺘﻄﻠﺒﺎﺕ ﺍﳊﻴﻮﻳﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺿﺮﻭﺭﺓ ﺗﻔﻌﻴﻠﻬﺎ ، ﻻ ﺑﺪ ﻟﻨﺎ ﻣﻦ ﺍﳋﺮﻭﺝ ﺑﺘﻮﺻﻴﺎﺕ ﻣﺒﺴﻄﺔ ﺗﻜﻮﻥ ﺍﳕﻮﺫﺟﺎﹰ ﻟﻠﺘﻌﺎﻣـﻞ ﻣـﻊ ﻫﺬﻩ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺑﺸﻜﻞ ﻭﺛﻴﻖ ﰲ ﺳﺒﻴﻞ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ، ﻭﻫـﻲ : ﻛﺎﻻﰐ ٤- ﳏﺪﻭﺩﻳﺔ ﺍﻟﺪﻭﺭ ﻛﺄﺣﺪﻯ ﻣﻌﻮﻗﺎﺕ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﻓﺎﻟﺪﻭﺭ ﺍﻟـﺬﻱ ﺗﻠﻌﺒـﻪ ﺑﻌﺾ ﺍﳌﺆﺳﺴﺎﺕ ﺍﻟﺮﲰﻴﺔ ﻻ ﳝﻜﻦ ﺃﻥ ﲤﺎﺭﺳﻪ ﺍﳌﺆﺳﺴﺎﺕ ﺍﻷﺧﺮﻯ ﳑﺎ ﳚﻌﻠﻬﺎ ﺣﻜﺮﺍﹰ ﻋﻠـﻰ ﺑﻌﺾ ﺍﳌﺆﺳﺴﺎﺕ ﺩﻭﻥ ﺍﻷﺧﺮﻯ ﻭﺑﺎﻟﺘﺎﱄ ﻳﺴﻮﺩ ﻣﺒﺪﺃ ﻋﺪ ﺍﻟﺸﺮﺍ ﻛﺔ ﻭﺗﻮﺯﻳﻊ ﺍﻻﺩﻭﺍﺭ ﻓﺘﺼﺪﺭ ﺎ ﺃﻥ ﺗﻜﻮﻥ ﻋﺎﺋﻘﺎﹰ ﺍﻣﺎﻡ ﺑﻨﺎﺀﺍﻟﻘﺮﺍﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﱵ ﺗﺴﻤﻰ ) ﺍﳌﺼﻠﺤﻴﺔ ( ﺍﻟﱵ ﻣﻦ ﺷﺄ . ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻧﺘﻴﺠﺔ ﻟﻄﺒﻴﻌﺔ ﺍﻟﺘﺤﺪﻳﺎﺕ ﻭﺃﳕﺎﻃﻬﺎ ﺍﳌﺨﺘﻠﻔﺔ ﻭﺣﺎﺟﺔ ﺍﻟﺒﻠـﺪ ﺇﱃ ﺍﻟﺒﺤـﺚ ﻭﺍﻟﺪﺭﺍﺳـﺔ ﻭﺍﻟﺘﺤﻠﻴﻞ ﰲ ﺑﻌﺾ ﺍﳌﻌﻄﻴﺎﺕ ﻭﺍﳌﻘ ﻮﻣﺎﺕ ﻭﺍﳌﺆﺛﺮﺍﺕ ﻭﺍﳌﻜﻮﻧﺎﺕ ﻭﺍﳌﻬﺪﺩﺍﺕ ﻭﺍﻟﺘﺤﺪﻳﺎﺕ ﰲ ﺍﻟﺒﻴﺌﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺪﺍﺧﻠﻴﺔ ﻭﺍﳋﺎﺭﺟﻴﺔ ، ﻭﺑﻌﺪ ﺍﻟﻮﻗﻮﻑ ﻋﻠﻰ ﻣﺴﺘﻮﻯ ﺍﻟﺘﺨﻄﻴﻂ ﰲ ﺑﻌﺾ ، ﻭﺯﺍﺭﺍﺕ ﻭﻣﺆﺳﺴﺎﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ، ﻭﺍﻟﻨﻈﺮ ﰲ ﺍﻻﺑﻌﺎﺩ ) ﺍﻟﺴﻴﺎﺳـﻴﺔ ، ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﺍﻻﺟﺘﻤﺎﻋﻴﺔ ، ﺍﻟﺜﻘﺎﻓﻴﺔ ( ﻭﻣﺪﻯ ﺃﳘﻴﺔ ﻫﺬﺍ ﺍﻻ ﺑﻌﺎﺩ ﻟﻠﻤﺘﻄﻠﺒﺎﺕ ﺍﳊﻴﻮﻳﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺿﺮﻭﺭﺓ ﺗﻔﻌﻴﻠﻬﺎ ، ﻻ ﺑﺪ ﻟﻨﺎ ﻣﻦ ﺍﳋﺮﻭﺝ ﺑﺘﻮﺻﻴﺎﺕ ﻣﺒﺴﻄﺔ ﺗﻜﻮﻥ ﺍﳕﻮﺫﺟﺎﹰ ﻟﻠﺘﻌﺎﻣـﻞ ﻣـﻊ ﻫﺬﻩ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺑﺸﻜﻞ ﻭﺛﻴﻖ ﰲ ﺳﺒﻴﻞ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ، ﻭﻫـﻲ : ﻛﺎﻻﰐ ١- ﺿﺮﻭﺭﺓ ﺍﻻﻋﺘﺮﺍﻑ ﺑﺎﳌﺸﻜﻠﺔ ﺍﻟﻘﺎﺋﻤﺔ ﻣﻦ ﺍﳌﻨﻈﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ، ﻭﺍﻟﻌﻤ ﻞ ﻋﻠﻰ ﺗﺸﻜﻴﻞ ﻓﺮﻳﻖ ﺧﱪﺍﺀ ﳝﺘﻠﻚ ﺭﺅﻳﺔ ﻭﺧﱪﺓ ﻗﻴﺎﺩﻳﺔ ﻭﺳﻴﺎﺳﻴﺔ ﻣﺆﺳﺴﻴﺔ ﻭﻓﺮﺩﻳـﺔ ﺫﺍﺕ ﻗـﺪﺭﺍﺕ ﺗﻨﻈﻴﻤﻴـﺔ ، ﻭﺗﻨﺴﻴﻘﻴﺔ ، ﲟﻌﲎ ﺁﺧﺮ ﳝﺘﻠﻚ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﻟﺘﻐﻴﲑ ﻭﺍﻟﺘﻌﺎﻣﻞ ﻣﻊ ﺍﳌﻤﺎﻧﻌﺎﺕ ﺑﺸﻜﻞ ﻭﺛﻴـﻖ ﻣﻦ ﺧﻼﻝ ﲢﻠﻴﻞ ﺍﳌﺸﺎﻛﻞ ﻭﲢﺪﻳﺪ ﺍﻻﻃﺎﺭ ﺍﻟﻌﺎﻡ ﻭﺍﳌﹸﻔﺼﻞ ﻟﻠﺮﺅﻳﺔ ﺍﻹﺳـﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸـﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ، ﻭ . ﺍﺟﺮﺍﺀ ﺣﺼﺮ ﺷﺎﻣﻞ ﻷﻫﻢ ﺍﻟﺘﺤﺪﻳﺎﺕ ﻭﺍﻟﺘﻬﺪﻳﺪﺍﺕ ﺍﻟﱵ ﺗﻮﺍﺟﻪ ﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ٢- . ﺎﻻﺕ ﺍﳌﺨﺘﻠﻔﺔﺍﻷﺧﺬ ﺑﺎﻻﳕﺬﺟﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺗﻔﻌﻴﻠﻬﺎ ﻭﺗﻮﻇﻴﻔﻬﺎ ﰲ ﺍ ٣- ﺧﻠﻖ ﻧﻮﻉ ﻣﻦ ﺍﻟﺘﻮﺍﻓﻖ ﻭﺍﻻﻧﺴﺠﺎﻡ ﺑﲔ ﺍﻟﻘﻴﺎﺩﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻣﻦ ﺧﻼﻝ ﺍﳌﺰﻳﺪ ﻣـﻦ ﺍﳌﻨﺘﺪﻳﺎﺕ ﺍﻟﻌﻠﻤﻴﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍﻷﻣﻨﻴﺔ ، ﻟﺘﻔﻌﻴﻞ ﺍﻟﻨﺴﻴﺞ ﺍﻷﻣـﲏ ﺍﻟﻌﺮﺍﻗـﻲ ﻭﺗﻘﺮﻳـﺐ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ""ﺍﳌﺼﻠﺤﺔ ﺍﻟﻮﻃﻨﻴﺔ * ﲢﺴﲔ ﺍﻟﻮﺿﻊ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺍﻟﺴﻌﻲ ﻟﺘﻐﻴﲑﻩ ﳓﻮ ﺍﻷﻓﻀﻞ ﺑﺪﺭﺟﺔ ﻣﻌﻘﻮﻟـﺔ ﻣـﻦ ﺍﳊﺮﻳـﺔ ﻭﺍﳌﻤﺎﺭﺳﺔ ، ﻭﺯﻳﺎﺩﺓ ﺍﺳﺘﻘﺮﺍﺭﻩ ﻋﱪ ﺷﺮﺍﻛﺔ ﻭﻃﻨﻴﺔ ﺣﻘﻴﻘﻴﺔ ﲤﺘﻠﻚ ﺭﺅﻳﺔ ﻣﻮﺣـﺪﺓ ﻭﺍﺿـﺤﺔ ﺍﳌﻌﺎﱂ ﲞﺼﻮﺹ ﲨﻴ. ﻊ ﺍﻟﻘﻀﺎﻳﺎ ﺍﻟﱵ ﺗﺆﺭﻕ ﺃﻓﻜﺎﺭﻫﺎ * ﺍﻻﺭﺗﻘﺎﺀ ﺑﺎﻟﻌﻤﻠﻴﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻟﺘﻜﻮﻥ ﻗﺎﺩﺭﺓ ﻋﻠﻰ ﻣﻮﺍﺟﻬﺔ ﺍﻟﺘﺤﺪﻳﺎﺕ ﻭﺍﳌﺘﻐﲑﺍﺕ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴ ﺔ ﺑﺎﻻﻋﺘﻤﺎﺩ ﻋﻠﻰ ﻧﻔﺴﻬﺎ ، ﺩﻭﻥ ﺣﺎﺟﺔ ﻟﺮﻋﺎﻳﺔ ﺩﻭﻟﻴﺔ ﺃﻭ ﺇﻗﻠﻴﻤﻴﺔ ، ﺑﻮﺻﻠﺘﻬﺎ ﰲ ﺫﻟﻚ . ""ﺍﳌﺼﻠﺤﺔ ﺍﻟﻮﻃﻨﻴﺔ * ﲢﺴﲔ ﺍﻟﻮﺿﻊ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺍﻟﺴﻌﻲ ﻟﺘﻐﻴﲑﻩ ﳓﻮ ﺍﻷﻓﻀﻞ ﺑﺪﺭﺟﺔ ﻣﻌﻘﻮﻟـﺔ ﻣـﻦ ﺍﳊﺮﻳـﺔ ﻭﺍﳌﻤﺎﺭﺳﺔ ، ﻭﺯﻳﺎﺩﺓ ﺍﺳﺘﻘﺮﺍﺭﻩ ﻋﱪ ﺷﺮﺍﻛﺔ ﻭﻃﻨﻴﺔ ﺣﻘﻴﻘﻴﺔ ﲤﺘﻠﻚ ﺭﺅﻳﺔ ﻣﻮﺣـﺪﺓ ﻭﺍﺿـﺤﺔ ﺍﳌﻌﺎﱂ ﲞﺼﻮﺹ ﲨﻴ. ﻊ ﺍﻟﻘﻀﺎﻳﺎ ﺍﻟﱵ ﺗﺆﺭﻕ ﺃﻓﻜﺎﺭﻫﺎ ٦٩٣       * ﺍﻟﺘﺨﻠﺺ ﻣﻦ ﺍﻟﻄﺎﺑﻊ ﺍﳌﺬﻫﱯ ﻟﻠﻨﻈﺎﻡ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﻌﺮﺍﻗﻲ ، ﲟﻌﲎ ﺗﺸﻜﻴﻞ ﺃﺣﺰﺍﺏ ﺳﻴﺎﺳـﻴﺔ ﻋﻠﻰ ﺃﺳﺲ ﻭﻃﻨﻴﺔ ، ﻭﻟﻴﺲ ﻋﻠﻰ ﺃﺳﺲ ﻗﻮﻣﻴﺔ ﺃﻭ ﺩﻳﻨﻴﺔ ﺃﻭ ﻃﺎﺋﻔﻴﺔ . ﻓﺎﻷﺣﺰﺍﺏ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺗﻌﺪ ﻋﺎﻣﻼﹰ ﻓﻌﺎﻻﹰ ﰲ ﺇﺩﺍﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﲟﻨﻄﻖ ﺇﺩﺍﺭﺓ ﺩﻭﻟﺔ ﻭﻟﻴﺲ ﲟ ﻨﻄـﻖ ﺇﺩﺍﺭﺓ . ﻣﺼﺎﱀ ﺣﺰﺑﻴﺔ ﺃﻭ ﻓﺌﻮﻳﺔ ﺃﻭ ﻣﻨﺎﻃﻘﻴﺔ * ﺗﻌﺰﻳﺰ ﻣﻜﺎﻧﺔ ﺍﻟﻌﺮﺍﻕ ﺇﻗﻠﻴﻤﻴﺎﹰ ﻭﺩﻭﻟﻴﺎﹰ ﻣﻦ ﺧﻼﻝ ﺍﻻﻧﻔﺘﺎﺡ ﻋﻠﻰ ﺍﻟﻌـﺎﱂ ﺍﳋـﺎﺭﺟﻲ ﻭﺑﻨـﺎﺀ . ﻋﻼﻗﺎﺕ ﻋﻠﻰ ﺍﺳﺎﺱ ﺍﳌﺼﺎﱀ ﺍﳌﺸﺘﺮﻛﺔ ﲟﺎ ﻳﻀﻤﻦ ﲢﻘﻴﻖ ﺍﻷﻣﻦ ﻭﺍﻻﺳﺘﻘﺮﺍﺭ ﻟﻠﺒﻠﺪ * ﺗﺮﺳﻴﺦ ﻣﻔﻬﻮﻡ ﺍﳌﻮﺍﻃﻨﺔ ﻋﱪ ﺇﻋﺎﺩﺓ ﺻﻴﺎﻏﺔ ﺍﻟﻌﻘﺪ ﺍﻻﺟﺘﻤﺎﻋﻲ ﺍﳌﺘﻔﻖ ﻋﻠﻴﻪ ﺑـﲔ ﺍﻟﺪﻭﻟـﺔ ﺎ ﺍﻻﺟﺘﻤﺎﻋﻴﺔ ، ﺑﺼﻮﺭﺓ ﻗﻮﺍﻧﲔ ﺗﻨﻈﻢ ﺗﻠﻚ ﺍﻟﻌﻼﻗﺔ ﻭﻣﺎ ﺗﻨﻈﻤﻪ ﻣـﻦ ﺣﻘـﻮﻕﻭﲨﻴﻊ ﻣﻜﻮﻧﺎ . ﻭﻭﺍﺟﺒﺎﺕ ﻭﲢﺪﻳﺪ ﻟﻠﻤﺴﺆﻭﻟﻴﺎﺕ ﺍﻟﺘﺒﺎﺩﻟﻴﺔ * ﺍﻟﺘﺨﻠﺺ ﻣﻦ ﺍﻟﻄﺎﺑﻊ ﺍﳌﺬﻫﱯ ﻟﻠﻨﻈﺎﻡ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﻌﺮﺍﻗﻲ ، ﲟﻌﲎ ﺗﺸﻜﻴﻞ ﺃﺣﺰﺍﺏ ﺳﻴﺎﺳـﻴﺔ ﻋﻠﻰ ﺃﺳﺲ ﻭﻃﻨﻴﺔ ، ﻭﻟﻴﺲ ﻋﻠﻰ ﺃﺳﺲ ﻗﻮﻣﻴﺔ ﺃﻭ ﺩﻳﻨﻴﺔ ﺃﻭ ﻃﺎﺋﻔﻴﺔ . ﻓﺎﻷﺣﺰﺍﺏ ﺍﻟﺴﻴﺎﺳـﻴﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺗﻌﺪ ﻋﺎﻣﻼﹰ ﻓﻌﺎﻻﹰ ﰲ ﺇﺩﺍﺭﺓ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﲟﻨﻄﻖ ﺇﺩﺍﺭﺓ ﺩﻭﻟﺔ ﻭﻟﻴﺲ ﲟ ﻨﻄـﻖ ﺇﺩﺍﺭﺓ ﻣﺼﺎﱀﺣﺰﺑﺔﺃﻭﻓﺌﻮﻳﺔﺃﻭﻣﻨﺎﻃﻘﺔ . ﻣﺼﺎﱀ ﺣﺰﺑﻴﺔ ﺃﻭ ﻓﺌﻮﻳﺔ ﺃﻭ ﻣﻨﺎﻃﻘﻴﺔ * ﺗﻌﺰﻳﺰ ﻣﻜﺎﻧﺔ ﺍﻟﻌﺮﺍﻕ ﺇﻗﻠﻴﻤﻴﺎﹰ ﻭﺩﻭﻟﻴﺎﹰ ﻣﻦ ﺧﻼﻝ ﺍﻻﻧﻔﺘﺎﺡ ﻋﻠﻰ ﺍﻟﻌـﺎﱂ ﺍﳋـﺎﺭﺟﻲ ﻭﺑﻨـﺎﺀ . ﻋﻼﻗﺎﺕ ﻋﻠﻰ ﺍﺳﺎﺱ ﺍﳌﺼﺎﱀ ﺍﳌﺸﺘﺮﻛﺔ ﲟﺎ ﻳﻀﻤﻦ ﲢﻘﻴﻖ ﺍﻷﻣﻦ ﻭﺍﻻﺳﺘﻘﺮﺍﺭ ﻟﻠﺒﻠﺪ * ﺗﺮﺳﻴﺦ ﻣﻔﻬﻮﻡ ﺍﳌﻮﺍﻃﻨﺔ ﻋﱪ ﺇﻋﺎﺩﺓ ﺻﻴﺎﻏﺔ ﺍﻟﻌﻘﺪ ﺍﻻﺟﺘﻤﺎﻋﻲ ﺍﳌﺘﻔﻖ ﻋﻠﻴﻪ ﺑـﲔ ﺍﻟﺪﻭﻟـﺔ ﺎ ﺍﻻﺟﺘﻤﺎﻋﻴﺔ ، ﺑﺼﻮﺭﺓ ﻗﻮﺍﻧﲔ ﺗﻨﻈﻢ ﺗﻠﻚ ﺍﻟﻌﻼﻗﺔ ﻭﻣﺎ ﺗﻨﻈﻤﻪ ﻣـﻦ ﺣﻘـﻮﻕﻭﲨﻴﻊ ﻣﻜﻮﻧﺎ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﻭﻭﺍﺟﺒﺎﺕ ﻭﲢﺪﻳﺪ ﻟﻠﻤﺴﺆﻭﻟﻴﺎﺕ ﺍﻟﺘﺒﺎﺩﻟﻴﺔ ) : ﻋﺴﻜﺮﻳﺎ / ﻭﻣﻨﻬﺎ ٠٥( * ﺇﻥ ﺑﻨﺎﺀ ﺍﻻﻗﺘﺼﺎﺩ ﻻ ﻳﻨﺴﺤﺐ ﻋﻠﻰ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ﻓﺤﺴﺐ ، ﻭﺇﳕﺎ ﻳـﺪﺧﻞ ﰲ ﺑﻨﺎﺀ ﻣﺆﺳﺴﺎﺕ ﺍﻟﺪﻭﻟﺔ ﺑ ، ﺸﻜﻞ ﻋﺎﻡ ﻭﻣﻦ ﺿﻤﻨﻬﺎ ﺑﻨﺎﺀ ﺍﻟﻘﻮﺍﺕ ﺍﳌﺴﻠﺤﺔ ) ﺍﻟﱪﻳﺔ ، ﺍﳉﻮﻳـﺔ ﺍﻟﺒﺤﺮﻳﺔ ، ﺍﻟﺪﻓﺎﻉ ﺍﳉﻮﻱ ، ﻃﲑﺍﻥ ﺍﳉﻴﺶ ( ﻋﻠﻰ ﺃﺳﺲ ﺍﳌﻮﺍﻃﻨﺔ ﻭﺍﻻﻧﺘﻤﺎﺀ ﺍﻟﻮﻃﲏ ، ، ﻭﺗﺄﻫﻴﻠﻬﺎ ﻣﻦ ﻧﺎﺣﻴﺔ : ) ﺍﻟﺘﺪﺭﻳﺐ ﺍﻟﻌﺴﻜﺮﻱ ، ﺍﻟﺘﺮﺑﻴﺔ ﺍﻟﻌﺴـﻜﺮﻳﺔ ، ﺍﻟﻀـﺒﻂ ﺍﳉﻴـﺪ ﻭﺍﻟﻘﻴﺎﺩﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ)( ( ، ﻭﺭﻓﻊ ﻛﻔﺎﺋﺘﻬﺎ ﻭﺩﻋ ﻢ ﻭﺍﺳﻨﺎﺩ ﻋﻤﻠﻬﺎ ﺍﻹﺳﺘﺨﺒﺎﺭﺍﰐ ﻭﻣﺴـﺘﻮﺍﻫﺎ ﺪﻳﺪ ﻳﺴﺘﻬﺪﻑ ﻛﻴـﺎﻥ ﺍﻟﺪﻭﻟـﺔ ﻭﺍﺳـﺘﻘﺮﺍﺭﻫﺎ ﺍﻟﺘﻜﻨﻮﻟﻮﺟﻲ ﻣﻦ ﺃﺟﻞ ﺻﺪ ﻭﻣﻮﺍﺟﻬﺔ ﺃﻱ . ﺍﻟﺴﻴﺎﺳﻲ ﺿﺮﻭﺭﺓ ﺍﻟﺘﻐﻠﺐ ﻋﻠﻰ ﺍﻟﺘﺤﺴﺲ ﻣﻦ ﺍﳉﻴﺶ "ﺍﻟﺴﺎﺑﻖ" ، ﻣﻦ ﺧﻼﻝ ﺍﻟﺘﺮﻛﻴﺰ ﻭﺍﻻﺳـﺘﻔﺎﺩﺓ . ﻣﻦ ﺍﶈﺎﺳﻦ ﺍﻟﺘﺎﺭﳜﻴﺔ ﻟﻠﺠﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ * ﺇﻋﺎﺩﺓ ﺍﻟﻌﻤﻞ ﺑﺎﻟﺘﺼﻨﻴﻊ ﺍﻟﻌﺴﻜﺮﻱ. ﻟﺴﺪ ﺍﻟﻨﻘﺺ ﺍﳊﺎﺻﻞ ﺑﺎﳌﻌﺪﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ، * ﺍﻟﻌﻮﺩﺓ ﻟﻨﻈﺎﻡ ﺍﻟﺘﺠﻨﻴﺪ ﺍﻻﻟﺰﺍﻣﻲ)*( ﻟﺮﻓﺪ ﺍﻟﺪﻭﻟﺔ ﺑﺎﻟﻘﺪﺭﺍﺕ ﺍﻟﻼﺯﻣﺔ ﻟﻠﺪﻓﺎﻉ ﻋﻦ ﺍﻟﺒﻠﺪ ﻭﻣﺎ ﻟﻪ ﻣﻦ ﺃﺛﺮ "ﺍﻟﺘﺠﻨﻴﺪ ﺍﻻﻟﺰﺍﻣﻲ" ﰲ ﺗﺮﺳﻴﺦ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻮﻃﻨﻴﺔ ﺑﺎﻻﺑﺘﻌﺎﺩ ﻋـﻦ ﺍﶈﺎﺻﺼـﺔ ﻭﺍﻟﻄﺎﺋﻔﻴﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﻮﻓﲑ ﺍﻟﻌﻨﺼﺮ ﺍﻟﺒﺸﺮﻱ ﺍﳌﺘﻨﻮﻉ ﻣﻦ ﻧﺎﺣﻴﺔ ﺍﻟﺘﻌﻠﻴﻢ ﻣﻦ ﻫﻢ ﰲ ﻣﺴﺘﻮﻯ ﺟﻴﺪ ﻣﻦ ﺍﻻﺧﺘﺼﺎﺻﺎﺕ ﺍﻟﻌﻠﻤﻴﺔ ﻛﻤﺎ ﻛﺎﻥ ﻣﻌﻤﻮﻻﹰ ﺑﻪ ﻗﺒﻞ ﺍﻻﺣﺘﻼﻝ ﺍﻷﻣﺮﻳﻜﻲ ﻟﻠﻌﺮﺍﻕ ﻋﺎﻡ ٣٠٠٢). ﻡ ١٥( ) : ﻋﺴﻜﺮﻳﺎ / ﻭﻣﻨﻬﺎ ٠٥( ﺇﻥ ﺑﻨﺎﺀ ﺍﻻﻗﺘﺼﺎﺩ ﻻ ﻳﻨﺴﺤﺐ ﻋﻠﻰ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ﻓﺤﺴﺐ ، ﻭﺇﳕﺎ ﻳـﺪﺧﻞ ﰲ ﺑﻨﺎﺀ ﻣﺆﺳﺴﺎﺕ ﺍﻟﺪﻭﻟﺔ ﺑ ، ﺸﻜﻞ ﻋﺎﻡ ﻭﻣﻦ ﺿﻤﻨﻬﺎ ﺑﻨﺎﺀ ﺍﻟﻘﻮﺍﺕ ﺍﳌﺴﻠﺤﺔ ) ﺍﻟﱪﻳﺔ ، ﺍﳉﻮﻳـﺔ ﺍﻟﺒﺤﺮﻳﺔ ، ﺍﻟﺪﻓﺎﻉ ﺍﳉﻮﻱ ، ﻃﲑﺍﻥ ﺍﳉﻴﺶ ( ﻋﻠﻰ ﺃﺳﺲ ﺍﳌﻮﺍﻃﻨﺔ ﻭﺍﻻﻧﺘﻤﺎﺀ ﺍﻟﻮﻃﲏ ، ، ﻭﺗﺄﻫﻴﻠﻬﺎ ﻣﻦ ﻧﺎﺣﻴﺔ : ) ﺍﻟﺘﺪﺭﻳﺐ ﺍﻟﻌﺴﻜﺮﻱ ، ﺍﻟﺘﺮﺑﻴﺔ ﺍﻟﻌﺴـﻜﺮﻳﺔ ، ﺍﻟﻀـﺒﻂ ﺍﳉﻴـﺪ ﻭﺍﻟﻘﻴﺎﺩﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ)( ( ، ﻭﺭﻓﻊ ﻛﻔﺎﺋﺘﻬﺎ ﻭﺩﻋ ﻢ ﻭﺍﺳﻨﺎﺩ ﻋﻤﻠﻬﺎ ﺍﻹﺳﺘﺨﺒﺎﺭﺍﰐ ﻭﻣﺴـﺘﻮﺍﻫﺎ ﺪﻳﺪ ﻳﺴﺘﻬﺪﻑ ﻛﻴـﺎﻥ ﺍﻟﺪﻭﻟـﺔ ﻭﺍﺳـﺘﻘﺮﺍﺭﻫﺎ ﺍﻟﺘﻜﻨﻮﻟﻮﺟﻲ ﻣﻦ ﺃﺟﻞ ﺻﺪ ﻭﻣﻮﺍﺟﻬﺔ ﺃﻱ ﺍﻟﺎﺳ ﺇﻋﺎﺩﺓ ﺍﻟﻌﻤﻞ ﺑﺎﻟﺘﺼﻨﻴﻊ ﺍﻟﻌﺴﻜﺮﻱ. ﻟﺴﺪ ﺍﻟﻨﻘﺺ ﺍﳊﺎﺻﻞ ﺑﺎﳌﻌﺪﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ، * ﺍﻟﻌﻮﺩﺓ ﻟﻨﻈﺎﻡ ﺍﻟﺘﺠﻨﻴﺪ ﺍﻻﻟﺰﺍﻣﻲ)*( ﻟﺮﻓﺪ ﺍﻟﺪﻭﻟﺔ ﺑﺎﻟﻘﺪﺭﺍﺕ ﺍﻟﻼﺯﻣﺔ ﻟﻠﺪﻓﺎﻉ ﻋﻦ ﺍﻟﺒﻠﺪ ﻭﻣﺎ ﻟﻪ ﻣﻦ ﺃﺛﺮ "ﺍﻟﺘﺠﻨﻴﺪ ﺍﻻﻟﺰﺍﻣﻲ" ﰲ ﺗﺮﺳﻴﺦ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻮﻃﻨﻴﺔ ﺑﺎﻻﺑﺘﻌﺎﺩ ﻋـﻦ ﺍﶈﺎﺻﺼـﺔ ﻭﺍﻟﻄﺎﺋﻔﻴﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﻮﻓﲑ ﺍﻟﻌﻨﺼﺮ ﺍﻟﺒﺸﺮﻱ ﺍﳌﺘﻨﻮﻉ ﻣﻦ ﻧﺎﺣﻴﺔ ﺍﻟﺘﻌﻠﻴﻢ ﻣﻦ ﻫﻢ ﰲ ﻣﺴﺘﻮﻯ ﺟﻴﺪ ﻣﻦ ﺍﻻﺧﺘﺼﺎﺻﺎﺕ ﺍﻟﻌﻠﻤﻴﺔ ﻛﻤﺎ ﻛﺎﻥ ﻣﻌﻤﻮﻻﹰ ﺑﻪ ﻗﺒﻞ ﺍﻻﺣﺘﻼﻝ ﺍﻷﻣﺮﻳﻜﻲ ﻟﻠﻌﺮﺍﻕ ﻋﺎﻡ ٣٠٠٢). ﻡ ١٥( ٧٩٣              ﺍﻟﻨﻮﻋﻴﺔ ﰲ ﺍﺧﺘﻴﺎﺭ ﺍﻻﺷﺨﺎﺹ ﻭﺍﳌﻌﺪﺍﺕ ، ﻭﻋﺪﻡ ﳏ ﺎﻭﻟﺔ ﺯﻳﺎﺩﺓ ﺍﻟﻜﻢ ﻋﻠﻰ ﺣﺴﺎﺏ ﺍﻟﻨﻮﻉ. * ﺍﻟﻌﻘﻼﻧﻴﺔ ﰲ ﺗﻘﺪﻳﺮ ﺍﳌﻮﺍﻗﻒ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ ﻣﻦ ﺃﺟﻞ ﺇﳚﺎﺩ ﻋﻼﻗﺎﺕ ﺧﺎﺭﺟﻴﺔ ﻣﺘﻮﺍﺯﻧﺔ. ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ * ﺍﺑﻌﺎﺩ ﺍﳌﺆﺳﺴﺔ ﺍﻟﻌﺴﻜﺮﻳﺔ ﻋﻦ ﺍﻟﺘﺄﺛﲑﺍﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﳊﺰﺑﻴﺔ ﺍﻟﺪﺍﺧﻠﻴﺔ ﻭﺍﳋﺎﺭﺟﻴﺔ ، ﲟﻌﲎ ﺎﻝ ﺍﻟﺴﻴﺎﺳﻲ ، ﻭﺿﺮﻭﺭﺓ ﺍﺳﺘﺒﻌﺎﺩ ﻛﻞ ﻣﻦ ﻳﺜﺒﺖ ﻟـﻪ ﺻـﻠﺔﺍﻟﺘﺤﺮﱘ ﺍﻟﻘﺎﻃﻊ ﻟﻠﻌﻤﻞ ﰲ ﺍ . ﺑﺎﻷﺣﺰﺍﺏ ﻭﺍﻟﻜﺘﻞ ﺍﻟﺴﻴﺎﺳﻴﺔ )* ﺣﻞ ﺍﳌﻴﻠﻴﺸﻴﺎﺕ ٢٥ ﺎ ﺍﻟﺴﻴﺎﺳﻴﺔ( ﻣﻬﻤﺎ ﻛﺎﻧﺖ ﺍﺭﺗﺒﺎﻃﺎ ﺎ ﺣﺠـﺮﺎ ﺍﳌﺨﺘﻠﻔﺔ ﻛﻮﻭﺗﺴﻤﻴﺎ . ﻋﺜﺮﺓ ﰲ ﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ﻭﺍﺳﺘﻘﺮﺍﺭ ﺍﻟﺒﻼﺩ ﰲ : ﺃﻣﻨﻴﺎﹰ / ﻋﻦ ﻃﺮﻳﻖ * ﲢﺪﻳﺪ ﺍﻷﻫﺪﺍﻑ ﻭﺍﳌﺼﺎﱀ ﺍﻷﻣﻨﻴﺔ ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴـﺔ ﺑﺸـﻜﻞ ﻭﺍﺿـﺢ ﰲ ﺇﻃـﺎﺭ . ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﻃﻨﻴﺔ ﻋﻠﻴﺎ * ﺇﻳﻼﺀ ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻷﻣﲏ ﺃﳘﻴﺔ ﻗﺼﻮﻯ ، ﻟﻠﻮﻗﻮﻑ ﻋﻠﻰ ﻧﻘﺎﻁ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻀﻌﻒ . ﻭﲢﺪﻳﺪ ﺍﻟﻔﺮﺹ ﺍﳌﺘﺎﺣﺔ ﻟﺘﻮﻇﻴﻔﻬﺎ ﲞﺪﻣﺔ ﺍﳌﺼﺎﱀ ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ * ﺍﻟﺘﺮﻛﻴﺰ ﻋﻠﻰ ﺍﻟﻌﻤﻞ ﻭﺍﳉﻬﺪ ﺍﻹﺳﺘﺨﺒﺎﺭﺍﰐ ﻭﺍﻷﻣﲏ ﻭﺭﻓﻊ ﻛﻔﺎﺀﺓ ﺍﺩﺍﺅﻩ ﳌﻌﺮﻓﺔ ﺍﻟﺘﻬﺪﻳﺪﺍﺕ . ﺍﻷﻣﻨﻴﺔ ﻭﺍﶈﺘﻤﻠﺔ ﻭﻛﻴﻔﻴﺔ ﻣﻮﺍﺟﻬﺘﻬﺎ ﰲ ﺍﳌﺴﺘﻘﺒﻞ * ﺇﻋﺎﺩﺓ ﺻﻴﺎﻏﺔ ﺍﻟﻌﻘﻴﺪﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻟﺘﻨﺴـﺠﻢ ﻭﺍﳌـ ﺘﻐﲑ ﺍﺕ ﺍﶈﻠﻴـﺔ ﻭﺍﻹﻗﻠﻴﻤﻴـﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ . : ﺃﻣﻨﻴﺎ / ﻋﻦ ﻃﺮﻳﻖ * ﲢﺪﻳﺪ ﺍﻷﻫﺪﺍﻑ ﻭﺍﳌﺼﺎﱀ ﺍﻷﻣﻨﻴﺔ ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴـﺔ ﺑﺸـﻜﻞ ﻭﺍﺿـﺢ ﰲ ﺇﻃـﺎﺭ . ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﻃﻨﻴﺔ ﻋﻠﻴﺎ . ﺇﺮﻴﺠﻴ ﻭﻴ ﻴ * ﺇﻳﻼﺀ ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻷﻣﲏ ﺃﳘﻴﺔ ﻗﺼﻮﻯ ، ﻟﻠﻮﻗﻮﻑ ﻋﻠﻰ ﻧﻘﺎﻁ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻀﻌﻒ . ﻭﲢﺪﻳﺪ ﺍﻟﻔﺮﺹ ﺍﳌﺘﺎﺣﺔ ﻟﺘﻮﻇﻴﻔﻬﺎ ﲞﺪﻣﺔ ﺍﳌﺼﺎﱀ ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ * ﺍﻟﺘﺮﻛﻴﺰ ﻋﻠﻰ ﺍﻟﻌﻤﻞ ﻭﺍﳉﻬﺪ ﺍﻹﺳﺘﺨﺒﺎﺭﺍﰐ ﻭﺍﻷﻣﲏ ﻭﺭﻓﻊ ﻛﻔﺎﺀﺓ ﺍﺩﺍﺅﻩ ﳌﻌﺮﻓﺔ ﺍﻟﺘﻬﺪﻳﺪﺍﺕ . ﺍﻷﻣﻨﻴﺔ ﻭﺍﶈﺘﻤﻠﺔ ﻭﻛﻴﻔﻴﺔ ﻣﻮﺍﺟﻬﺘﻬﺎ ﰲ ﺍﳌﺴﺘﻘﺒﻞ * ﺇﻋﺎﺩﺓ ﺻﻴﺎﻏﺔ ﺍﻟﻌﻘﻴﺪﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻟﺘﻨﺴـﺠﻢ ﻭﺍﳌـ ﺘﻐﲑ ﺍﺕ ﺍﶈﻠﻴـﺔ ﻭﺍﻹﻗﻠﻴﻤﻴـﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ . * ﺇﻳﻼﺀ ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻷﻣﲏ ﺃﳘﻴﺔ ﻗﺼﻮﻯ ، ﻟﻠﻮﻗﻮﻑ ﻋﻠﻰ ﻧﻘﺎﻁ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻀﻌﻒ . ﻭﲢﺪﻳﺪ ﺍﻟﻔﺮﺹ ﺍﳌﺘﺎﺣﺔ ﻟﺘﻮﻇﻴﻔﻬﺎ ﲞﺪﻣﺔ ﺍﳌﺼﺎﱀ ﺍﻟﻌﻠﻴﺎ ﻟﻠﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ * ﺍﻟﺘﺮﻛﻴﺰ ﻋﻠﻰ ﺍﻟﻌﻤﻞ ﻭﺍﳉﻬﺪ ﺍﻹﺳﺘﺨﺒﺎﺭﺍﰐ ﻭﺍﻷﻣﲏ ﻭﺭﻓﻊ ﻛﻔﺎﺀﺓ ﺍﺩﺍﺅﻩ ﳌﻌﺮﻓﺔ ﺍﻟﺘﻬﺪﻳﺪﺍﺕ . ﺍﻷﻣﻨﻴﺔ ﻭﺍﶈﺘﻤﻠﺔ ﻭﻛﻴﻔﻴﺔ ﻣﻮﺍﺟﻬﺘﻬﺎ ﰲ ﺍﳌﺴﺘﻘﺒﻞ * ﺇﻋﺎﺩﺓ ﺻﻴﺎﻏﺔ ﺍﻟﻌﻘﻴﺪﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﻟﺘﻨﺴـﺠﻢ ﻭﺍﳌـ ﺘﻐﲑ ﺍﺕ ﺍﶈﻠﻴـﺔ ﻭﺍﻹﻗﻠﻴﻤﻴـﺔ ﻭﺍﻟﺪﻭﻟﻴﺔ . * ﺍﻟﻌﻤﻞ ﻋﻠﻰ ﺇﺷﺮﺍﻙ ﺍﻟﻘﻴﺎﺩﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ﻭﺍﻷﻣﻨﻴﺔ ﰲ ﺩﻭﺭﺍﺕ ﻣﻊ ﺩﻭﻝ ﺃﺧﺮﻯ ﻣﻦ ﺃﺟـﻞ . ﺗﺒﺎﺩﻝ ﺍﳋﱪﺍﺕ ﻭﺗﻄﻮﻳﺮ ﺍﳌﻬﺎﺭﺍﺕ ﺎﺀ ﻇﺎﻫﺮﺓ ﺍﻧﺘﺸﺎﺭ ﺍﻟﺴﻼﺡ ﺧﺎﺭﺝ ﻧﻄﺎﻕ ﺍﻃﺎﺭ ﺍﻟﺪﻭﻟـﺔ ، ﻟـﻴﻌﻢ* ﺍﻟﻌﻤﻞ ﻋﻠﻰ ﺍ ﺍﻟﻨﻈـﺎﻡ ﻭﺍﻷﻣﻦ. /ﺍﻗﺘﺼﺎﺩﻳﺎﹰ : ﺑﺎﻟﻌﻤﻞ ﻋﻠﻰ * ﺇﻋﺎﺩ. ﺓ ﺗﺄﻫﻴﻞ ﺍﻟﺒﻨﻴﺔ ﺍﻟﺘﺤﺘﻴﺔ ﺍﻟﻜﻠﻴﺔ ﰲ ﳐﺘﻠﻒ ﺍﻟﻘﻄﺎﻋﺎﺕ ﻣﻦ ﺃﺟﻞ ﺗﻮﻓﲑ ﺍﳋﺪﻣﺎﺕ . * ﺧﻠﻖ ﻓﺮﺹ ﺍﻟﻌﻤﻞ ﻭﻣﻌﺎﳉﺔ ﺍﻟﺒﻄﺎﻟﺔ . * ﺗﻮﻓﲑ ﻣﺴﺘﻠﺰﻣﺎﺕ ﺍﳊﻴﺎﺓ ﻛﺎﻓﺔ ﻭﺍﺷﺒﺎﻉ ﺍﳊﺎﺟﺎﺕ ﻭﲡﻨﺐ ﺍﳌﻐﺎﻻﺓ ﰲ ﺍﻷﺳﻌﺎﺭ . * ﺻﻴﺎﻏﺔ ﻭﺇﻗﺮﺍﺭ ﺍﻷﻃﺮ ﺍﻟﻘﺎﻧﻮﻧﻴﺔ ﺍﳌﻨﻈﻤﺔ ﻟﻘﻄﺎﻉ ﺍﻟﻨﻔﻂ /ﺍﻗﺘﺼﺎﺩﻳﺎﹰ : ﺑﺎﻟﻌﻤﻞ ﻋﻠﻰ * ﺇﻋﺎﺩ. ﺓ ﺗﺄﻫﻴﻞ ﺍﻟﺒﻨﻴﺔ ﺍﻟﺘﺤﺘﻴﺔ ﺍﻟﻜﻠﻴﺔ ﰲ ﳐﺘﻠﻒ ﺍﻟﻘﻄﺎﻋﺎﺕ ﻣﻦ ﺃﺟﻞ ﺗﻮﻓﲑ ﺍﳋﺪﻣﺎﺕ . * ﺧﻠﻖ ﻓﺮﺹ ﺍﻟﻌﻤﻞ ﻭﻣﻌﺎﳉﺔ ﺍﻟﺒﻄﺎﻟﺔ ٨٩٣       * ﺑﻨﺎﺀ ﻣﺼﺎﻑ ﺟﺪﻳﺪﺓ ﻟﺮﻓﻊ ﺍﻟﻘ. ﺪﺭﺓ ﺍﻻﻧﺘﺎﺟﻴﺔ ﺎﺀ ﺍﳋﻼﻑ ﺍﻟﺴﻴﺎﺳﻲ ﺍﳌﺘﻜﺮﺭ ﺑﲔ ﺍﳊﻜﻮﻣﺔ ﺍﻻﲢﺎﺩﻳﺔ ﻭﺣﻜﻮﻣﺔ ﺇﻗﻠﻴﻢ ﻛﻮﺭﺩﺳﺘﺎﻥ ﺍﳌﺘﻌﻠﻖ* ﺇ . ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﺎ ﻣﻠﻚ ﻟﻠﺸﻌﺐ" ، ﻭﺣﺼﺔ ﺍﻹﻗﻠﻴﻢ ﻣﻦ ﺍﳌﻮﺍﺯﻧﺔ ﺍﻻﲢﺎﺩﻳﺔﺑﺎﳊﻘﻮﻝ ﺍﻟﻨﻔﻄﻴﺔ "ﻟﻜﻮ ﻤﺎ ﺭﻛﻨﺎﻥ ﺃﺳﺎﺳـﻴﺎﻥ ﰲ ﺗﻨﺸـﻴﻂ* ﲢﺴﲔ ﻭﺗﻄﻮﻳﺮ ﻗﻄﺎﻋﻲ ) ﺍﻟﺰﺭﺍﻋﺔ ، ﻭﺍﻟﺼﻨﺎﻋﺔ ( ﻛﻮ ﺍﻻﻗﺘﺼﺎﺩ. ﺍﻟﻮﻃﲏ . * ﺩﻋﻢ ﻭﺗﻨﺸﻴﻂ ﺍﻟﻘﻄﺎﻉ ﺍﳋﺎﺹ ﻟﻼﺳﻬﺎﻡ ﰲ ﺍﻟﺘﻨﻤﻴﺔ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ . * ﺗﻔﻌﻴﻞ ﻧﻈﺎﻡ ﻋﺎﺩﻝ ﻟﻠﻀﺮﺍﺋﺐ ﻟﺘﺨﻄﻲ ﺍﻟﺼﻔﺔ ﺍﻟﺮﻳﻌﻴﺔ ﺑﺎﻻﻋﺘﻤﺎﺩ ﻋﻠﻰ ﺍﻟﻨﻔﻂ ﺇﺩﺍﺭﻳﺎﹰ / ﲢﺴﲔ ﻗﺪﺭﺍﺕ ) ﻭﺯﺍﺭﺍﺕ ، ﻭﻣﺆﺳﺴﺎﺕ ، ﻭﺩﻭﺍﺋﺮ ﺍﻟﺪﻭﻟﺔ ( ﻛﺎﻓﺔ ﻭﺭﻓﻊ ﻛﻔﺎﺋﻴﺘﻬﺎ ﻟﺘﻤﻜﻴﻨﻬﺎ ﻣﻦ ﺍﳒﺎﺯ ﻣﻬﺎﻣﻬﺎ ﻭﲢﻘﻴﻖ ﺃﻫﺪﺍﻓﻬﺎ ﺑﺎﻟ ﺸﻜﻞ ﺍﻷﻣﺜﻞ . ﻓﺎﻟﻌﻤﻞ ﺍﳌﺆﺳﺴﻲ ﺍﳌﺘﻜﺎﻣـﻞ . ﻭﺍﳌﺘﻨﺎﺳﻖ ﻋﺎﻣﻞ ﺣﻴﻮﻱ ، ﻭﻫﻮ ﻣﻔﺘﺎﺡ ﺍﻻﺳﺘﻘﺮﺍﺭ ﻭﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ﲟﺎ ﻳﻀﻤﻦ ﻛﻔﺎﺀﺓ ﺍﻻﺩﺍﺀ / ﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﺘﻤﻊ ﺍﻟﻌﺮﺍﻗﻲ ﺑﺎﻃﻴﺎﻓﻪ ﻛﺎﻓﺔ ﻣﻦ ﻋﺎﺩﺍﺕ ﻭﺗﻘﺎﻟﻴﺪ ﻭﺗـﻮﻓﲑ* ﺑﺎﶈﺎﻓﻈﺔ ﻋﻠﻰ ﻛﻞ ﻣﺎ ﻳﺘﻌﻠﻖ ﺑﺎ . ﺍﳉﻮﺍﻧﺐ ﺍﻟﺘﺮﺑﻮﻳﺔ ﻭﺍﻟﺘﻌﻠﻴﻤﻴﺔ ﻭﺍﻟﺼﺤﻴﺔ * ﺿﺮﻭﺭﺓ ﺃﻥ ﻳﻨﻤﻲ ﺷﻌﻮﺭ ﺍﻟﻔﺮﺩ ﺑﺎﻻﻧﺘﻤﺎﺀ ﻟﻠﻤﺠﺘﻤﻊ ﻭﺍﻟﻮﻃﻦ ، ﳌﺎ ﻟﻪ ﻣﻦ ﺁﺛﺎﺭ ﺇﳚﺎﺑﻴـﺔ ﰲ . ﲢﻘﻴﻖ ﺃﻫﺪﺍﻑ ﺍﻟﺪﻭﻟﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ * ﺍﻟﻌﻤﻞ ﲜﺪﻳﺔ ﻋﻠﻰ ﺿﺮﻭﺭﺓ ﺇﻋﺎﺩﺓ ﺍﻟﻨﺎﺯﺣﲔ ﻭﺍﳌﻬﺠﺮﻳﻦ ﺇﱃ ﻣﻨﺎﻃﻖ ﺳﻜﻨﺎﻫﻢ ، ﻭﺗﻌﻮﻳﻀﻬﻢ . ﻢ ﻣﻦ ﺃﻭﺿﺎﻉ ﻣﺄﺳﺎﻭﻳﺔ ﻋﻠﻰ ﺍﻟﺼﻌﻴﺪ ﺍﻻﻗﺘﺼﺎﺩﻱ ﻭﺍﻻﺟﺘﻤﺎﻋﻲﳌﺎ ﺍﺻﺎ ﺇﻋﻼ ﻣﻴﺎﹰ / ﺿﺮﻭﺭﺓ ﻭﺟﻮﺩ ﺇﻋﻼﻡ ﺟﻴﺪ ﻭﺭﺻﲔ ﻭﻫﺎﺩﻑ ﻗﺎﺩﺭ ﻋﻠﻰ ﻧﺸـﺮ ﺍﻟـﻮﻋﻲ ﺍﻟﺜﻘـﺎﰲ . ﻭﺍﻻﺧﻼﻗﻲ : ﺑﻴﺌﻴﺎﹰ / ﻣﻦ ﺧﻼﻝ . * ﺍﻟﻌﻤﻞ ﻋﻠﻰ ﺗﻮﻓﲑ ﺑﻴﺌﺔ ﺁﻣﻨﺔ ﺑﻌﻴﺪﺓ ﻋﻦ ﺍﳌﺨﺎﻃﺮ ﻭﺍﺿﺮﺍﺭ ﺍﻟﻜﻮﺍﺭﺙ ﺍﻟﻄﺒﻴﻌﻴﺔ * ﺗﻄﻮﻳﺮ ﺑﺮﺍﻣﺞ ﳌﺮﺍﻗﺒﺔ ﺍﳌﻴﺎﻩ ﻭﺍﳊﺪ ﻣﻦ ﺗﻠﻮﺛﻬﺎ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﻘﻠﻴﻞ ﻧﺴﺒﺔ ﺍﻟﺸـﺤﺔ ﰲ ﻣﻴـﺎﻩ ﺍﻟﺸﺮ. ﺏ ﻭﲢﺴﲔ ﻧﻮﻋﻴﺘﻪ ﺧﺼﻮﺻﺎﹰ ﰲ ﺍﶈﺎﻓﻈﺎﺕ ﺍﳉﻨﻮﺑﻴﺔ * ﺑﻨﺎﺀ ﻣﺼﺎﻑ ﺟﺪﻳﺪﺓ ﻟﺮﻓﻊ ﺍﻟﻘ. ﺪﺭﺓ ﺍﻻﻧﺘﺎﺟﻴﺔ ﺎﺀ ﺍﳋﻼﻑ ﺍﻟﺴﻴﺎﺳﻲ ﺍﳌﺘﻜﺮﺭ ﺑﲔ ﺍﳊﻜﻮﻣﺔ ﺍﻻﲢﺎﺩﻳﺔ ﻭﺣﻜﻮﻣﺔ ﺇﻗﻠﻴﻢ ﻛﻮﺭﺩﺳﺘﺎﻥ ﺍﳌﺘﻌﻠﻖ* ﺇ . ﺎ ﻣﻠﻚ ﻟﻠﺸﻌﺐ" ، ﻭﺣﺼﺔ ﺍﻹﻗﻠﻴﻢ ﻣﻦ ﺍﳌﻮﺍﺯﻧﺔ ﺍﻻﲢﺎﺩﻳﺔﺑﺎﳊﻘﻮﻝ ﺍﻟﻨﻔﻄﻴﺔ "ﻟﻜﻮ ﻤﺎ ﺭﻛﻨﺎﻥ ﺃﺳﺎﺳـﻴﺎﻥ ﰲ ﺗﻨﺸـﻴﻂ* ﲢﺴﲔ ﻭﺗﻄﻮﻳﺮ ﻗﻄﺎﻋﻲ ) ﺍﻟﺰﺭﺍﻋﺔ ، ﻭﺍﻟﺼﻨﺎﻋﺔ ( ﻛﻮ ﺍﻻﻗﺘﺼﺎﺩ. ﺍﻟﻮﻃﲏ . * ﺩﻋﻢ ﻭﺗﻨﺸﻴﻂ ﺍﻟﻘﻄﺎﻉ ﺍﳋﺎﺹ ﻟﻼﺳﻬﺎﻡ ﰲ ﺍﻟﺘﻨﻤﻴﺔ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ . * ﺗﻔﻌﻴﻞ ﻧﻈﺎﻡ ﻋﺎﺩﻝ ﻟﻠﻀﺮﺍﺋﺐ ﻟﺘﺨﻄﻲ ﺍﻟﺼﻔﺔ ﺍﻟﺮﻳﻌﻴﺔ ﺑﺎﻻﻋﺘﻤﺎﺩ ﻋﻠﻰ ﺍﻟﻨﻔﻂ ﺇﺩﺍﺭﻳﺎﹰ / ﲢﺴﲔ ﻗﺪﺭﺍﺕ ) ﻭﺯﺍﺭﺍﺕ ، ﻭﻣﺆﺳﺴﺎﺕ ، ﻭﺩﻭﺍﺋﺮ ﺍﻟﺪﻭﻟﺔ ( ﻛﺎﻓﺔ ﻭﺭﻓﻊ ﻛﻔﺎﺋﻴﺘﻬﺎ ﻟﺘﻤﻜﻴﻨﻬﺎ ﻣﻦ ﺍﳒﺎﺯ ﻣﻬﺎﻣﻬﺎ ﻭﲢﻘﻴﻖ ﺃﻫﺪﺍﻓﻬﺎ ﺑﺎﻟ ﺸﻜﻞ ﺍﻷﻣﺜﻞ . ﻓﺎﻟﻌﻤﻞ ﺍﳌﺆﺳﺴﻲ ﺍﳌﺘﻜﺎﻣـﻞ . ﻭﺍﳌﺘﻨﺎﺳﻖ ﻋﺎﻣﻞ ﺣﻴﻮﻱ ، ﻭﻫﻮ ﻣﻔﺘﺎﺡ ﺍﻻﺳﺘﻘﺮﺍﺭ ﻭﺇﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ﲟﺎ ﻳﻀﻤﻦ ﻛﻔﺎﺀﺓ ﺍﻻﺩﺍﺀ ﻝ ﺑﻴﻴ /ﻦ . * ﺍﻟﻌﻤﻞ ﻋﻠﻰ ﺗﻮﻓﲑ ﺑﻴﺌﺔ ﺁﻣﻨﺔ ﺑﻌﻴﺪﺓ ﻋﻦ ﺍﳌﺨﺎﻃﺮ ﻭﺍﺿﺮﺍﺭ ﺍﻟﻜﻮﺍﺭﺙ ﺍﻟﻄﺒﻴﻌﻴﺔ * ﺗﻄﻮﻳﺮ ﺑﺮﺍﻣﺞ ﳌﺮﺍﻗﺒﺔ ﺍﳌﻴﺎﻩ ﻭﺍﳊﺪ ﻣﻦ ﺗﻠﻮﺛﻬﺎ ، ﻓﻀﻼﹰ ﻋﻦ ﺗﻘﻠﻴﻞ ﻧﺴﺒﺔ ﺍﻟﺸـﺤﺔ ﰲ ﻣﻴـﺎﻩ ﺍﻟﺸﺮ. ﺍﳌﻄﻠـﺐ ﺍﻟﺜـﺎﻧـﻲ / ﺃﻧـﻮﺍﻉ ﺍﻟﻘـﺪ. ﺭﺓ ) : ﻟﻠﻘﺪﺭﺓ ﺃﻧﻮﺍﻉ ﻋﺪﺓ ﺗﺘﻤﺜﻞ ﺑﺎﻵﰐ ٤١(  ) ، ( ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ : ﻫﻲ ﳎﻤﻮﻋﺔ ﻣﻦ ﺍﻻﻣﻜﺎﻧﺎﺕ ) ﺍﳌﺎﺩﻳﺔ ، ﻭﻏﲑ ﺍﳌﺎﺩﻳـﺔ ﺎ ﺍﻟﺘﻘﻠﻴﺪﻳﺔﺍﳌﺘﺎﺣﺔ ، ﻭﻏﲑ ﺍﳌﺘﺎﺣﺔ ( ﻟﻠﺪﻭﻟﺔ ، ﻭﺗﺘﻤﺜﻞ ﺍﻻﻣﻜﺎﻧﺎﺕ ﺍﳌﺎﺩﻳﺔ ﰲ ﺻﻮﺭ ﺑﺎﳌﻮﺍﺭﺩ ﺍﳌﺘﺎﺣﺔ ، ﺍﻣﺎ ﻏﲑ ﺍﳌﺎﺩﻳﺔ ﻓﺘﺸﻤﻞ ﻧﻈﻢ ﺍﳌﻌﻠﻮﻣﺎﺕ ﺍ ﻻﺩﺍﺭﻳﺔ ﻭﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺑﻮﺻﻔﻬﺎ ﻋﻨﺎﺻﺮ ﻣﺘﻔﺎﻋﻠﺔ ﻣﻊ ﺍﻟﻌﻨﺎﺻﺮ ﺍﳌﺎﺩﻳﺔ ﰲ ﺍﻃﺎﺭ ﺍﻟﻘﻮﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺳﺎﺣﺎﺕ ﺗﺄﺛﲑﻫﺎ ﺍﳋﺎﺭﺟﻲ ، ﺧﺎﺻﺔ ﻭﺍﻥ ﻫﻴﻜﻞ ﺍﻟﻘﻮﺓ ﰲ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﱂ ﻳﻌﺪ ﻫـﻴﻜﻼﹰ ﻋﺴﻜﺮﻳﺎﹰ ﺑﺎﻟﺪﺭﺟﺔ ﺍﻻﺳﺎﺱ ﻛﻤﺎ ﻛﺎﻥ ﰲ ﺍﻟﺴﺎﺑﻖ ﺑﻞ ﻋﺪ ﻫﻴﻜﻼﹰ ﺛﻼﺛﻴﺎﹰ ﻣﻜﻮﻧﺎﹰ ﻣﻦ ﺍﻻﻗﺘﺼﺎﺩ ﻭﺍﻟﺘﻜﻨﻮﻟﻮﺟﻴ ﺎ ﻭﺍﻟﻘﻮﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ﻭﺗﻌﺪ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﺭﻛﻴـﺰﺓ . ﺍﺳﺎﺱ ﻣﻦ ﺭﻛﺎﺋﺰ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺇﻥ ﺍﻟﻘﻴﺎﺱ ﺍﻟﺸﺎﻣﻞ ﺍﻟﻌﺎﻡ ﻟﻠﻘﺪﺭﺓ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻷﻱ ﺩﻭﻟﺔ ﻳﻜﻮﻥ ﺿﻤﻦ ﺁﻃﺎﺭ ﻣـﺎ ﻳﻄﻠـﻖ ﻋﻠﻴــﻪ ) ﺇﲨــﺎﱄ ﺍﻟﻨــﺎﺗﺞ ﺍﻟﻘــﻮﻣﻲGENERAL NATIONAL PRODUCTION ( ﻣﻦ ﺧﻼﻝ ﺗﻮﻓﲑﻩ ﺍﻟﺪﻋﻢ ﻭﺍﻻﺳﻨﺎﺩ ﻛﻮﻥ ﺍﻟﻘﺪﺭﺓ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ . ﺗﺘﺴﻢ ﺑﺴﻬﻮﻟﺔ ﺍﻻﻧﺪﻣﺎﺝ ﰲ ﺍﳕﺎﻁ ﻗﺪﺭﺍﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻷﺧﺮﻯ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ : ﻭﺍﻟﱵ ﲤﺜﻞ ﺍﳌﻈﻠﺔ ﺍﻟﱵ ﳝﻜﻦ ﺃﻥ ﺗﻮﻓﺮﻫﺎ ﺍﻟﺪﻭﻟﺔ ﳊﻤﺎﻳﺔ ﻗـﻮﻯ ﺩﻓﺎﻋﻬﺎ ﺍﻟﻮﻃﲏ ﺳﻴﺎﺳﻴﺎﹰ ﻭﺍﻗﺘﺼﺎﺩﻳﺎﹰ ﻭﺍﺟﺘﻤﺎﻋﻴﺎﹰ ﻭﺃﻣﻨﻴﺎﹰ ﻭﻣﻌﻨﻮﻳﺎﹰ ﻭﺑﺎﻟﺘﺎﱄ ﲢﻘﻴﻖ ﺃﻣﻨﻬﺎ ﺎ ﺍﳌﺴﻠﺤﺔ ﺍﻟﱪﻳـﺔ ﻭﺍﳉﻮﻳـﺔ ﻭﺍﻟﺒﺤﺮﻳـﺔﺍﻟﻘﻮﻣﻲ ، ﻭﺗﺸﻤﻞ ﺍﳌﻮﺍﺭﺩ ﻭﻛﺎﻓﺔ ﻗﻮﺍ ﺍﳌﻮﺟﻮﺩﺓ ﻭﺍﻟﱵ ﻫﻲ ﺟﺎﻫﺰﺓ ﻟﻼﺳﺘﺨﺪﺍﻡ ، ﻭﺗﻘﺴﻢ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺇﱃ ﻗﺴـﻤﲔ . ﺗﻘﻠﻴﺪﻳﺔ ﻭﻏﲑ ﺗﻘﻠﻴﺪﻳﺔ ، ﻓﻀﻼﹰ ﻋﻦ ﻣﻌﻨﻮﻳﺎﺕ ﻭﺩﻭﺍﻓﻊ ﺍﳌﻮﺍﻃﻨﲔ  ﺍﻟﻘﺪﺭﺓ ﺍﻟﺪﺑﻠﻮﻣﺎﺳﻴﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ( : ﺗﻌﺪ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻷﻱ ﺩﻭﻟـﺔ ﻫﻲ ﺍﶈﻮﺭ ﺍﻟﺮﺋﻴﺲ ﻟﻘﻴﺎﻡ ﺍﻟﺘﻌﺎﻭﻥ ﺑﻴﻨﻬﺎ ﻭﺑﲔ ﺍﻵﺧﺮﻳﻦ ﰲ ﻛﻞ ﻣﻦ ﺍﻟﺒﻴﺌﺔ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺪﻑ ﺍﻟﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﺇﱃ ﲪﺎﻳﺔ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ ﻟﻠﺪﻭﻟـﺔ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ . ﺇﺫ ﻭﺑﻘﺎﺋﻬﺎ ﻭﺗﻌﺰﻳﺰ ﺭﻓﺎﻫﻴﺔ ﺷﻌﺒﻬﺎ . ﻭﳝﻜﻦ ﺭﺳـﻢ ﺍﻟﺴﻴﺎﺳـﺔ ﺍﳋﺎﺭﺟﻴـﺔ ﺑﺘﺤﺪﻳـﺪ ﺏ ﻭﲢﺴﲔ ﻧﻮﻋﻴﺘﻪ ﺧﺼﻮﺻﺎﹰ ﰲ ﺍﶈﺎﻓﻈﺎﺕ ﺍﳉﻨﻮﺑﻴﺔ ٩٩٣       * ﺗﻨﻔﻴﺬ ﺑﺮﺍﻣﺞ ﻣﻜﺎﻓﺤﺔ ﺍﻟﺘﺼﺤﺮ ﻭﺍﳊﺪ ﻣﻦ ﺗﺪﻫﻮﺭ ﺍﻷﺭﺍﺿﻲ ﰲ ﺍﳌﻨﺎﻃﻖ ﺍﳉﺎﻓﺔ ﻭﺷﺒﻪ ﺍﳉﺎﻓﺔ ﰲ ﺍﻟﻌﺮﺍﻕ ، ﻭﺍﳊﻔﺎﻅ ﻋﻠﻰ ﺍﻟﻐﻄﺎﺀ ﺍﻟﻨﺒﺎﰐ ، ﻣﻦ ﺧﻼﻝ ﺗﺄﻣﲔ ﺍﻻﺣﺘﻴﺎﺟﺎﺕ ﺍﳌﺎﺋﻴﺔ ﺍﻟﱵ ﺗﺆﺩﻱ . ﺇﱃ ﺯﻳﺎﺩﺓ ﻣﺴﺎﺣﺔ ﺍﻷﺭﺍﺿﻲ ﺍﻟﺰﺭﺍﻋﻴﺔ * ﺿﻤﺎﻥ ﺣﺼ. ﺔ ﺍﻟﻌﺮﺍﻕ ﻣﻦ ﺍﳌﻴﺎﻩ ﺍﳌﺸﺘﺮﻛﺔ ﻣﻊ ﺩﻭﻝ ﺍﳉﻮﺍﺭ ٦- ) : ﻋﻠﻰ ) ﺍﻟﺼﻌﻴﺪ ﺍﻹﻗﻠﻴﻤﻲ ﻭﺍﻟﺪﻭﱄ ( ، ﻭﻣﻦ ﺫﻟﻚ ٣٥( * ﺭﻓﺾ ﺍﻟﺘﺪﺧﻞ ﺍﻹﻗﻠﻴﻤﻲ ﰲ ﺍﻟﺸﺄﻥ ﺍﻟﻌﺮﺍﻗﻲ "ﺍﻟﺪﺍﺧﻠﻲ" ، ﻭﻋـﺪﻡ ﺍﻟﺴـﻤﺎﺡ ﻟﻠﻘﻴـﺎﺩﺍﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ﰲ ﺍﻟﻌﺮﺍﻕ ﺍﻟﺘﻌﺎﻭﻥ ﻣﻊ ﻫﺬﻩ ﺍﻟﻘﻮﻯ ﺍﻹﻗﻠﻴﻤﻴـﺔ ، ﻭﺍﻟـﱵ ﺗـﺮﺗﺒﻂ "ﺍﻟﻘﻴـﺎﺩﺍﺕ "ﺍﻟﺴﻴﺎﺳﻴﺔ. ﻣﺼﺎﳊﻬﺎ ﺍﻟﺬﺍﺗﻴﺔ ﺍﻟﻀﻴﻘﺔ ﲟﺼﺎﱀ ﺍﻟﺪﻭﻝ ﺍﻹﻗﻠﻴﻤﻴﺔ ﺍﻟﺪﺍﻋﻤﺔ ﳍﺎ * ﲣﻔﻴﻒ ﺍﻟﺪﻭﺭ ﺍﻷﻣﺮﻳﻜﻲ ﺍﳌﺆﺛﺮ ﻣﻦ ﺧﻼﻝ ﺍﻻﺳﺘﻔﺎﺩﺓ ﻣﻦ ﺃﻳﺔ ﻓﺮﺻـﺔ ﺳـﺎﳓﺔ ﻟﺘﺤﺴـﲔ ﺍﻟﻌﻼﻗﺎﺕ ﺑﺪﻭﻝ ﺍﳉﻮﺍﺭ ﺍﻹﻗﻠﻴﻤﻲ ﻭﺑﻘﻴﺔ ﺩﻭﻝ ﺍﻟﻌﺎﱂ ، ﻷﻥ ﺃﻱ ﺗﻮﺗﺮ ﰲ ﻋﻼﻗﺎﺕ ﺍﻟﻌﺮﺍﻕ ﻣﻊ ﺍﻵﺧﺮﻳﻦ ﻳﺪﱘ ﺍﳊﺎﺟﺔ ﺇﱃ ﺍﻟﺘﺪﺧﻞ ﺍﻷﻣﺮﻳﻜﻲ ﰲ . ﺍﻟﺸﺄﻥ ﺍﻟﻌﺮﺍﻗﻲ ﺩﺍﺧﻠﻴﺎﹰ ﻭﺧﺎﺭﺟﻴﺎﹰ * ﺍﻟﺴﻌﻲ ﻟﻠﻮﺻﻮﻝ ﺇﱃ ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍﺳﺎﻟﻴﺐ ﻋﻤﻞ ﺟﺪﻳﺪﺓ ﺗﻌﺘﻤﺪ ﻋﻠﻰ ﻗﺮﺍﺀﺓ ﺟﺮﻳﺌﺔ ﻟﻠﻮﺍﻗﻊ ﻭﺍﻟﺒﻴﺌﺔ ﺍﻟﺪﻭﻟﻴﺔ ، ﻣﻦ ﺃﺟﻞ ﺗﺼﻮﻳﺐ ﺃﻱ ﺍﺧﻄﺎﺀ ﺭﺍﻓﻘﺖ ﺍﻟﻌﻤﻞ ﺍﻟﺴﻴﺎﺳـﻲ ﺍﻟﻌﺮﺍﻗـﻲ ﳓـﻮ . ﺍﳋﺎﺭﺝ ، ﻭﺗﻨﻄﻠﻖ ﻣﻦ ﺗﺄﻛﻴﺪ ﻭﺣﺪﺓ ﻣﺮﻛﺰ ﺍﻟﻘﺮﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ * ﺍﻟﺴﻌﻲ ﻟﻜﺴﺐ ﺍﻻﺻ ﺪﻗﺎﺀ ﰲ ﺍﻟﻮﺳﻂ ﺍﻹﻗﻠﻴﻤﻲ ﻭﺍﻟﺪﻭﱄ ، ﻷﻥ ﺍﻟﻌﺮﺍﻕ ﺃﻣـﺎﻡ ﻣﻬﻤـﺎﺕ ﺛﻘﻴﻠﺔ ﻻ ﻳﺴﺘﻄﻴﻊ ﺍﳒﺎﺯﻫﺎ ﲟﻔﺮﺩﻩ ، ﻭﻗﺎﺋﻤﺔ ﺍﳌﻬﺎﻡ ﺍﳌﻄﻠﻮﺑﺔ ﻻ ﺗﻨﺘﻬﻲ ﻋﻨﺪ ﺣﺪ ﻣﺎ ﺑﲔ ﺍﺑﻌـﺎﺩ ، ﺍﻟﺘﺪﺧﻼﺕ ﺍﳋﺎﺭﺟﻴﺔ ﺍﻟﱵ ﺗﻨﺘﻬﻚ ﺍﳉﺴﺪ ﺍﻟﻌﺮﺍﻗﻲ ﻭﺗﻀﻌﻔﻪ ﻭﺗﻀﻌﻒ ﻣﻮﻗﻔﻪ ﺃﻣـﺎﻡ ﺍﻟﻌـﺎﱂ ﻭﺍﻻﺗﻔﺎﻗﻴﺎﺕ ﺍﻟﺜﻨﺎﺋﻴﺔ ﻭﺍﳌﺴﺎﳘﺔ ﰲ ﺍﻟﺘﻨﻈﻴ ﻤﺎﺕ ﺍﻹﻗﻠﻴﻤﻴﺔ ﻭﺍﻟﺪﻭﻟﻴـﺔ ، ﻭﻣﺸـﺎﻛﻞ ﺍﳊـﺪﻭﺩ . ﻭﻣﺸﺎﻛﻞ ﺍﳌﻴﺎﻩ * ﺗﻨﻔﻴﺬ ﺑﺮﺍﻣﺞ ﻣﻜﺎﻓﺤﺔ ﺍﻟﺘﺼﺤﺮ ﻭﺍﳊﺪ ﻣﻦ ﺗﺪﻫﻮﺭ ﺍﻷﺭﺍﺿﻲ ﰲ ﺍﳌﻨﺎﻃﻖ ﺍﳉﺎﻓﺔ ﻭﺷﺒﻪ ﺍﳉﺎﻓﺔ ﰲ ﺍﻟﻌﺮﺍﻕ ، ﻭﺍﳊﻔﺎﻅ ﻋﻠﻰ ﺍﻟﻐﻄﺎﺀ ﺍﻟﻨﺒﺎﰐ ، ﻣﻦ ﺧﻼﻝ ﺗﺄﻣﲔ ﺍﻻﺣﺘﻴﺎﺟﺎﺕ ﺍﳌﺎﺋﻴﺔ ﺍﻟﱵ ﺗﺆﺩﻱ . ﺇﱃ ﺯﻳﺎﺩﺓ ﻣﺴﺎﺣﺔ ﺍﻷﺭﺍﺿﻲ ﺍﻟﺰﺭﺍﻋﻴﺔ "* ﺗﻄﻮﻳﺮ ﺷﺒﻜﺔ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺜﻘﺎﻓﻴﺔ ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻷﻣﻨﻴـﺔ ﻭﺍﻟﻔﻨﻴـﺔ ﻭﺣـﱴ "ﺍﻟﺮﻳﺎﺿـﻴﺔ ﻭﺗﻮﻇﻴﻔﻬﺎ ﻟﺘﺤﻘﻴﻖ ﺗﻌﺎﻭﻥ ﻭﺛﻴﻖ ﻻ ﳝﻜﻦ ﻟﻠﺨﻼﻓﺎﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ﺃﻥ ﺗﻘﺼﻢ ﻋﺮﺍﻫـﺎ . ﻭﻫـﺬﺍ ﻳﺘﻄﻠﺐ ﺍﻋﻼﻣﺎﹰ ﻧﺎﺿﺠﺎﹰ ﻭﺷﺒﻜﺔ ﺍﺗﺼﺎﻻﺕ ﻣﺘﻄﻮﺭﺓ ﻭﺍﺳﺘﻌﺪﺍﺩﺍﹰ . ﺫﻫﻨﻴﺎﹰ ﺭﻓﻴﻌﺎﹰ ﻟﻠﺤﻮﺍﺭ * ﺗﻮﻇﻴﻒ ﺍﻻﻣﻜﺎﻧﻴﺎﺕ ﻭﺍﻟﻘﺪﺭﺍﺕ ﺍﳌﺘﺎﺣﺔ ﻭﺑﻮﺟﻮﺩ ﺇﺭﺍﺩﺓ ﻭﻃﻨﻴﺔ ﻟﺘﻮﻇﻴﻒ ﻋﻨﺎﺻـﺮ ﺍﻟﻘـﻮﺓ ﻭﺗﺪﻋﻴﻤﻬﺎ ﻭﺧﻠﻖ ﻓﺮﺹ ﻟﻠﺘﻌﺎﻭﻥ ﻣﻊ ﺍﳉﻤﻴﻊ ، ﺣﻴﺚ ﳝﻜﻦ ﳌﺎ ﳝﺘﻠﻜﻪ ﺍﻟﻌﺮﺍﻕ ﻣﻦ ﺍﻣﻜﺎﻧﻴﺎﺕ ﻭﻗﺪﺭﺍﺕ ﺍﻗﺘﺼﺎﺩﻳﺔ ﻭﺛﺮﻭﺍﺕ ﺿﺨﻤﺔ ﺃﻥ ﻳﻜﻮﻥ ﺍﺩﻭﺍﺕ ﺟﺬﺏ ﲣﺪﻡ ﺍﻟﺪﺑﻠﻮﻣﺎﺳـﻴﺔ ﻋـﱪ ٠٠٤        ﺍﺗﻔﺎﻗﻴﺎﺕ ﺍﻻﺳ ﺘﺜﻤﺎﺭ ﻭﺍﻟﺘﺒﺎﺩﻝ ﺍﻟﺘﺠﺎﺭﻱ ﻭﻏﲑﻫﺎ ﻣﻦ ﺍﻻﻧﺸـﻄﺔ ﺍﻻﻗﺘﺼـﺎﺩﻳﺔ ﺍﻟﻮﺍﻋـﺪﺓ ﰲ . ﺍﻟﻌﺮﺍﻕ       Abstract: The issue of rebuilding the strategic capacity of Iraq after the occupation and the US invasion in 2003 has been one of the most contentious issues of debate and debate. It raises a number of political, social, economic, military and security problems. We say political primarily because the climate and political conditions play a large and active role along with other factors complementary to them. The important question that arises here is: How can the ruling political blocs move to the stage of reconstruction without reservations? In order to answer this question, we divided the study into three main axes. The first deals with the concept of power and its types. The second axis deals with the relationship between strategic capacity and its dimensions. The third axis deals with the internal and external constraints facing Iraq's strategic capacity building after 2003. The study concludes with conclusions that include recommendations to enhance Iraqi strategic capacity. ﺍﳌﺼ: ﺎﺩﺭ )١ ( ﺇﺑﺮﺍﻫﻴﻢ ﻣﺼﻄﻔﻰ ﻭﺁﺧﺮﻭﻥ، ﳎﻤﻊ ﺍﻟﻠﻐﺔ ﺍﻟﻌﺮﺑﻴﺔ ، ﺍﻹﺩﺍﺭﺓ ﺍﻟﻌﺎﻣﺔ ﻟﻠﻤﻌﺠﻤﺎﺕ ﻭﺇﺣﻴﺎﺀ ﺍﻟﺘـﺮﺍﺙ ، ﺍﳌﻜﺘﺒـﺔ ﺍﻹﺳﻼﻣﻴﺔ ﻟﻄﺒﺎﻋﺔ ﻭﺍﻟﻨﺸﺮ ﻭﺍﻟﺘﻮﺯﻳﻊ ، ﺍﻟﻘﺎﻫﺮﺓ ، ﺹ ٨١٧. )٢ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﳌﺎﺋﺪﺓ ، ﺍﻵﻳﺔ ٧١ . ( )٣ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﳌﻠﻚ ، ﺍﻵﻳﺔ ١ . ( )٤ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﻓﺎﻃﺮ ، ﺍﻵﻳﺔ ٤٤ . ( )٥ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﻟﺮﻭﻡ ، ﺍﻵﻳﺔ ٤٥ . ( )٦ ( ﺍﻟﻌﻤﻴﺪ ﺍﻟﺮﻛﻦ ﺯﺍﻳﺪ ﺑﻦ ﳏﻤﺪ ﺣﺴﻦ ﺍﻟﻌﻤﺮﻱ ، ﻣﻔﻬﻮﻡ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻘﺪﺭﺓ ﰲ ﺍﻟﻔﻜﺮ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ، ﳎﻠﺔ ﺍﻟﺪﻓﺎﻉ ﺍﳉ ) ﻮﻱ ﺍﳌﻠﻜﻲ ﺍﻟﺴﻌﻮﺩﻱ ، ﺍﻟﻌﺪﺩ ٣ ( ، ﺍﳌﻤﻠﻜﺔ ﺍﻟﻌﺮﺑﻴﺔ ﺍﻟﺴﻌﻮﺩﻳﺔ ، ﺳﺒﺘﻤﱪ ٠١٠٢ ﻡ ، ﺹ ٨١ ٩١ ـ . )٧ : ﺎ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ( ﺍﻟﻘﻮﻯ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺣﺴﺎﺑﺎ http://www.moqatel.com/openshare/Behoth/Askria6/KowaShamla/sec03.doc_cvt.htm )٨ ( ﻧﻘﻼﹰ ﻋﻦ : ﺍﻟﻠﻮﺍﺀ ﺍﻟﻄﻴﺎﺭ ﺍﻟﺮﻛﻦ ﻋﻠﻮﺍﻥ ﺣﺴﻮﻥ ﻋﻠﻮﺍﻥ ﺍﻟﻌﺒﻮﺳﻲ ، ﺍﻟﻘﺪﺭﺍﺕ ﻭﺍﻷﺩﻭﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺴﻼﺡ ﺍﳉﻮ ﺍﻟﻌﺮﺍﻗﻲ ﰲ ﺍﻟﻔﺘﺮﺓ ١٣٩١ ٣٠٠٢ ﻡ ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﺍﻻﻛﺎﺩ ، ﳝﻴﻮﻥ ﻟﻠﻨﺸﺮ ﻭﺍﻟﺘﻮﺯﻳﻊ ، ﺍﻷﺭﺩﻥ ٤١٠٢ ، ٢٣ﺹ . )٩ ، ( ﺩ. ﳏﻤﺪ ﺣﺴﲔ ﺃﺑﻮ ﺻﺎﱀ ، ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻟﻘﻮﻣﻲ ، ﺍﻟﻄﺒﻌﺔ ﺍﻟﺜﺎﻧﻴﺔ ، ﳎﻤﻮﻋﺔ ﺍﻟﻨﻴـﻞ ﺍﻟﻌﺮﺑﻴـﺔ ، ﺍﻟﻘﺎﻫﺮﺓ ٩٠٠٢ ، ﺹ ١٠٤ ٣٠٤ . )٠١ (ﻧﻘﻼﹰﻋﻦ :ﺩﻗﺤﻄﺎﻥﺃﲪﺪﺳﻠﻴﻤﺎﻥ ،ﺍﻷﺳﺎﺱﰲﺍﻟﻌﻠﻮﻡﺍﻟﺴﻴﺎﺳﻴﺔ ،ﺍﻟﻄﺒﻌﺔﺍﻷﻭﱃ ،ﺩﺍﺭﳎﺪﻻﻭﻱﻟﻠﻨﺸﺮ ﺍﳌﺼ: ﺎﺩﺭ )١ ( ﺇﺑﺮﺍﻫﻴﻢ ﻣﺼﻄﻔﻰ ﻭﺁﺧﺮﻭﻥ، ﳎﻤﻊ ﺍﻟﻠﻐﺔ ﺍﻟﻌﺮﺑﻴﺔ ، ﺍﻹﺩﺍﺭﺓ ﺍﻟﻌﺎﻣﺔ ﻟﻠﻤﻌﺠﻤﺎﺕ ﻭﺇﺣﻴﺎﺀ ﺍﻟﺘـﺮﺍﺙ ، ﺍﳌﻜﺘﺒـﺔ ﺍﻹﺳﻼﻣﻴﺔ ﻟﻄﺒﺎﻋﺔ ﻭﺍﻟﻨﺸﺮ ﻭﺍﻟﺘﻮﺯﻳﻊ ، ﺍﻟﻘﺎﻫﺮﺓ ، ﺹ ٨١٧.     )١١ ، ( ﻧﻌﻴﻢ ﺇﺑﺮﺍﻫﻴﻢ ﺍﻟﻈﺎﻫﺮ ، ﺳﻴﺎﺳﺔ ﺑﻨﺎﺀ ﺍﻟﻘﻮﺓ ﰲ ﺍﻷﺭﺩﻥ ، ﺩﺍﺭ ﳎﺪﻻﻭﻱ ﻟﻠﻨﺸﺮ ، ﻋﻤﺎﻥ ٣٠٠٢ ، ﺹ ٧٢ . )٢١ ، ( ﺳﻌﺪ ﺣﻘﻲ ﺗﻮﻓﻴﻖ ، ﻣﺒﺎﺩﻱﺀ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﺩﺍﺭ ﻭﺍﺋﻞ ﻟﻠﻄﺒﺎﻋﺔ ﻭﺍﻟﻨﺸﺮ ، ﻋﻤـﺎﻥ ٠٠٠٢ ، ﺹ ٣٩١ . )٣١ ، ( ﻓﺎﻳﻖ ﺣﺴﻦ ﺍﻟﺸﺠﲑﻱ ، ﳘﺎﻡ ﻃﻪ ، ﻛﻴﻒ ﲢﺪﺩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻗﻮﺓ ﺍﻟﺪﻭﻟﺔ ﺍﳋﺎﺭﺟﻴـﺔ ) ﺻﺤﻴﻔﺔ ) ﺍﻟﻌﺮﺏ ( ، ﺍﻟﺴﻨﺔ ٨٣ ) ( ، ﺍﻟﻌﺪﺩ ١٥١٠١ ( ، ﺹ ٦ . )٣١ ، ( ﻓﺎﻳﻖ ﺣﺴﻦ ﺍﻟﺸﺠﲑﻱ ، ﳘﺎﻡ ﻃﻪ ، ﻛﻴﻒ ﲢﺪﺩ ﺍﻟﻘﺪﺭﺓ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻗﻮﺓ ﺍﻟﺪﻭﻟﺔ ﺍﳋﺎﺭﺟﻴـﺔ ) ﺻﺤﻴﻔﺔ ) ﺍﻟﻌﺮﺏ ( ، ﺍﻟﺴﻨﺔ ٨٣ ) ( ، ﺍﻟﻌﺪﺩ ١٥١٠١ ( ، ﺹ ٦ . )٤١ ( ﺍﻟﻠﻮﺍﺀ ﺍﻟﻄﻴﺎﺭ ﺍﻟﺮﻛﻦ ﻋﻠﻮﺍﻥ ﺣﺴﻮﻥ ﻋﻠﻮﺍﻥ ﺍﻟﻌﺒﻮﺳﻲ ، ﺍﻟﻘﺪﺭﺍﺕ ﻭﺍﻷﺩﻭﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺴﻼﺡ ﺍﳉـﻮ ) ﺻﺤﻴﻔﺔ ) ﺍﻟﻌﺮﺏ ( ، ﺍﻟﺴﻨﺔ ٨٣ ) ( ، ﺍﻟﻌﺪﺩ ١٥١٠١ ( ، ﺹ ٦ . )٤١ ( ﺍﻟﻠﻮﺍﺀ ﺍﻟﻄﻴﺎﺭ ﺍﻟﺮﻛﻦ ﻋﻠﻮﺍﻥ ﺣﺴﻮﻥ ﻋﻠﻮﺍﻥ ﺍﻟﻌﺒﻮﺳﻲ ، ﺍﻟﻘﺪﺭﺍﺕ ﻭﺍﻷﺩﻭﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺴﻼﺡ ﺍﳉـﻮ ، ﺍﻟﻌﺮﺍﻗﻲ ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ ، ﺹ ٢٣ ٣٣ . ـ ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﺳﻌﺪ ﺣﻘﻲ ﺗﻮﻓﻴﻖ ، ﻣﺒﺎﺩﻱﺀ ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ، ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ ، ﺹ ٣٨٢ . )٦١ (ﺃﻧﺪﺭﻳﻪ ﺑﻮﻓﺮ ، ﻣﺪﺧﻞ ﺇﱃ ﺍﻻﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺴﻜﺮﻳﺔ ، ، ﺗﺮﲨﺔ : ﺍﻛﺮﻡ ﺩﻳﺮﻱ ﻭﺍﳍﻴﺜﻢ ﺍﻻﻳﻮﰊ ، ﺩﺍﺭ ﺍﻟﻄﻠﻴﻌﺔ ، ﺑﲑﻭﺕ ٠٧٩١ ، ﺹ ٨١ . )٨١ ( ﻧﻘﻼﹰ ﻋﻦ : ﻫﺎﺭﻱ ﺁﺭ. ﻳﺎﻏﺮ ، ﺍﻻﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﳏﺘﺮﻓﻮ ﺍﻷﻣﻦ ﺍﻟ ﻘﻮﻣﻲ : ﺍﻟﺘﻔﻜﲑ ﺍﻻﺳـﺘﺮﺍﺗﻴﺠﻲ ﻭﺻـﻴﺎﻏﺔ ﺍﻻﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﰲ ﺍﻟﻘﺮﻥ ﺍﳊﺎﺩﻱ ﻭﺍﻟﻌﺸﺮﻳﻦ ، ﺗﺮﲨﺔ : ﺭﺍﺟﺢ ﳏﺮﺯ ﻋﻠـﻲ ، ﺍﻟﻄﺒﻌـﺔ ﺍﻷﻭﱃ ، ﻣﺮﻛـﺰ ﺍﻹﻣـﺎﺭﺍﺕ ، ﻟﻠﺪﺭﺍﺳﺎﺕ ﻭﺍﻟﺒﺤﻮﺙ ﺍﻻﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﺩﻭﻟﺔ ﺍﻹﻣﺎﺭﺍﺕ ﺍﻟﻌﺮﺑﻴﺔ ﺍﳌﺘﺤﺪﺓ ١١٠٢ ، ﺹ ٠٦٢ . )٩١ ( ﻣﻌﻤﺮ ﻣﻨﻌﻢ ﺻﺎﺣﻲ ، ﺍﻟﺪﻭﻟﺔ ﻭﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺇﺩﺍﺭﺓ ﺍ ﻟﺘﻐﻴﲑ ، ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ ، ﺹ ٦٧ ٧٧ ـ . )٠٢ ( ﻣﻬﻨﺪ ﻋﻠﻲ ﻋﻤﺮﺍﻥ ، ﺃﺛﺮ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻘﺪﺭﺓ ﻭﺣﺮﻳﺔ ﺍﻟﻌﻤﻞ ﰲ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﺩﺭﺍﺳـﺔ ﺣﺎﻟـﺔ ﲨﻬﻮﺭﻳﺔ ﺃﳌﺎﻧﻴﺎ ﺍﻻﲢﺎﺩ ﻳﺔ، ﺭﺳﺎﻟﺔ ﻣﺎﺟﺴﺘﲑ "ﻏﲑ ﻣﻨﺸﻮﺭﺓ"، ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ، ﺟﺎﻣﻌـﺔ ﺍﻟﻨـﻬﺮﻳﻦ ،٣٠٠٢ ، ﺹ ٠٣١. )١٢ ( ﺃ.ﺩ. ﺻﻼﺡ ﺍﻟ ﺪﻳﻦ ﻋﻮﺍﺩ ﺍﻟﻜﺒﻴﺴﻲ ، ﺍﻟﺒﺎﺣﺚ : ﺗﻐﺮﻳﺪ ﺧﻠﻴﻞ ﺍﺑﺮﺍﻫﻴﻢ ، ﺍﻟﻘﺪﺭﺍﺕ ﺍﳌﻌﺮﻓﻴﺔ ﻭﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍﺛﺮﳘﺎ ﰲ ﺑﻨﺎﺀ ﺍﳌﻴﺰﺓ ﺍﻟﺘﻨﺎﻓﺴﻴﺔ : ﲝﺚ ﻣﻴﺪﺍﱐ ﻻﺭﺍﺀ ﻋﻴﻨﺔ ﻣﻦ ﺍﻟﻘﻴﺎﺩﺍﺕ ﺍﻻﺩﺍﺭﻳﺔ ﰲ ﺍﳌﺼﺎﺭﻑ ﺍﻷﻫﻠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ، ﳎﻠﺔ ) ﻠﺪﺍﻟﻌﻠﻮﻡ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻭﺍﻻﺩﺍﺭﻳﺔ ، ﺍ ٠٢ ) ( ، ﺍﻟﻌﺪﺩ ٩٧ ( ، ﻛﻠ ، ﻴﺔ ﺍﻻﺩﺍﺭﺓ ﻭﺍﻻﻗﺘﺼﺎﺩ ، ﺟﺎﻣﻌـﺔ ﺑﻐـﺪﺍﺩ ٤١٠٢ ﻡ ، ﺹ ٢٤ ٣٤ ـ . Abstract: )٢ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﳌﺎﺋﺪﺓ ، ﺍﻵﻳﺔ ٧١ . ( )٣ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﳌﻠﻚ ، ﺍﻵﻳﺔ ١ . ( )٤ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﻓﺎﻃﺮ ، ﺍﻵﻳﺔ ٤٤ . ( )٥ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﻟﺮﻭﻡ ، ﺍﻵﻳﺔ ٤٥ . ( )٢ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﳌﺎﺋﺪﺓ ، ﺍﻵﻳﺔ ٧١ . ( )٤ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﻓﺎﻃﺮ ، ﺍﻵﻳﺔ ٤٤ . ( )٥ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﻟﺮﻭﻡ ، ﺍﻵﻳﺔ ٤٥ . ( )٦ ( ﺍﻟﻌﻤﻴﺪ ﺍﻟﺮﻛﻦ ﺯﺍﻳﺪ ﺑﻦ ﳏﻤﺪ ﺣﺴﻦ ﺍﻟﻌﻤﺮﻱ ، ﻣﻔﻬﻮﻡ ﺍﻟﻘﻮﺓ ﻭﺍﻟﻘﺪﺭﺓ ﰲ ﺍﻟﻔﻜﺮ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ، ﳎﻠﺔ ﺍﻟﺪﻓﺎﻉ ﺍﳉ ) ﻮﻱ ﺍﳌﻠﻜﻲ ﺍﻟﺴﻌﻮﺩﻱ ، ﺍﻟﻌﺪﺩ ٣ ( ، ﺍﳌﻤﻠﻜﺔ ﺍﻟﻌﺮﺑﻴﺔ ﺍﻟﺴﻌﻮﺩﻳﺔ ، ﺳﺒﺘﻤﱪ ٠١٠٢ ﻡ ، ﺹ ٨١ ٩١ ـ . )٧ : ﺎ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ( ﺍﻟﻘﻮﻯ ﺍﻟﺸﺎﻣﻠﺔ ﻟﻠﺪﻭﻟﺔ ﻭﺣﺴﺎﺑﺎ ﻊ http://www.moqatel.com/openshare/Behoth/Askria6/KowaShamla/sec03.doc_cvt.htm ﹰ )٨ ( ﻧﻘﻼﹰ ﻋﻦ : ﺍﻟﻠﻮﺍﺀ ﺍﻟﻄﻴﺎﺭ ﺍﻟﺮﻛﻦ ﻋﻠﻮﺍﻥ ﺣﺴﻮﻥ ﻋﻠﻮﺍﻥ ﺍﻟﻌﺒﻮﺳﻲ ، ﺍﻟﻘﺪﺭﺍﺕ ﻭﺍﻷﺩﻭﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺴﻼﺡ ﺍﳉﻮ ﺍﻟﻌﺮﺍﻗﻲ ﰲ ﺍﻟﻔﺘﺮﺓ ١٣٩١ ٣٠٠٢ ﻡ ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﺍﻻﻛﺎﺩ ، ﳝﻴﻮﻥ ﻟﻠﻨﺸﺮ ﻭﺍﻟﺘﻮﺯﻳﻊ ، ﺍﻷﺭﺩﻥ ٤١٠٢ ، ٢٣ﺹ )٨ ( ﻧﻘﻼﹰ ﻋﻦ : ﺍﻟﻠﻮﺍﺀ ﺍﻟﻄﻴﺎﺭ ﺍﻟﺮﻛﻦ ﻋﻠﻮﺍﻥ ﺣﺴﻮﻥ ﻋﻠﻮﺍﻥ ﺍﻟﻌﺒﻮﺳﻲ ، ﺍﻟﻘﺪﺭﺍﺕ ﻭﺍﻷﺩﻭﺍﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻟﺴﻼﺡ ﺍﳉﻮ ﺍﻟﻌﺮﺍﻗﻲ ﰲ ﺍﻟﻔﺘﺮﺓ ١٣٩١ ٣٠٠٢ ﻡ ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﺍﻻﻛﺎﺩ ، ﳝﻴﻮﻥ ﻟﻠﻨﺸﺮ ﻭﺍﻟﺘﻮﺯﻳﻊ ، ﺍﻷﺭﺩﻥ ٤١٠٢ ، ٢٣ﺹ . )٩ ، ( ﺩ. ﳏﻤﺪ ﺣﺴﲔ ﺃﺑﻮ ﺻﺎﱀ ، ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻟﻘﻮﻣﻲ ، ﺍﻟﻄﺒﻌﺔ ﺍﻟﺜﺎﻧﻴﺔ ، ﳎﻤﻮﻋﺔ ﺍﻟﻨﻴـﻞ ﺍﻟﻌﺮﺑﻴـﺔ ، ﺍﻟﻘﺎﻫﺮﺓ ٩٠٠٢ ، ﺹ ١٠٤ ٣٠٤ . )٩ ، ( ﺩ. ﳏﻤﺪ ﺣﺴﲔ ﺃﺑﻮ ﺻﺎﱀ ، ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﺍﻟﻘﻮﻣﻲ ، ﺍﻟﻄﺒﻌﺔ ﺍﻟﺜﺎﻧﻴﺔ ، ﳎﻤﻮﻋﺔ ﺍﻟﻨﻴـﻞ ﺍﻟﻌﺮﺑﻴـﺔ ، ﺍﻟﻘﺎﻫﺮﺓ ٩٠٠٢ ، ﺹ ١٠٤ ٣٠٤ . )٠١ ( ﻧﻘﻼﹰ ﻋﻦ : ﺩ. ﻗﺤﻄﺎﻥ ﺃﲪﺪ ﺳﻠﻴﻤﺎﻥ ، ﺍﻷﺳﺎﺱ ﰲ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﺩﺍﺭ ﳎﺪﻻﻭﻱ ﻟﻠﻨﺸﺮ ، ، ﻋﻤﺎﻥ ٤٠٠٢ ، ﺹ ٩٣ . ، ، ﻋﻤﺎﻥ ٤٠٠٢ ، ﺹ ٩٣ . ١٠٤              )٢٢ : ( ﺍﳌﻌﺎﱐ ﺍﳉﺎﻣﻊ ، ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ https://www.almaany.com/ar/dict/ar- ar/%D9%85%D9%85%D8%A7%D9%86%D8%B9%D8%A9/ )٣٢ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﻟﻘﻠﻢ ، ﺍﻵﻳﺔ ٢١ . ( )٤٢ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﻕ ، ﺍﻵﻳﺔ ٥٢ . ( https://www.almaany.com/ar/dict/ar- ar/%D9%85%D9%85%D8%A7%D9%86%D8%B9%D8%A9/ )٣٢ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﺍﻟﻘﻠﻢ ، ﺍﻵﻳﺔ ٢١ . ( )٤٢ ( ) ﺍﻟﻘﺮﺁﻥ ﺍﻟﻜﺮﱘ ، ﺳﻮﺭﺓ ﻕ ، ﺍﻵﻳﺔ ٥٢ . ( )٥٢ ( Porter ،E.Michael, (1998), Competitive Strategy: Techniques for Analyzing Industries and Competitors, New Introduction Copyright, The Free Press, New York . ٢٠٤       )٦٢ ( ﺃﲪﺪ ﻋﺒﺪ ﺍﳍﺎﺩﻱ ﺣﺴﲔ ، ﺍﺳﺘﻘﺮﺍﺭ ﺍﻟﻨﻈﺎﻡ ﺍﻟﺴﻴﺎﺳﻲ ﻭﺃﺛﺮﻩ ﰲ ﺻﻴﺎﻏﺔ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﺸﺎﻣﻠﺔ : ﺍﻟﻌﺮﺍﻕ ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﺇﳕﻮﺫﺟﺎﹰ ، ﺃﻃﺮﻭﺣﺔ ﺩﻛﺘﻮﺭﺍﻩ ) ﻏﲑ ﻣﻨﺸﻮﺭﺓ ( ، ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﺟﺎﻣﻌﺔ ﺍﻟﻨﻬ ، ﺮﻳﻦ ، ﺑﻐـﺪﺍﺩ ٦١٠٢ ، ﺹ ٦١١. )٧٢ ( ﺍﳌﺼﺪﺭ ﻧﻔﺴﻪ ، ﺹ ٨١١ . )٨٢ : ( ﻓﻮﺯﻱ ﺣﺴﻦ ﺣﺴﲔ ، ﺍﻟﺘﺨﻄﻴﻂ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﻟﻠﺴﻴﺎﺳﺔ ﺍﳋﺎﺭﺟﻴﺔ ﻭﺑﺮﺍﻣﺞ ﺍﻷﻣـﻦ ﺍﻟﻘـﻮﻣﻲ ﻟﻠـﺪﻭﻝ ، ﺍﻟﻮﻻﻳﺎﺕ ﺍﳌﺘﺤﺪﺓ ﺍﻷﻣﺮﻳﻜﻴﺔ "ﺃﳕﻮﺫﺟﺎﹰ" ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﻣﻜﺘﺒﺔ ﻣﺪﺑﻮﱄ ، ﺍﻟﻘﺎﻫﺮﺓ ٣١٠٢ ﻡ ، ﺹ ٥٩ . )٩٢ ( ﻫﺎﺭﻱ ﺁﺭ. ﻳﺎ ﻏﺮ ، ﺍﻻﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﳏﺘﺮﻓﻮ ﺍﻷﻣﻦ ﺍﻟﻘﻮﻣﻲ : ﺍﻟﺘﻔﻜﲑ ﺍﻻﺳﺘﺮﺍﺗﻴﺠﻲ ﻭﺻﻴﺎﻏﺔ ﺍﻻﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﰲ ﺍﻟﻘﺮﻥ ﺍﳊﺎﺩﻱ ﻭﺍﻟﻌﺸﺮﻳﻦ ، ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ ، ﺹ ٣٠٢ . )٠٣ ( ﺃ.ﻡ.ﺩ. ﺍﺳﺮﺍﺀ ﻋﻼﺀ ﺍﻟﺪﻳﻦ ﻧﻮﺭﻱ ، ﺃ.ﻡ.ﺩ. ﻋﻠﻲ ﳏﻤﺪ ﻋﻠﻮﺍﻥ ، ﺃ.ﻡ.ﺩ. ﺧﻀﺮ ﻋﺒﺎﺱ ﻋﻄﻮﺍﻥ ، ﻣﻌﻀﻠﺔ ﺑﻨﺎﺀ ﺍﻟﺪﻭﻟﺔ ﰲ ﺍﻟﻌﺮﺍﻕ ، ﺍﳌﺮﻛﺰ ﺍﻟ ﺪﳝﻮﻗﺮﺍﻃﻲ ﺍﻟﻌﺮﰊ ﻟﻠﺪﺭﺍﺳﺎﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻﻗﺘﺼﺎﺩﻳﺔ ، ﻧﺸﺮ ﺑﺘـﺎﺭﻳﺦ ٥١/٤/٧١٠٢ : ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ http://democraticac.de/?p=45610 ) * ( ﺇﻥ ﺍﻟﺪﻭﻟﺔ ) ﻫﻲ ﻣﺸﺮﻭﻉ ﺳﻴﺎﺳﻲ ، ﺎ ﺍﻻﻧﺴﺎﻥ ﻧﻔﺴـﻪ ﻭﺍﻗﺘﺼﺎﺩﻱ ﻭﺍﺟﺘﻤﺎﻋﻲ ﺩﺍﺧﻠﻴﺎﹰ ، ﻭﻫﻲ ﻫﻮﻳﺔ ﻳﻌﺮﻑ ﻭﻫﻲ ﻣﺸﺮﻭﻉ ﺳﻴﺎﺳﻲ ﻣﻮﺟﻪ ﳓﻮ ﺍﳋﺎﺭﺝ ﺃﻳﻀﺎﹰ ، ﻳﺴﺘﻄﻴﻊ ﺍﻟﻔﺮﺩ ﺍﳌﻮﺍﻃﻦ ﺧﻼﳍﺎ ﺍﻟﻌﻴﺶ ﰲ ﻛﻨﻒ ﻣﺆﺳﺴﺎﺕ ﺗﻀﻤﻦ ﺳﻼﻣﺘﻪ ﻭﺗﻀﻤﻦ ﻏﺪﻩ ( ، ﺇﻻ ﺃﻥ ﻣﺎ ﺍﻗﺮ ﰲ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﻳﻜﺎﺩ ﻳﺘﻔﻖ ﻋﻠﻴﻪ ﺷﺮﳛﺔ ﻭﺍﺳﻌﺔ ﻣﻦ ﺍﻟﺒﺎﺣﺜﲔ ﺃﻧﻪ ﺩﺳﺘﻮ ﺭ . ﻻ ﻳﻀﻤﻦ ﺑﻨﺎﺀ ﺩﻭﻟﺔ ﺟﺪﻳﺪﺓ ﻭﻓﺎﻋﻠﺔ ﻭﺣﱴ ﺃﻥ ﻛﺎﻧﺖ ﻫﻨﺎﻙ ﻋﻤﻠﻴﺔ ﻹﻋﺎﺩﺓ ﺍﻟﺒﻨﺎﺀ ﺎﻓﺈ ﺗﻜﻮﻥ ﰲ ﺍﻟﻐﺎﻟﺐ ﻣﺴﻴﺴـﺔ ﺑﺎﳌﺴﺎﻭﻣﺎﺕ ﺍﻟﻄﺎﺋﻔﻴﺔ ﻭﺍﻟﻌﺮﻗﻴﺔ ﻭﻣﻨﻄﻠﻖ ﻟﻠﻤﺤﺎﺻﺼﺔ ﻭﻫﻲ ﻣﻦ ﺍﻟﻌﻮﺍﻣﻞ ﺍﻟﱵ ﺗﻌﺮﻗﻞ ﺇﻋﺎﺩﺓ ﺑﻨﺎﺀ ﺍﻟﻘﺪﺭﺓ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ .     )١٣ ( ﺗﻌﺘﱪ ﺍﻟﺴﻴﺎﺳﺎﺕ ﺍﳌﺘﺨﺬﺓ ﰲ ﺍﻟﺪﻭﻟﺔ ﻣﻦ ﺃﻫﻢ ﺍﻷﻣﻮﺭ ﺍﻟﱵ ﲢﻘﻖ ) ﺍﻟﻮﺣﺪﺓ ﺍﻟﻮﻃﻨﻴﺔ ( ، ﻓﺤﻴﻨﻤـﺎ ﻳﺘﻀـﻤﻦ ﺩﺳﺘﻮﺭ ﺍﻟﺪﻭﻟﺔ ﻧﺼﻮﺻﺎﹰ ﺟﺎﺩﺓ ﰲ ﺍﻟﺘﺄﻛﻴﺪ ﻋﻠﻰ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻮﻃﻨﻴﺔ ﻭﺍﻟﺘﺄﻛﻴﺪ ﻋﻠﻰ ﻣﻌﺎﻳﲑ ﺍﻟﱰﺍﻫـﺔ ﻭﺍﳌﺴـﺎﻭﺍﺓ ﺑـﲔ ﺍﳌﻮﺍﻃﻨﲔ ، ﻭﻣﺴﺄﻟﺔ ﻣﻦ ﻳﺘﻌﺮﺽ ﻟﻠﻮﺣﺪﺓ ﺍﻟﻮﻃﻨﻴﺔ ﺑﺎﻟﻘﻮﻝ ﺃﻭ ﺍﻟﻔﻌﻞ ، ﻭﺗﻨﻔﻴﺬ ﺗﻠﻚ ﺍﻟﺴﻴﺎﺳﺎﺕ ﻋﻠﻰ ﺃﺭﺽ ﺍﻟﻮﺍﻗـﻊ ﻓﺈﻥ ﺫﻟ ﻚ ﻳﻀﻤﻦ ﺑﻼ ﺷﻚ ﺃﻥ ﺗﺘﺤﻘﻖ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻮﻃﻨﻴﺔ ﰲ ﺍﻟﺒﻠﺪ . ﻟﻠﻤﺰﻳﺪ ﻋﻦ ﺫﻟﻚ ﻳﻨﻈﺮ : ﻃﻼﻝ ﻣﺸﻌﻞ ، ﲝـﺚ ﻋﻦ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻮﻃﻨﻴﺔ ، ﻧﺸﺮ ﺑﺘﺎﺭﻳﺦ ٧١/١/٦١٠٢ : ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ https://mawdoo3.com )٢٣ ( ﻡ.ﺩ. ﻓﺎﺗ ﻦ ﳏﻤﺪ ﺭﺯﺍﻕ ، ﺃ.ﻡ. ﻋﻼﺀ ﺟﺒﺎﺭ ﺃﲪﺪ ، ﺿﻌﻒ ﺍﳍﻮﻳﺔ ﺍﻟﻮﻃﻨﻴﺔ ﻭﺗﺄﺛﲑﻫﺎ ﻋﻠﻰ ﺍﻷﻣﻦ ﺍﻟﻮﻃﲏ ، ﳎﻠﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﻟﺪﻭﻟﻴﺔ ، ﺍﻟﻌﺪﺩ ٦٢ ٧٢ ، ( ، ﺍﳉﺎﻣﻌﺔ ﺍﳌﺴﺘﻨﺼﺮﻳﺔ ، ﺑﻐﺪﺍﺩ ٥١٠٢ ، ﺹ ٠٨٤ . )٢٣ ( ﻡ.ﺩ. ﻓﺎﺗ ﻦ ﳏﻤﺪ ﺭﺯﺍﻕ ، ﺃ.ﻡ. ﻋﻼﺀ ﺟﺒﺎﺭ ﺃﲪﺪ ، ﺿﻌﻒ ﺍﳍﻮﻳﺔ ﺍﻟﻮﻃﻨﻴﺔ ﻭﺗﺄﺛﲑﻫﺎ ﻋﻠﻰ ﺍﻷﻣﻦ ﺍﻟﻮﻃﲏ ، ﳎﻠﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﻟﺪﻭﻟﻴﺔ ، ﺍﻟﻌﺪﺩ ٦٢ ٧٢ ، ( ، ﺍﳉﺎﻣﻌﺔ ﺍﳌﺴﺘﻨﺼﺮﻳﺔ ، ﺑﻐﺪﺍﺩ ٥١٠٢ ، ﺹ ٠٨٤ . )٣٣ ( ﺳﺎﺭﺓ ﺍﻟﺴﻬﻴﻞ ، ﻧﺎﺯﺣﻮ ﺍﻟﻌﺮﺍﻕ ﺑﲔ ﺍﳌﻌﺎﻧﺎﺓ ﻭﺍﻟﻌﻮﺩﺓ ﻭﺍﻻﺳﺘﻐﻼﻝ ﺍﻟﺴﻴﺎﺳﻲ ، ﻗﻨﺎﺓ ) ﺭﻭﻭﺩﺍ ، ﻭ ( ﺍﻹﺧﺒﺎﺭﻳﺔ / /ﳌﻗ ﻠ ﻻ ﻟﻜﺔﻟﻟﺔﻟﻠﻠ )٣٣ ( ﺳﺎﺭﺓ ﺍﻟﺴﻬﻴﻞ ، ﻧﺎﺯﺣﻮ ﺍﻟﻌﺮﺍﻕ ﺑﲔ ﺍﳌﻌﺎﻧﺎﺓ ﻭﺍﻟﻌﻮﺩﺓ ﻭﺍﻻﺳﺘﻐﻼﻝ ﺍﻟﺴﻴﺎﺳﻲ ، ﻗﻨﺎﺓ ) ﺭﻭﻭﺩﺍ ، ﻭ ( ﺍﻹﺧﺒﺎﺭﻳﺔ ﺑﺘﺎﺭﻳﺦ ٧١/١/٨١٠٢ : ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ http://www.rudaw.net/arabic/opinion/17012018 )٤٣ ( ﺳﺎﻣﻲ ﺍﻟﺰﺑﻴﺪﻱ ، ﺃﺳﺒﺎﺏ ﺿﻌﻒ ﺍﻟﻘﻮﺍﺕ ﺍﳌﺴﻠﺤﺔ ﻭﺳﺒﻞ ﲡﺎﻭﺯﻫﺎ ، ﺻﺤﻴﻔﺔ ) ﺍﻟﺰﻣﺎﻥ ( ، ﻧﺸﺮﺕ ﺑﺘﺎﺭﻳﺦ ٣١/٦/٥١٠٢ : ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ http://www.rudaw.net/arabic/opinion/17012018 )٤٣ ( ﺳﺎﻣﻲ ﺍﻟﺰﺑﻴﺪﻱ ، ﺃﺳﺒﺎﺏ ﺿﻌﻒ ﺍﻟﻘﻮﺍﺕ ﺍﳌﺴﻠﺤﺔ ﻭﺳﺒﻞ ﲡﺎﻭﺯﻫﺎ ، ﺻﺤﻴﻔﺔ ) ﺍﻟﺰﻣﺎﻥ ( ، ﻧﺸﺮﺕ ﺑﺘﺎﺭﻳﺦ ٣١/٦/٥١٠٢ : ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ ﻊ ﻰ https://www.azzaman.com/index.php/archive/117219 ٣٠٤   - ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﻋﺼﺎﻡ ﺍﻟﻴﺎﺳﺮﻱ ، ﺍﻹﻋﻼﻡ ﻭﺩﻭﺭﻩ ﰲ ﺍﻟﺸﺄﻥ ﺍﻟﻌﺮﺍﻗﻲ ؟ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ : ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ http://www.iraqicp.com/index.php/sections/platform/31364-2015-07-31-10-32-20 - ، ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﻋﻠﻲ ﻋﺒﺪ ﺍﳍﺎﺩﻱ ﺍﳌﻌﻤﻮﺭﻱ ، ﺳﻴﺎﺳﺔ ﺍﻷﻣﻦ ﺍﻟﻮﻃﲏ ﰲ ﺍﻟﻌﺮﺍﻕ ، ﺍﻟﺪﺍﺭ ﺍﻟﻌﺮﺑﻴﺔ ﻟﻠﻌﻠﻮﻡ ﻧﺎﺷﺮﻭﻥ ، ﺑﲑﻭﺕ ٦١٠٢ ، ﺹ ٧٦٣ . )٥٣ ﻴﺎﺭ ﺍﻟﻘﺪﺭﺓ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﲤﺜﻠﺖ ﰲ ﻗﺮﺍﺭ ) ﺑﻮﻝ ﺑﺮﳝﺮ ( ﺍﳊﺎ( ﺃﺣﺪ ﺃﻫﻢ ﺍﺳﺒﺎﺏ ﺍ ﻛﻢ ﺍﳌﺪﱐ ﰲ ﺍﻟﻌﺮﺍﻕ ﺑﻌﺪ ﻋﺎﻡ ٣٠٠٢ ﻡ ﻭﺍﻟﺬﻱ ﻗﻀﻰ ﲝﻞ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ، ﳑﺎ ﻭﻟﺪ ﻓﺮﺍﻏﺎﹰ ﺃﻣﻨﻴﺎﹰ .     ـ ﻟﻠﻤﺰﻳﺪ ﻋﻦ ﺫﻟﻚ ﻳﻨﻈﺮ : ﳏﻤﻮﺩ ﺃﲪﺪﻋﺰﺕ ، ﺑﻨﺎﺀ ﺍﻟﻘﻮﺍﺕ ﺍﳌﺴﻠﺤﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ : ﺍﻗﺘﺮﺍﺣﺎﺕ ﻋﻤﻠﻴﺔ ، ﺑﻨﺎﺀ ﺩﻭﻟﺔ ﺍﻟﻌﺮﺍﻕ ، ﺃﻋﻤﺎﻝ ﺍﳌﺆﲤﺮ ﺍﻟﺴﻨﻮﻱ ﻟﺒﻴﺖ ﺍﳊﻜﻤﺔ ، ﺑﻐﺪﺍﺩ ، ﻛﺎﻧﻮﻥ ﺍﻟﺜﺎﱐ ٢١٠٢ ، ﺹ ٧٢ ﺎﻳﺎﺕ ﻏﲑ ﳏﺴـﻮﻣﺔ ، ﻗـﻮﺍﺕ ﺍﻷﻣـﻦ : . ـ ﻛﺬﻟﻚ ﻳﻨﻈﺮ ) ﺎ ، ﺗﻘﺮﻳﺮ ﳎﻤﻮﻋﺔ ﺍﻷﺯﻣﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ﺭﻗـﻢﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﲔ ﲣﻔﻴﺾ ﻋﺪﺩ ﺍﻟﻘﻮﺍﺕ ﺍﻷﻣﺮﻳﻜﻴﺔ ﻭﺍﻧﺴﺤﺎ ٩٩ ( ﺣـﻮﻝ ﺍﻟﺸﺮﻕ ﺍﻷﻭﺳﻂ ، ﺑﺘﺎﺭﻳﺦ ٦٢/٠١/٠١٠٢ ﻡ ، ﺹ ١ . ) * ( ﻣﺜﻞ "ﺍﻛﺎﺩﳝﻴﺔ ﺳﻴﺪ ﺍﻟﻜﻮﻧﲔ" ﻟﺘﺨﺮﻳﺞ ﺍﻟﻘﺎﺩﺓ ، ﺗﻘﻊ ﰲ ﺷﺮﻕ ﺍﻟﻌﺎﺻﻤﺔ ﺑﻐﺪﺍﺩ ، ﰎ ﺇﻧﺸﺎﺀ ﺍﻷﻛﺎﺩﳝﻴـﺔ ﺑﻌـﺪ ﺃﺣﺪﺍﺙ ﺣﺰﻳﺮﺍﻥ ﻋﺎﻡ ٤١٠٢ ﻡ ، ﻭﺗﺒﻠﻎ ﻣﺪﺓ ﻣﺎ ﺗﻮﺻﻒ ﺏ "ﺍﻟﺪﺭﺍﺳﺔ" ﺷﻬﺮﻳﻦ ﻓﻘﻂ ، ﻭﳝﻨﺢ ﺍﳌﺘﺨـﺮﺝ ﺷـﺎﺭﺓ "ﺍﻟﻘﺎﺩﺓ" ﻭﺷﻬﺎﺩﺓ ﺗﻮﺿﺢ ﺩﺧﻮﻟﻪ ﺩﻭﺭﺓ ﲣﺼﺼﻴﺔ ﰲ ﺍﻟﻘﻴﺎﺩﺓ ﻭﺧﻮﺽ ﺍﳌﻌﺎﺭﻙ ، ﻭﺗﺘﺒﻊ ﳌﻠﻴﺸﻴﺎ "ﺳﺮﺍﻳﺎ ﺍﻟﺴﻼﻡ" ﺍﻟﺘﺎﺑﻌﺔ ﺎ ، ﻭﻫﻢ ﺑﺎﻟﻀﺮﻭﺭﺓ ﻣﻦ ﻗﺎﺩﺓ ﺍﳌﻠﻴﺸـﻴﺎﺕﻟﺮﺟﻞ ﺍﻟﺪﻳﻦ "ﻣﻘﺘﺪﻯ ﺍﻟﺼﺪﺭ" ، ﺇﺫ ﺗﻘﻮﻡ ﲟﻨﺢ ﺍﻟﺮﺗﺐ ﻭﺍﻟﺸﺎﺭﺍﺕ ﻟﻄﻼ ﻭﻋﻨﺎﺻﺮﻫﺎ ﺍﻟﺒﺎﺭﺯﻳﻦ ، ﰎ ﺍﻟﻐﺎﺅﻫﺎ ﺑﺘﺎﺭﻳﺦ ٦١/٧/٧١٠٢ ﻡ ﰲ ﺍﲡﺎﻩ ﺗﻘﻮﻳﺔ ﻣﺆﺳﺴﺎﺕ ﺍﻟﺪﻭﻟﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ . ـ ﻟﻠﻤﺰﻳﺪ ﻋﻦ ﺫﻟﻚ ﻳﻨﻈﺮ : ﺃﲪﺪ ﺍﻟﻨﻌﻴﻤﻲ ، ﺃ ﻛﺎﺩﳝﻴﺔ ﺧﺎﺻﺔ ﰲ ﺑﻐﺪﺍﺩ ﻟﺘﺨﺮﻳﺞ ﻗﺎﺩﺓ ﺍﳌﻠﻴﺸﻴﺎﺕ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ : ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊhttps://www.alaraby.co ) ** ( ﺣﺴﺐ ) ﺍﳌﺎﺩﺓ ٩ ( ) ﻓﻘﺮﺓ ﻫـ ( ﻣﻦ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﺍﻟﺪﺍﺋﻢ ﻟﻌﺎﻡ ٥٠٠٢ ﻡ ، ﻓﺮﺽ ﺍﳉﺎﻧﺐ ﺍﻷﻣﺮﻳﻜﻲ ﻗﻴﻮﺩﺍﹰ ﻋﻠﻰ ﺃﻱ ﺣﻜﻮﻣﺔ ﻋﺮﺍﻗﻴﺔ ﻭﻣﻨﻌﻬﺎ ﻣﻦ ﺍﻟﺘﻌﺎﻃﻲ ﻣﻊ ﺍﻷﺳﻠﺤﺔ ) ﺍﻟﻨﻮﻭﻳﺔ ، ﻭﺍﻟﻜﻴﻤﻴﺎﻭﻳﺔ ، ﻭﺍﻟﺒﻴﻮﻟﻮﺟﻴﺔ ( ﻭﻛﻞ ﻣﺎ ﳝﺖ ﻟﺬﻟﻚ ﺑﺼﻠﺔ . ﻭﺑﺬﻟﻚ ﺷﻠﺖ ﺍﻟﻘﺪﺭﺍﺕ ﺍﻟﻌﺴﻜﺮﻳﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﰲ ﻋﺎﱂ ﻳﺘﺴﺎﺑﻖ ﺣﻮﻝ ﺗﻠﻚ ﺍﻷﺳﻠﺤﺔ ﲟﺎ ﻓﻴﻬﺎ ﺩﻭﻝ ﺍﻟﺸﺮﻕ ﺍﻷﻭﺳﻂ . ـ ﻟﻠﻤﺰﻳﺪ ﻋﻦ ﺫﻟﻚ ﻳﻨﻈﺮ : ﺩ. ﻧﺪﱘ ﺍﳉﺎﺑﺮﻱ ، ﻣ ، ﻼﺣﻈﺎﺕ ﺣﻮﻝ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﺍﻟـﺪﺍﺋﻢ ﺻﺤﻴﻔﺔ ) ﺍﻟﺼﺒﺎﺡ ﺍﳉﺪﻳﺪ (، ﻧﺸﺮﺕ ﺑﺘﺎﺭﻳﺦ ٣١/٦/٥١٠٢. ﻡ )٦٣ ( ﻡ.ﺩ. ﺣﺎﻣﺪ ﻋﺒﻴﺪ ﺣﺪﺍﺩ ، ﺍﻟﺘﺤﺪﻳﺎﺕ ﺍﻻﻗﺘﺼﺎﺩﻳﺔ ﻟﻠﻌﺮﺍﻕ ﺑﻌﺪ ﺍﻻﻧﺴﺤﺎﺏ ﺍﻷﻣﺮﻳﻜﻲ ، ﳎﻠﺔ ﺩﺭﺍﺳﺎﺕ ﺩﻭﻟﻴﺔ ) ، ﺍﻟﻌﺪﺩ ٢٥ ( ، ﻣﺮﻛﺰ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ، ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ ، ﺑﻐﺪﺍﺩ ، ﻧﻴﺴﺎﻥ ٢١٠٢ ، ﺹ ١٥ ٤٧ ـ . )٧٣ ( ﺪﺩ ﺃﻣﻦ ﻭﺍﺳﺘﻘﺮﺍﺭ ﺍﻟﺒﻼﺩ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﺃﲪﺪ ﲨﻌﺔ ، ﻓﺘﻨﺔ ﺍﳉﻨﻮﺏ ﺍﻟﻌﺮﺍﻗﻲ : ﻋﻠﻰ ﺍﳌﻮﻗﻊ.com/story/2018/7/15 https://www.youm7 )٨٣ ( ﻋﺎﻣﺮ ﺍﻟﻌﻤﺮﺍﻥ ، ﺍﻷﺯﻣﺔ ﺍﻻ ﻗﺘﺼﺎﺩﻳﺔ ﰲ ﻛﻮﺭﺩﺳﺘﺎﻥ ﺍﻟﻌﺮﺍﻕ .. http://democraticac.de/?p=34554 http://democraticac.de/?p=34554 )٤٤ ( ﺃﺑﻮ ﺑﻜﺮ ﺍﻟﺪﺳﻮﻗﻲ ، ﺇﻗﻠﻴﻢ ﺟﺪﻳﺪ ﻳﺘﺸﻜﻞ : ﺍﻟﺘﺤﻮﻻﺕ ﺍﳍﻴﻜﻠﻴﺔ ﺍﻟﻜﱪﻯ ﰲ ﺍﻟﺸﺮﻕ ﺍﻷﻭﺳـﻂ ، ﳎﻠـﺔ ) ﺍﻟﺴﻴﺎﺳﺔ ﺍﻟﺪﻭﻟﻴﺔ، ﺍﻟﻌﺪﺩ ٦٨١ ( ، ﺍﻟﺴﻨﺔ ﺍﻟﺴﺎﺑﻌﺔ ﻭﺍﻷﺭﺑﻌﻮﻥ ، ﺍﻟﻘﺎﻫﺮﺓ ، ﺃﻛﺘﻮﺑﺮ ١١٠٢ ، ﺹ ١٦ . ) * ( ﻣﺎ ﺣﺪﺙ ﰲ ﺍﻟﻌﺮﺍﻕ ﺑﻌﺪ ﺍﻻﺣﺘﻼﻝ ﺍﻷﻣﺮ ﻳﻜﻲ ﻋﺎﻡ ٣٠٠٢ ﻡ ، ﻭﻛﻞ ﻣﺎ ﺻﺪﺭ ﻣﻦ ﺍﳊﻜﻮﻣﺎﺕ ﺍﳌﺘﻌﺎﻗﺒﺔ ، ﳍﻮ ﺎﺍﻟﺪﻟﻴﻞ ﺍﻟﻘﺎﻃﻊ ﻋﻠﻰ ﺃﻥ ﻫﺬﻩ ﺍﳊﻜﻮﻣﺎﺕ ﻫﻲ ﻧﺘﺎﺝ ﻭﺗﻄﺒﻴﻖ ﻋﻤﻠﻲ ﳌﺎ ﻳﺴﻤﻰ ﺍﻟﻌﻤﻠﻴﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﻣﻨﻬﺎﺟﻬﺎ ﻭﺗﻮﺟﻬﺎ ﺎ ﺍﳌﺮﺳﻮﻣﺔ ﻣﻦ ﻟﺪﻥ ﻣﻦ ﺃﻭﺟﺪﻫﺎ ﻭﺯﺭﻋﻬﺎ ، ﻓﻠﻢ ﻳﺄﺕ ﻣﻦ ﻫﺬﻩ ﺍﻟﻌﻤﻠﻴﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﻛﻞ ﺍﳊﻜﻮﻣﺎﺕ ﺍﻟـﱵﻭﻏﺎﻳﺎ ﻣﺜﻠﺘﻬ ـﺎ ﰲﺎ ﻭﺧﻼﻝ ﻛﻞ ﺍﻷﻋﻮﺍﻡ ﺍﳌﺎﺿﻴﺔ ﺃﻱ ﻋﻤﻞ ﺃﻭ ﺧﻄﻮﺓ ﳝﻜﻦ ﺃﻥ ﻳﻌﺘﱪﻫﺎ ﺃﻭ ﻳﻘﺒﻬﺎ ﺷﻌﺐ ﺍﻟﻌﺮﺍﻕ ﻋﻠـﻰ ﺃ ﺎ ﳋﺪﻣﺔ ﺍﻟﻌﺮﺍﻕ ﻭﻣﺼﺎﳊﻪﺍﻻﲡﺎﻩ ﺍﻟﺼﺤﻴﺢ ، ﺃﻭ ﺃﻥ ﻳﻔﺴﺮﻫﺎ ﺃﺣﺪ ﺃﺑﻨﺎﺀ ﺷﻌﺐ ﺍﻟﻌﺮﺍﻕ ﺑﺄ ، ﺑﻞ ﻋﻠﻰ ﺍﻟﻌﻜﺲ ﲤﺎﻣﺎﹰ ، ﻓﻠﻢ ﻳﻜﻦ ﻣﻦ ﻫﺬﻩ ﺍﳊﻜﻮﻣﺎﺕ ﺇﻻ ﺍﻟﺘﻄﺒ ﻴﻖ ﺍﻟﻌﻤﻠﻲ ﻷﻫﺪﺍﻑ ﺍﻟﺪﻭﻝ ﺍﶈﺘﻠﺔ ﻟﻠﻌﺮﺍﻕ . ـ ﻟﻠﻤﺰﻳﺪ ﻋﻦ ﺫﻟﻚ ﻳﻨﻈﺮ : ﺩ. ﻳﺎﺳﲔ ﺍﻟﻜﻠﻴﺪﺍﺭ ﺍﳊﺴﻴﲏ ، ﺍﻟﻌﻤﻠﻴﺔ ﺍﻟﺴﻴﺎﺳﻴﺔ ﰲ ﺍﻟﻌﺮﺍﻕ .. ﺍﻟﺒﺎﻃﻞ ﺍﻟﺬﻱ ﳚﺐ ﺇﺳﻘﺎﻃﻪ ، ﻧﺸﺮﺕ ﺑﺘﺎﺭﻳﺦ ٣٢/٦/٦١٠٢: ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ )٥٤ ( ﻣﲎ ﺣﺴﲔ ﻋﺒﻴﺪ ، ﺧﻠﻮﺩ ﳏﻤﺪ ﲬﻴﺲ ، ﺍﻟﻌﻼﻗﺎﺕ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ﰲ ﺿﻮﺀ ﺍﺗﻔﺎﻗﻴﺔ ﺍﻹﻃﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ، ، ﻛﺘﺎﺏ : ﺇﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺑﻨﺎﺀ ﺩﻭﻟﺔ ﺍﻟﻌﺮﺍﻕ ﺑﻌﺪ ﺍﻻﻧﺴﺤﺎﺏ ﺍﻷﻣﺮﻳﻜﻲ ، ﺑﻴﺖ ﺍﳊﻜﻤﺔ ١١٠٢ ، ﺹ ٢٣ . - ، ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﺍﻟﻌﻤﻴﺪ ﺑﺮﻫﺎﻥ ﺇﺑﺮﺍﻫﻴﻢ ﻛﺮﱘ ، ﺍﻹﺗﻔﺎﻗﻴﺔ ﺍﻷﻣﻨﻴﺔ ﺑﲔ ﺍﻟﻌﺮﺍﻕ ﻭﺍﻟﻮﻻﻳﺎﺕ ﺍﳌﺘﺤـﺪﺓ ﺍﻷﻣﺮﻳﻜﻴـﺔ : ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ ﻊ http://www.zamanalwsl.net/news/article/6899     ﺇﱃ ﺃﻳﻦ ؟ ، ﻣﺮﻛﺰ ﺍﻟـﺮﻭﺍﺑﻂ ﻟﻠﺪﺭﺍﺳـﺎﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍﻟﺴﻴﺎﺳﻴﺔ ، ﻧﺸﺮﺕ ﺑﺘﺎﺭﻳﺦ ٠٢/٣/٦١٠٢ ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠـﻰ : ﺍﳌﻮﻗﻊm http://webcache.googleusercontent.co )٩٣ ( ﺃﲪﺪ ﻏﺎﻟﺐ ﳏﻲ ﺟﻌﻔﺮ ، ﺍﳍﻮﻳﺔ ﺍﻟﻮﻃﻨﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ : ﺩﺭﺍﺳﺔ ﰲ ﺍﺷﻜﺎﻟﻴﺔ ﺍﻟﺒﻨﺎﺀ ﻭﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﺍﻃﺮﻭﺣـﺔ ، ﺩﻛﺘﻮﺭﺍﻩ "ﻏﲑ ﻣﻨﺸﻮﺭﺓ" ، ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ ٠١٠٢ ، ﺹ ٦٢٣ . ٤٠٤        http://www.iraqicp.com/index.php/sections/platform/31364-2015-07-31-10-32-20 ـ ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﻋﺒﺪ ﺍﻟﺴﻼﻡ ﺃﲪﺪ ﺍﻟﺴﺎﻣﺮ ، ﺍﻻﻋﻼﻡ ﻭﺍﳍﻮﻳﺔ ﺍﻟﻮﻃﻨﻴﺔ ﰲ ﺍﻟﻌﺮﺍﻕ ، ﻧﺪﻭﺓ ﺟﺎﻣﻌﺔ ﺑ ﻐﺪﺍﺩ : ) ﺍﻟﺘﻌﻠﻴﻢ ، ( ﻭﺗﻌﺰﻳﺰ ﺍﳍﻮﻳﺔ ﺍﻟﻮﻃﻨﻴﺔ ﰲ ﺍﻟﻌﺮﺍﻕ ٧١ ﺷﺒﺎﻁ ٠١٠٢ ، ﺹ ٨٨ . )٢٤ ( ﻋﺒﺪ ﺍﻻﻟﻪ ﺍﻟﺒﻠﺪﺍﻭﻱ ، ﺍﻟﺘﻬﺪﻳﺪﺍﺕ ﻭﺍﻟﺘﺤﺪﻳﺎﺕ ﺍﳋﺎﺭﺟﻴﺔ ﻭﺍﻟﺪﺍﺧﻠﻴﺔ ﻟﻸﻣﻦ ﺍﻟﻘـﻮﻣﻲ ﺍﻟﻌﺮﺍﻗـﻲ ﻭﻛﻴﻔﻴـﺔ . ﻣﻮﺍﺟﻬﺘﻬﺎ ، ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ )٣٤ ( ﺩﻳﻨﺎ ﳏﺴﻦ ﻋﺒﻮﺩ ﻋﺒﺪﻩ ، ﺍﻻﲡﺎﻫﺎﺕ ﺍﻟﻌﺎﻣﺔ ﻟﻠﻤﺼﺎﱀ ﺍﻹﻗ ﻠﻴﻤﻴﺔ ﻹﻳﺮﺍﻥ ﰲ ﺍﳌﻨﻄﻘﺔ ﺍﻟﻌﺮﺑ ﻴﺔ ﺩﺭﺍﺳﺔ ﻣﻘﺎﺭﻧـﺔ ""ﺳﻮﺭﻳﺎ ﻭﺍﻟﻴﻤﻦ ، ﺍﳌﺮﻛﺰ ﺍﻟﺪﳝﻮﻗﺮﺍﻃﻲ ﺍﻟﻌﺮﰊ ﻟﻠﺪﺭﺍﺳﺎﺕ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﻭﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻻ ﻗﺘﺼﺎﺩﻳﺔ ، ﻧﺸـﺮ ﺑﺘـﺎﺭﻳﺦ ٥٢/٧/٦١٠٢ ﻡ ، ﺍﻟﺸﺒ ﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ )ﺍﻻﻧﺘﺮﻧﺖ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ : http://www.zamanalwsl.net/news/article/6899 . ) ** ( ﻛﻤﺎ ﺣﺼﻞ ﰲ ﻓﻴﺘﻨﺎﻡ ﺍﳉﻨﻮﺑﻴﺔ ﻭﻛﻮﺭﻳﺎ ﺍﳉﻨﻮﺑﻴﺔ ، ﺃﻭ ﺑﻌﺾ ﺩﻭﻝ ﺃﻣﺮﻳﻜﺎ ﺍﳉﻨﻮﺑﻴﺔ ﻭﺍﻟﻮﺳﻄﻰ )٦٤ ) ( ﳏﻤﺪ ﺍﻟﺴﻌﻴﺪ ﺇﺩﺭﻳﺲ ، ﺍﻻﺗﻔﺎﻗﻴﺔ ﺍﻷﻣﻨﻴﺔ ﺍﻟﻌﺮﺍﻗﻴﺔ ﺑﲔ ﻃﻬﺮﺍﻥ ﻭﻭﺍﺷﻨﻄﻦ ، ﳎﻠﺔ ﺍﻟﺴﻴﺎﺳﺔ ﺍﻟﺪﻭﻟﻴﺔ ، ﺍﻟﻌﺪﺩ ٥٧١ ( ، ﻣﺮﻛﺰ ﺍﻷﻫ ، ﺮﺍﻡ ﻟﻠﺪﺭﺍﺳﺎﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ﻭﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ، ﺍﻟﻘﺎﻫﺮﺓ ٩٠٠٢ ، ﺹ ١٩١ . ٥٠٤       )٧٤ ، ( ﻋﻼﺀ ﻳﻮﺳﻒ ، ﺗﺮﺩﺩ ﺃﻣﺮﻳﻜﻲ ﰲ ﺗﺴﻠﻴﺢ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ، ﺗﻘﺎﺭﻳﺮ ﻭﺣﻮﺍﺭﺍﺕ ، ﺷﺒﻜﺔ ﺍﳉﺰﻳﺮﺓ ﺍﻹﻋﻼﻣﻴﺔ ﻧﺸﺮﺕ ﺑﺘﺎﺭﻳﺦ ٧١/٤/٥١٠٢ : ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ )٧٤ ، ( ﻋﻼﺀ ﻳﻮﺳﻒ ، ﺗﺮﺩﺩ ﺃﻣﺮﻳﻜﻲ ﰲ ﺗﺴﻠﻴﺢ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ، ﺗﻘﺎﺭﻳﺮ ﻭﺣﻮﺍﺭﺍﺕ ، ﺷﺒﻜﺔ ﺍﳉﺰﻳﺮﺓ ﺍﻹﻋﻼﻣﻴﺔ ﻧﺸﺮﺕ ﺑﺘﺎﺭﻳﺦ ٧١/٤/٥١٠٢ : ﻡ ، ﺍﻟﺸﺒﻜﺔ ﺍﻟﺪﻭﻟﻴﺔ ﻟﻠﻤﻌﻠﻮﻣﺎﺕ ) ﺍﻻﻧﺘﺮﻧﺖ ( ﻋﻠﻰ ﺍﳌﻮﻗﻊ http://www.aljazeera.net )٨٤ ( ﻋﻤﺎﺩ ﻫﺎﺩﻱ ﺍﻟﺮﺑﻴﻌﻲ ، ﺍﻟﻌﺮﺍﻕ ﻭﺍﻟﺘﺤﺎﻟﻒ ﺍﻟﻐﺮﰊ ١٩٩١ ٣٠٠٢ ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﺩﺍﺭ ﺯﻫﺮﺍﻥ ﻟﻠﻨﺸﺮ ، ﻭﺍﻟﺘﻮﺯﻳﻊ ، ﻋﻤﺎﻥ ٣١٠٢ ، ﺹ ٨١٢ . )٩٤ ( ﻋﺒﺎﺱ ﻋﻠﻲ ﳏﻤﺪ ، ﺍﻷﻣﻦ ﻭﺍﻟﺘﻨﻤﻴﺔ : ﺩﺭﺍﺳﺔ ﺣﺎﻟﺔ ﺍﻟﻌﺮﺍﻕ ﻟﻠﻤﺪﺓ ٠٧٩١ ٧٠٠٢ ، ﺍﻟﻄ ، ﺒﻌـﺔ ﺍﻷﻭﱃ ، ﻣﺮﻛﺰ ﺍﻟﻌﺮﺍﻕ ﻟﻠﺪﺭﺍﺳﺎﺕ ، ﺑﻐﺪﺍﺩ ٣١٠٢ ، ﺹ ١٢ ٢٢ ـ ، . ـ ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﺻﻼﺡ ﺣﺴﻦ ﺍﻟﺸـﻤﺮﻱ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺣﻴﺎﻝ ﺍﻟﻌﺮﺍﻕ : ﻗﺮﺍﺀﺓ ﰲ ﻣﻼﻣﺢ ﺍﻟﺘﻐﻴﲑ ، ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ ، ﺹ ٨٢٤ ـ ٩٢٤ . )٠٥ ( ﳏﻤﻮﺩ ﺃﲪﺪ ﻋﺰﺕ ، ﺗﺪﺍﻋﻴﺎﺕ ﺍﻻﺗﻔﺎﻗﻴﺔ ﺍﻷﻣﻨﻴﺔ ﻋﻠﻰ ﺻﻴﺎﻏﺔ ﺇﺳﺘﺮﺍﺗﻴﺠﻴ ﺔ ﻟﻠﻌﺮﺍﻕ ، ﻧﺪﻭﺓ ﻓﻜﺮﻳﺔ ، ﳎﻠـﺔ ) ﺩﺭﺍﺳﺎﺕ ﺳﻴﺎﺳﻴﺔ ، ﺍﻟﻌﺪﺩ ٥١ ، ( ، ﻗﺴﻢ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﺑﻴﺖ ﺍﳊﻜﻤﺔ ، ﺑﻐﺪﺍﺩ ٠١٠٢ ، ﺹ ٨٠١ . ـ ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﻭﻟﻴﺪ ﻋﺒﺪ ﺍﳉﺒﺎﺭ ﺍﻟﻌﻨﺒﻜﻲ ، ﺍﻻﻃﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﰲ ﺑﻨﺎﺀ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗـﻲ ﺑﻌـﺪ ﺍﻻﻧﺴـﺤﺎﺏ ﺍﻷﻣﺮﻳﻜﻲ ، ﺍﳌﺆﲤﺮ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﺴﻨﻮﻱ ﺍﻷﻭﻝ ، ﺑ ، ﻴﺖ ﺍﳊﻜﻤﺔ ، ﺑﻐﺪﺍﺩ ١١٠٢ ، ﺹ ٥١١ . ) * ( ﻭﻟﻴﺪ ﻋﺒﺪ ﺍﳉﺒﺎﺭ ﺍﻟﻌﻨﺒﻜﻲ ، ﺍﻹﻃﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﰲ ﺑﻨﺎﺀ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ﺑﻌﺪ ﺍﻻﻧﺴﺤﺎﺏ ﺍﻷﻣﺮﻳﻜﻲ ، ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ، ﺹ ٨٨٣ ـ ٠٩٣ . ) ** ( ﻧﺺ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﺍﻟﺪﺍﺋﻢ ﻟﻌﺎﻡ ٥٠٠٢ ) ﻡ ، ﺍﳌﺎﺩﺓ ٩ ( / ) ﺃﻭﻻ ﺃ ( ﻋﻠﻰ ﺗﺸﻜﻴﻞ ﺍﻟﻘﻮﺍﺕ ﺍﳌﺴـﻠﺤ ﺔ ) ﺍﻟﻌﺮﺍﻗﻴﺔ ﻣﻦ ﻣﻜﻮﻧﺎﺕ ﺍﻟﺸﻌﺐ ﺍﻟﻌﺮﺍﻗﻲ ﺩﻭﻥ ﲤﻴﻴﺰ ﺃﻭ ﺇﻗﺼﺎﺀ ، ﺃﻣﺎ ﺍﳌﺎﺩﺓ ٩ ( / ) ﺛﺎﻧﻴﺎﹰ ( ﻓﻘﺪ ﺍﺷﺎﺭﺕ ﺇﱃ ﺗﻨﻈـﻴﻢ ﻗﺎﻧﻮﻥ ﺧﺪﻣﺔ ﺍﻟﻌﻠﻢ . ﻧﻘﻼﹰ ﻋﻦ : ﺩﺳﺘﻮﺭ ﲨﻬﻮﺭﻳﺔ ﺍﻟﻌﺮﺍﻕ ﻟﺴﻨﺔ ٥٠٠٢ ) ﻡ ، ﺍﳌﺎﺩﺓ ٩ ) ( / ) ﺃﻭﻻ ﺃ ( ﻭ ﺍﳌﺎﺩﺓ ٩ . http://www.zamanalwsl.net/news/article/6899 ( ( / ) ﺛﺎﻧﻴﺎﹰ )١٥ ( ﻫﺎﻧﺰ ﻓﻮﻥ ﺳﺒﻮﻧﻴﻚ ) ، ﻣﺎ ﺑﻌﺪ ﺍﳊﺮﺏ ﻭﻣﺎ ﻗﺒﻞ ﺍﻟﺴﻼﻡ ﺍﻟﻌﺮﺍﻕ ﺇﱃ ﺃﻳﻦ ، ﳎﻠﺔ ﺍﳌﺴﺘﻘﺒﻞ ﺍﻟﻌﺮﰊ ، ﺍﻟﻌـﺪﺩ ٧١٣ ، ( ، ﻣﺮﻛﺰ ﺩﺭﺍﺳﺎﺕ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻌﺮﺑﻴﺔ ، ﺑﲑﻭﺕ ٥٠٠٢ ، ﺹ ٠٤ . )٢٥ ) ( ﺍﳌﺎﺩﺓ ٩ ( ﻓﻘﺮﺓ ) ﺏ ( ﻣﻦ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﻟﻌﺎﻡ ٥٠٠٢. ﻡ )٣٥ ( ﻛﻮﺛﺮ ﻋﺒﺎﺱ ﺍﻟﺮﺑﻴﻌﻲ ، ﺳﻴﺎﺳﺔ ﺍﻟﻌﺮﺍﻕ ﺍﳋﺎﺭﺟﻴﺔ ﺑﲔ ﺍﻟﻘﻴﻮﺩ ﻭﺍﻟﻔﺮﺹ ، ﺩﺭﺍﺳﺎﺕ ﺩﻭﻟﻴﺔ ، ﻣﺮﻛﺰ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ، ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ ، ﺍﻟﻌﺪﺩ ) ﺍﻟﺮﺍﺑﻊ ﻭﺍﻷﺭﺑﻌﻮﻥ ( ، ﻧﻴﺴﺎﻥ ٠١٠٢ ، ﺹ ١١ . )٨٤ ( ﻋﻤﺎﺩ ﻫﺎﺩﻱ ﺍﻟﺮﺑﻴﻌﻲ ، ﺍﻟﻌﺮﺍﻕ ﻭﺍﻟﺘﺤﺎﻟﻒ ﺍﻟﻐﺮﰊ ١٩٩١ ٣٠٠٢ ، ﺍﻟﻄﺒﻌﺔ ﺍﻷﻭﱃ ، ﺩﺍﺭ ﺯﻫﺮﺍﻥ ﻟﻠﻨﺸﺮ ، ﻭﺍﻟﺘﻮﺯﻳﻊ ، ﻋﻤﺎﻥ ٣١٠٢ ، ﺹ ٨١٢ . )٩٤ ( ﻋﺒﺎﺱ ﻋﻠﻲ ﳏﻤﺪ ، ﺍﻷﻣﻦ ﻭﺍﻟﺘﻨﻤﻴﺔ : ﺩﺭﺍﺳﺔ ﺣﺎﻟﺔ ﺍﻟﻌﺮﺍﻕ ﻟﻠﻤﺪﺓ ٠٧٩١ ٧٠٠٢ ، ﺍﻟﻄ ، ﺒﻌـﺔ ﺍﻷﻭﱃ ، ﻣﺮﻛﺰ ﺍﻟﻌﺮﺍﻕ ﻟﻠﺪﺭﺍﺳﺎﺕ ، ﺑﻐﺪﺍﺩ ٣١٠٢ ، ﺹ ١٢ ٢٢ ـ ، . ـ ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﺻﻼﺡ ﺣﺴﻦ ﺍﻟﺸـﻤﺮﻱ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻴﺔ ﺍﻷﻣﺮﻳﻜﻴﺔ ﺣﻴﺎﻝ ﺍﻟﻌﺮﺍﻕ : ﻗﺮﺍﺀﺓ ﰲ ﻣﻼﻣﺢ ﺍﻟﺘﻐﻴﲑ ، ﻣﺼﺪﺭ ﺳﺒﻖ ﺫﻛﺮﻩ ، ﺹ ٨٢٤ ـ ٩٢٤ . )٠٥ ( ﳏﻤﻮﺩ ﺃﲪﺪ ﻋﺰﺕ ، ﺗﺪﺍﻋﻴﺎﺕ ﺍﻻﺗﻔﺎﻗﻴﺔ ﺍﻷﻣﻨﻴﺔ ﻋﻠﻰ ﺻﻴﺎﻏﺔ ﺇﺳﺘﺮﺍﺗﻴﺠﻴ ﺔ ﻟﻠﻌﺮﺍﻕ ، ﻧﺪﻭﺓ ﻓﻜﺮﻳﺔ ، ﳎﻠـﺔ ) ﺩﺭﺍﺳﺎﺕ ﺳﻴﺎﺳﻴﺔ ، ﺍﻟﻌﺪﺩ ٥١ ، ( ، ﻗﺴﻢ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﺴﻴﺎﺳﻴﺔ ، ﺑﻴﺖ ﺍﳊﻜﻤﺔ ، ﺑﻐﺪﺍﺩ ٠١٠٢ ، ﺹ ٨٠١ . ـ ﻛﺬﻟﻚ ﻳﻨﻈﺮ : ﻭﻟﻴﺪ ﻋﺒﺪ ﺍﳉﺒﺎﺭ ﺍﻟﻌﻨﺒﻜﻲ ، ﺍﻻﻃﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﰲ ﺑﻨﺎﺀ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗـﻲ ﺑﻌـﺪ ﺍﻻﻧﺴـﺤﺎﺏ ﺍﻷﻣﺮﻳﻜﻲ ، ﺍﳌﺆﲤﺮ ﺍﻟﺴﻴﺎﺳﻲ ﺍﻟﺴﻨﻮﻱ ﺍﻷﻭﻝ ، ﺑ ، ﻴﺖ ﺍﳊﻜﻤﺔ ، ﺑﻐﺪﺍﺩ ١١٠٢ ، ﺹ ٥١١ . ) * ( ﻭﻟﻴﺪ ﻋﺒﺪ ﺍﳉﺒﺎﺭ ﺍﻟﻌﻨﺒﻜﻲ ، ﺍﻹﻃﺎﺭ ﺍﻹﺳﺘﺮﺍﺗﻴﺠﻲ ﰲ ﺑﻨﺎﺀ ﺍﳉﻴﺶ ﺍﻟﻌﺮﺍﻗﻲ ﺑﻌﺪ ﺍﻻﻧﺴﺤﺎﺏ ﺍﻷﻣﺮﻳﻜﻲ ، ﻣﺼﺪﺭ ) ** ( ﻧﺺ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﺍﻟﺪﺍﺋﻢ ﻟﻌﺎﻡ ٥٠٠٢ ) ﻡ ، ﺍﳌﺎﺩﺓ ٩ ( / ) ﺃﻭﻻ ﺃ ( ﻋﻠﻰ ﺗﺸﻜﻴﻞ ﺍﻟﻘﻮﺍﺕ ﺍﳌﺴـﻠﺤ ﺔ ) ﺍﻟﻌﺮﺍﻗﻴﺔ ﻣﻦ ﻣﻜﻮﻧﺎﺕ ﺍﻟﺸﻌﺐ ﺍﻟﻌﺮﺍﻗﻲ ﺩﻭﻥ ﲤﻴﻴﺰ ﺃﻭ ﺇﻗﺼﺎﺀ ، ﺃﻣﺎ ﺍﳌﺎﺩﺓ ٩ ( / ) ﺛﺎﻧﻴﺎﹰ ( ﻓﻘﺪ ﺍﺷﺎﺭﺕ ﺇﱃ ﺗﻨﻈـﻴﻢ ﻗﺎﻧﻮﻥ ﺧﺪﻣﺔ ﺍﻟﻌﻠﻢ . ﻧﻘﻼﹰ ﻋﻦ : ﺩﺳﺘﻮﺭ ﲨﻬﻮﺭﻳﺔ ﺍﻟﻌﺮﺍﻕ ﻟﺴﻨﺔ ٥٠٠٢ ) ﻡ ، ﺍﳌﺎﺩﺓ ٩ ) ( / ) ﺃﻭﻻ ﺃ ( ﻭ ﺍﳌﺎﺩﺓ ٩ . ( ( / ) ﺛﺎﻧﻴﺎﹰ )١٥ ( ﻫﺎﻧﺰ ﻓﻮﻥ ﺳﺒﻮﻧﻴﻚ ) ، ﻣﺎ ﺑﻌﺪ ﺍﳊﺮﺏ ﻭﻣﺎ ﻗﺒﻞ ﺍﻟﺴﻼﻡ ﺍﻟﻌﺮﺍﻕ ﺇﱃ ﺃﻳﻦ ، ﳎﻠﺔ ﺍﳌﺴﺘﻘﺒﻞ ﺍﻟﻌﺮﰊ ، ﺍﻟﻌـﺪﺩ ٧١٣ ، ( ، ﻣﺮﻛﺰ ﺩﺭﺍﺳﺎﺕ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻌﺮﺑﻴﺔ ، ﺑﲑﻭﺕ ٥٠٠٢ ، ﺹ ٠٤ . http://www.zamanalwsl.net/news/article/6899 )٢٥ ) ( ﺍﳌﺎﺩﺓ ٩ ( ﻓﻘﺮﺓ ) ﺏ ( ﻣﻦ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﻟﻌﺎﻡ ٥٠٠٢. ﻡ )٣٥ ( ﻛﻮﺛﺮ ﻋﺒﺎﺱ ﺍﻟﺮﺑﻴﻌﻲ ، ﺳﻴﺎﺳﺔ ﺍﻟﻌﺮﺍﻕ ﺍﳋﺎﺭﺟﻴﺔ ﺑﲔ ﺍﻟﻘﻴﻮﺩ ﻭﺍﻟﻔﺮﺹ ، ﺩﺭﺍﺳﺎﺕ ﺩﻭﻟﻴﺔ ، ﻣﺮﻛﺰ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ، ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ ، ﺍﻟﻌﺪﺩ ) ﺍﻟﺮﺍﺑﻊ ﻭﺍﻷﺭﺑﻌﻮﻥ ( ، ﻧﻴﺴﺎﻥ ٠١٠٢ ، ﺹ ١١ . )١٥ ( ﻫﺎﻧﺰ ﻓﻮﻥ ﺳﺒﻮﻧﻴﻚ ) ، ﻣﺎ ﺑﻌﺪ ﺍﳊﺮﺏ ﻭﻣﺎ ﻗﺒﻞ ﺍﻟﺴﻼﻡ ﺍﻟﻌﺮﺍﻕ ﺇﱃ ﺃﻳﻦ ، ﳎﻠﺔ ﺍﳌﺴﺘﻘﺒﻞ ﺍﻟﻌﺮﰊ ، ﺍﻟﻌـﺪﺩ ٧١٣ ، ( ، ﻣﺮﻛﺰ ﺩﺭﺍﺳﺎﺕ ﺍﻟﻮﺣﺪﺓ ﺍﻟﻌﺮﺑﻴﺔ ، ﺑﲑﻭﺕ ٥٠٠٢ ، ﺹ ٠٤ . )٢٥ ) ( ﺍﳌﺎﺩﺓ ٩ ( ﻓﻘﺮﺓ ) ﺏ ( ﻣﻦ ﺍﻟﺪﺳﺘﻮﺭ ﺍﻟﻌﺮﺍﻗﻲ ﻟﻌﺎﻡ ٥٠٠٢. ﻡ )٣٥ ( ﻛﻮﺛﺮ ﻋﺒﺎﺱ ﺍﻟﺮﺑﻴﻌﻲ ، ﺳﻴﺎﺳﺔ ﺍﻟﻌﺮﺍﻕ ﺍﳋﺎﺭﺟﻴﺔ ﺑﲔ ﺍﻟﻘﻴﻮﺩ ﻭﺍﻟﻔﺮﺹ ، ﺩﺭﺍﺳﺎﺕ ﺩﻭﻟﻴﺔ ، ﻣﺮﻛﺰ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﺪﻭﻟﻴﺔ ، ﺟﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ ، ﺍﻟﻌﺪﺩ ) ﺍﻟﺮﺍﺑﻊ ﻭﺍﻷﺭﺑﻌﻮﻥ ( ، ﻧﻴﺴﺎﻥ ٠١٠٢ ، ﺹ ١١ . ٦٠٤
https://openalex.org/W2966917845	https://hal.archives-ouvertes.fr/hal-02157195/file/bbigap2019.pdf	English	null	Metadata Systems for Data Lakes: Models and Features	Communications in computer and information science	2,019	cc-by	5,816	Metadata Systems for Data Lakes: Models and Features Pegdwendé Nicolas Sawadogo, Etienne Scholly, Cécile Favre, Eric Ferey, Sabine Loudcher, Jérôme Darmont To cite this version: Pegdwendé Nicolas Sawadogo, Etienne Scholly, Cécile Favre, Eric Ferey, Sabine Loudcher, et al.. Metadata Systems for Data Lakes: Models and Features. 1st International Workshop on BI and Big Data Applications (BBIGAP@ADBIS 2019), Sep 2019, Bled, Slovenia. pp.440-451, 10.1007/978-3- 030-30278-8. hal-02157195 Distributed under a Creative Commons Attribution 4.0 International License Metadata Systems for Data Lakes: Models and Features Pegdwendé N. Sawadogo1, Étienne Scholly1,2, Cécile Favre1, Éric Ferey2, Sabine Loudcher1, and Jérôme Darmont1 Pegdwendé N. Sawadogo1, Étienne Scholly1,2, Cécile Favre1, Éric Ferey2, Sabine Loudcher1, and Jérôme Darmont1 1 Université de Lyon, Lyon 2, ERIC EA 3083 https://eric.ish-lyon.cnrs.fr/ 2 BIAL-X https://www.bial-x.com/ 1 Université de Lyon, Lyon 2, ERIC EA 3083 https://eric.ish-lyon.cnrs.fr/ 2 BIAL-X https://www.bial-x.com/ https://www.bial-x.com/ Abstract. Over the past decade, the data lake concept has emerged as an alternative to data warehouses for storing and analyzing big data. A data lake allows storing data without any predeﬁned schema. There- fore, data querying and analysis depend on a metadata system that must be eﬃcient and comprehensive. However, metadata management in data lakes remains a current issue and the criteria for evaluating its eﬀective- ness are more or less nonexistent. In this paper, we introduce MEDAL, a generic, graph-based model for metadata management in data lakes. We also propose evaluation criteria for data lake metadata systems through a list of expected features. Even- tually, we show that our approach is more comprehensive than existing metadata systems. Keywords: Data lakes · Metadata modeling · Metadata management Keywords: Data lakes · Metadata modeling · Metadata management HAL Id: hal-02157195 https://hal.science/hal-02157195v1 Submitted on 20 Sep 2019 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 P.N. Sawadogo et al. To address these issues, we ﬁrst identify a set of features that should ide- ally be proposed by the metadata system of a data lake. By comparing several metadata systems with respect to these features, we hint that none of them of- fers all expected features. Thus, we propose a new metadata model that is more complete and generic. Our graph-based metadata model is named MEtadata model for DAta Lakes (MEDAL). It is also based on a typology distinguishing intra-object, inter-object and global metadata. The remainder of this paper is organized as follows. Section 2 introduces the concept of data lake. Section 3 details the expected features of a data lake’s metadata system and compares several works on the organization of metadata w.r.t. these features. Section 4 presents the metadata typology on which MEDAL is based. Section 5 formalizes MEDAL and introduces its graph representation. Finally, Section 6 concludes the paper and hint at research perspectives. 2.1 Deﬁnitions from the Literature James Dixon introduces the data lake concept as an alternative to data marts, which are subsets of data warehouses that store data into silos. A data lake is a large repository of heterogeneous raw data, supplied by external data sources and from which various analyses can be performed [6]. Thereafter, data lakes are associated with the Hadoop technology [7, 21]. Data lake design may notably be viewed as a methodology for using free or low-cost technologies, typically Hadoop, for storing, processing and exploring raw data within a company [7]. However, this view is becoming minority in the literature, as the data lake concept is now also associated with proprietary solutions such as Azure or IBM [18, 25]. A more consensual deﬁnition is to see a data lake as a central repository where data of all formats are stored without a strict schema [14, 16, 19]. This deﬁnition is based on two key characteristics of data lakes: data variety and the schema-on-read (or late binding) approach, which consists in deﬁning the data schema at analysis time [20]. However, the variety/schema-on-read deﬁnition provides little detail about the characteristics of a data lake. Thus, a more complete deﬁnition by Madera and Laurent views data lake as a logical view of all data sources and datasets in their raw format, accessible by data scientists or statisticians for knowledge extraction [18]. This deﬁnition is complemented by a list of key features: 1) data quality is provided by a set of metadata; 2) the lake is controlled by data gov- ernance policy tools; 3) usage of the lake is limited to statisticians and data scientists; 4) the lake integrates data of all types and formats; 5) the data lake has a logical and physical organization. 1 Introduction Since the beginning of the 21st century, the usages of organizations in decision- making processes have been disrupted by the availability of large amounts of data, i.e., big data. Mainly issued from social media and the Internet of things, big data bring about great opportunities for organizations, but also issues related to data volume, velocity and variety, which surpass the capabilities of traditional storage and data processing systems [20]. In this context, the concept of data lake [6] appears as a solution to big data heterogeneity problems. A data lake provides integrated data storage without predeﬁned schema [11]. In the absence of a data schema, an eﬀective metadata system becomes essential to make data queryable and thus prevent the lake from turning into a data swamp, i.e., an inexploitable data lake [1, 11, 26]. While the literature seems unanimous about the importance of the metadata system in a data lake, questions and uncertainties remain about its implementa- tion methodology. Several approaches help organize metadata, but most concern only structured and semi-structured data [8, 11, 17, 22]. Moreover, the eﬀective- ness of a metadata system is diﬃcult to measure because, to the best of our knowledge, there are no widely shared and accepted evaluation criteria. 2 P.N. Sawadogo et al. 2.2 Discussion and New Deﬁnition Some points in Madera and Laurent’s deﬁnition of data lakes [18] are debat- able. The authors indeed reserve the use of the lake to data specialists and, as a Metadata Systems for Data Lakes Metadata Systems for Data Lakes 3 consequence, exclude business experts for security reasons. Yet, in our opinion, it is entirely possible to allow controlled access to this type of users through a navigation or analysis platform. Moreover, we do not share the vision of the data lake as a logical view over data sources, since some data sources may be external to an organization, and therefore to the data lake. Dixon also speciﬁes that lake data come from data sources [6]. Including data sources into the lake may therefore be considered contrary to the spirit of data lakes. Finally, although quite complete, Madera and Laurent’s deﬁnition omits an essential property of data lakes: scalability [20]. Since a data lake is intended for big data storage and processing, it is indeed essential to address this issue. Thence, we amend Madera and Laurent’s deﬁnition to bring it in line with our vision and introduce scalability. Deﬁnition 1. A data lake is a scalable storage and analysis system for data of any type, retained in their native format and used mainly by data specialists (statisticians, data scientists or analysts) for knowledge extraction. Its charac- teristics include: 1) a metadata catalog that enforces data quality; 2) data gover- nance policies and tools; 3) accessibility to various kinds of users; 4) integration of any type of data; 5) a logical and physical organization; 6) scalability. 3.1 Expected Features Versioning also allows to support a branched evolution of data, especially in their schema [13]. Usage tracking (UT) records the interactions between users and the data lake. Interactions are generally operations of data creation, update and access. The integration of this information into the metadata system makes it possible to understand and explain possible inconsistencies in the data [3]. It can also be used to manage sensitive data, by detecting intrusions [26]. Usage tracking and data versioning are closely linked, because interactions lead in some cases to the creation of new versions or representations of the data. Thus, such features are often integrated together in a provenance tracking module [12, 13, 27]. Yet, we still consider that they remain diﬀerent features since they are not systematically proposed together [3, 5, 26]. 3.1 Expected Features We identify in the literature six main functionalities that should ideally be provided by the metadata system of a data lake. Semantic enrichment (SE), also called semantic annotation [11] or semantic proﬁling [2], consists in generating a description of the context of data, e.g., with tags, to make them more interpretable and understandable [27]. It is done using knowledge bases such as ontologies. Semantic annotation plays a key role in data exploitation, by summarizing the datasets contained in the lake so that they are more understandable to the user. It can also be used as a basis for identifying data links. For instance, data associated with the same tags can be considered linked. Data indexing (DI) consists in setting up a data structure to retrieve datasets based on speciﬁc characteristics (keywords or patterns). This requires the con- struction of forward or inverted indexes. Indexing makes it possible to optimize data querying in the lake through keyword ﬁltering. It is particularly useful for textual data management, but can also be used in a semi-structured or struc- tured data context [24]. Link generation and conservation (LG) is the process of detecting similarity relationships or integrating preexisting links between datasets. The integration of data links can be used to expand the range of possible analyses from the lake by recommending data related to those of interest to the user [17]. Data links P.N. Sawadogo et al. 4 can also be used to identify data clusters, i.e., groups where data are strongly linked to each other and signiﬁcantly diﬀerent from other data [9]. g y [ ] We deﬁne data polymorphism (DP) as storing multiple representations of the same data. Each representation corresponds to the initial data, modiﬁed or reformatted for a speciﬁc need. For example, a textual document can be represented without stopwords or as a bag of words. It is essential in the context of data lakes to at least partially structure unstructured data to allow their automated analysis [5]. Simultaneously storing several representations of the same data notably avoids repeating preprocessings and thus speeds up analyses. Data versioning (DV) refers to the ability of the metadata system to support data changes while conserving previous states. This ability is essential in data lakes, as it ensures the reproducibility of analyses and supports the detection and correction of possible errors or inconsistencies. 3.2 Comparison of Metadata Systems We consider in this comparison two types of metadata systems: metadata models and data lake implementations. Metadata models refer to conceptual sys- tems for organizing metadata. They have the advantage of being more detailed and more easily reproducible than data lake implementations, which lie at a more operational level. Data lake implementations focus on operation and function- ality, with little detail on the conceptual organization of metadata. Eventually, we include in this study systems (models or implementations) not explicitly as- sociated with the concept of data lake by their authors, but that may be used in a data lake context, e.g., the Ground metadata model [13]. [ ] Table 1 provides a synthetic comparison of 15 metadata systems for data lakes (and assimilated). It shows that the most complete systems in terms of functionality are the GOODS and CoreKG data lakes, with ﬁve out of six features available. These systems notably support polymorphism and data versioning. However, they are also black boxes providing little detail on the conceptual organization of metadata. Ground may therefore be preferred, since it is much more detailed and almost as complete (4/6). In terms of functionalities, we note an almost unanimous agreement on the relevance of semantic enrichment, with 12 out of 15 systems oﬀering this feature and, to a lesser extent, of data indexing (9/15) and data link generation (8/15). Metadata Systems for Data Lakes 5 Table 1: Features provided by data lake metadata systems System Type SE DI LG DP DV UT SPAR (Fauduet and Peyrard, 2010) [10] ♦♯ ✓ ✓ ✓ ✓ Alrehamy and Walker (2015) [1] ♦ ✓ ✓ Terrizzano et al. (2015) [27] ♦ ✓ ✓ ✓ ✓ Constance (Hai et al., 2016) [11] ♦ ✓ ✓ GEMMS (Quix et al., 2016) [22] ♦ ✓ CLAMS (Farid et al., 2016) [8] ♦ ✓ Suriarachchi and Plale (2016) [26] ♦ ✓ ✓ Singh et al. (2016) [24] ♦ ✓ ✓ ✓ ✓ Farrugia et al. (2016) [9] ♦ ✓ GOODS (Halevy et al., 2016) [12] ♦ ✓ ✓ ✓ ✓ ✓ CoreDB (Beheshti et al., 2017) [3] ♦ ✓ ✓ Ground (Hellerstein et al., 2017) [13] ♦♯ ✓ ✓ ✓ ✓ KAYAK (Maccioni and Torlone, 2018) [17] ♦ ✓ ✓ ✓ CoreKG (Beheshti et al., 2018) [4] ♦ ✓ ✓ ✓ ✓ ✓ Diamantini et al. 3.2 Comparison of Metadata Systems (2018) [5] ♦ ✓ ✓ ✓ ♦: Data lake implementation ♦: Metadata model ♯: Model or implementation assimilable to a data lake Table 1: Features provided by data lake metadata systems Table 1: Features provided by data lake metadata systems System Type SE DI LG DP DV UT SPAR (Fauduet and Peyrard, 2010) [10] ♦♯ ✓ ✓ ✓ ✓ Alrehamy and Walker (2015) [1] ♦ ✓ ✓ Terrizzano et al. (2015) [27] ♦ ✓ ✓ ✓ ✓ Constance (Hai et al., 2016) [11] ♦ ✓ ✓ GEMMS (Quix et al., 2016) [22] ♦ ✓ CLAMS (Farid et al., 2016) [8] ♦ ✓ Suriarachchi and Plale (2016) [26] ♦ ✓ ✓ Singh et al. (2016) [24] ♦ ✓ ✓ ✓ ✓ Farrugia et al. (2016) [9] ♦ ✓ GOODS (Halevy et al., 2016) [12] ♦ ✓ ✓ ✓ ✓ ✓ CoreDB (Beheshti et al., 2017) [3] ♦ ✓ ✓ Ground (Hellerstein et al., 2017) [13] ♦♯ ✓ ✓ ✓ ✓ KAYAK (Maccioni and Torlone, 2018) [17] ♦ ✓ ✓ ✓ CoreKG (Beheshti et al., 2018) [4] ♦ ✓ ✓ ✓ ✓ ✓ Diamantini et al. (2018) [5] ♦ ✓ ✓ ✓ ♦: Data lake implementation ♦: Metadata model ♯: Model or implementation assimilable to a data lake ♯: Model or implementation assimilable to a data lake On the other hand, other features are much less shared, especially data poly- morphism (4/15) and data versioning (3/15). In our opinion, this rarity does not indicate a lack of relevance, but rather implementation complexity. Such features are indeed mainly found in the most complete systems (GOODS, CoreKG and Ground) and can therefore be considered as advanced features. 4 Metadata Typology The comparison results from Section 3.2 show that no metadata system oﬀers all expected functionalities. To bridge this gap, we propose in the following a metadata model that supports all six key functionalities. Beforehand, we need a generic concept that represents any set of homogeneous data that the model can process. In the literature, we ﬁnd data units [22], entities [3], datasets [17] and objects [5]. We adopt objects, which seem more appropriate to represent a dataset in an abstract way. More precisely, an object may be a relational table or a physical ﬁle (spreadsheet document, XML or JSON document, text document, tweet collection, image, video, etc.). The deﬁnition of a metadata model for data lakes also involves identifying the metadata to be considered. To this end, we extend a medatata typology that categorizes metadata into intra-object, inter-object and global metadata [23] with new types of inter-object (relationships) and global (index, event logs) metadata. P.N. Sawadogo et al. 6 4.1 Intra-object Metadata This category refers to metadata associated with a given object. Properties provide a general description of an object, in the form of key-value pairs. Such metadata are usually obtained from the ﬁlesystem: object title, size, date of last modiﬁcation, access path, etc. Summaries and previews provide an overview of the content or structure of an object. They can take the form of a data schema in a structured or semi- structured data context, or a word cloud for textual data. Raw data in the lake are often modiﬁed through updates that result in the creation of new versions of the initial data, which can be considered as meta- data. Similarly, raw data (especially unstructured data) can be reformatted for a speciﬁc use, inducing the creation of new representations of an object. Semantic metadata are annotations that help understand the meaning of data. More concretely, they are descriptive tags, textual descriptions or business categories. Semantic metadata are often used for detecting object relationships. 4.2 Inter-object Metadata Inter-object metadata account for relationships between at least two objects. Objects groupings organize objects into collections, each object being able to belong simultaneously to several collections. Such groups can be automatically derived from semantic metadata such as tags and business categories. Some properties can also be used for generating groups, e.g., objects can be grouped w.r.t. format or language. Similarity links reﬂect the strength of the similarity between two objects. Unlike object groupings, similarity relationships refer to the intrinsic properties of objects, such as their content or structure. For example, it may be the common word rate between two textual documents, a measure of the compatibility of the schemas of two structured or semi-structured objects [17], or other common similarity measures. Parenthood relationships, which we add to our reference typology [23], reﬂect the fact that an object can be the result of joining several others. In such a case, there is a “parenthood” relationship between the combined objects and the resulting object, and a “co-parenthood” relationship between the merged objects. This type of relationship thus makes it possible to take advantage of the processing carried out in the data lake to identify objects that can be used together, in addition to maintaining traceability of the origin of the objects generated inside the lake. 4.3 Global Metadata Global metadata are data structures designed to provide a contextual layer to the lake’s data, to facilitate and optimize its analysis. Unlike intra and inter- object metadata, global metadata potentially concern the entire data lake. In 7 Metadata Systems for Data Lakes Metadata Systems for Data Lakes addition to the semantic resources identiﬁed in our reference typology [23], we propose two new types of global metadata. Semantic resources are essentially knowledge bases (ontologies, taxonomies, thesauri, dictionaries) used to generate other metadata and improve analyses. For example, a thesaurus can help extend a query by associating synonyms of the terms typed by the user. Similarly, a thesaurus can be used while generating object groupings, to merge collections from diﬀerent but equivalent tags. Semantic resources are generally coming from external sources. This is typi- cally the case for ontologies that are provided by knowledge bases on the Inter- net. However, in some cases, semantic resources can be created and customized speciﬁcally for the management and analysis of lake data. For instance, a busi- ness ontology can thus be used to deﬁne abstract tags allowing to group together several equivalent or close tags during analysis. Indexes are data structures that help ﬁnd an object quickly. They establish (or measure) the correspondence between characteristics such as keywords, patterns or colors, with the objects contained in the data lake. Indexes can be simple (textual indexing) or more complex (e.g., on images or sound content). They are mainly used to search for data in the lake. Logs are used to track user interactions with the data lake. This involves the sequential recording of events such as users logging in, viewing or modifying an object. Such metadata help analyze data lake usage by identifying the most consulted objects or studying user behaviour. From the above typology, we can now formally deﬁne a data lake. Deﬁnition 2. A data lake is a pair DL = ⟨D, M⟩, where D is a set of raw data and M a set of metadata describing the objects of D. Objects in D can take the form of structured (relational database tables, CSV ﬁles, etc.), semi- structured (JSON, XML, YAML documents, etc.) and unstructured data (im- ages, textual documents, videos, etc.). Metadata are subdivided into three com- ponents: M = ⟨Mintra, Minter, Mglob⟩, where Mintra is the set of intra-object metadata, Minter the set of inter-object metadata and Mglob the set of global metadata. 4.4 Formal Deﬁnition of a Data Lake From the above typology, we can now formally deﬁne a data lake. 5 Metadata Model MEDAL adopts a logical metadata representation based on the hypergraph, nested graph and attributed graph notions. We represent an object by an hy- pernode containing various elements (versions and representations, properties, etc.). Hypernodes can be linked together (similarity, parenthood, etc.). P.N. Sawadogo et al. 8 5.1 Intra-object Metadata Each hypernode contains representations, reﬂecting the fact that data as- sociated with an object can be presented in diﬀerent ways. There is at least one representation per hypernode, corresponding to raw data. Other representations all derive from this initial representation. Each representation corresponds to a node bearing attributes, simple or complex. These are the properties of the rep- resentation. A representation can be associated with an object actually stored in the lake or be a view calculated on demand. The transition from one representation to another is done via a transfor- mation. It takes the form of a directed edge connecting two representation nodes. This edge also bears attributes, which are the properties describing the transformation process from the ﬁrst representation to the second (full script or description, in case of manual transformation). A hypernode can also contain versions, which are used to manage the evo- lution of lake data over time. We also associate versions with nodes bearing attributes. The creation of a new version node is not necessarily systematic at the slightest change. Depending on the nature and frequency of data evolution, it is possible to implement various strategies, such as those used to manage slowly changing dimensions in data warehouses [15]. The creation of a new version is done via an update similar to a transformation, since it is also translated by a directed edge and possesses some attributes. Finally, a hypernode also bears attributes such as the origin of the object or aggregates of the attributes of the representations and versions it contains (num- ber of versions, representations, total size, etc.). Thus, a hypernode contains a tree whose nodes are representations or versions and directed edges are transfor- mations or updates. One representation (resp. version) is derived from another by a transformation (resp. update). A version can lead to a representation via a transformation, but a version cannot be derived from a representation. Thus, the root of the tree is the initial raw representation of the hypernode and each version has its own subtree of representations. Deﬁnition 3. Let N be a set of nodes. 5.1 Intra-object Metadata The set of intra-object metadata Mintra is the set of hypernodes such that ∀h ∈Mintra, h = ⟨N, E⟩, where N ⊂N is the set of nodes (representations and versions) carrying attributes of h and E = {r(transformation \| update) ∈N × N} is the set of edges (transformations and updates) carrying attributes of h. Deﬁnition 3. Let N be a set of nodes. The set of intra-object metadata Mintra is the set of hypernodes such that ∀h ∈Mintra, h = ⟨N, E⟩, where N ⊂N is the set of nodes (representations and versions) carrying attributes of h and E = {r(transformation \| update) ∈N × N} is the set of edges (transformations and updates) carrying attributes of h. Let us illustrate these notions with an example (Figure 1). Imagine a com- pany selling various products. Information on these products (name, unit price, description, etc.) is stored in the lake as an XML ﬁle. A hypernode describes this dataset and has a version node that corresponds to the initially ingested XML ﬁle. To assist in querying product information, a user decides to extract the XML ﬁle’s schema. This generates a new representation. Now suppose that the price of some products changes and new products are added to the catalogue. This change in data generates a new version, linked to the ﬁrst version by an 9 Metadata Systems for Data Lakes Metadata Systems for Data Lakes update. Finally, if the user wants to obtain the schema of the most recent data, this creates a new representation coming from the second version. Fig. 2: Sample interconnected hypernodes Fig. 1: Sample hypernode and its representation tree Fig. 2: Sample interconnected hypernodes Fig. 1: Sample hypernode and its representation tree Fig. 1: Sample hypernode and its representation tree Fig. 1: Sample hypernode and its representation tree Fig. 2: Sample interconnected hypernodes Deﬁnition 4. The set of inter-object metadata Minter is deﬁned by three pairs ⟨H, Eg⟩, ⟨H′, Es⟩and ⟨H′′, Ep⟩, where H ⊂Mintra, H′ ⊂Mintra and H′′ ⊂ Mintra are sets of hypernodes carrying attributes; Eg = {Eparam g \| Eparam g : H →P(H)} is the set of functions grouping hypernodes in collections w.r.t. a given parameter (often an attribute); Es = {s \| s ∈H′ × H′} is the set of edges (similarity links) carrying attributes; and Ep = {(h1, ..., hn, hchild) \| (h1, ..., hn, hchild) ∈ (H′′)n+1} is the set of parenthood relationships, with (h1, ..., hn) being the parent hypernodes (n ≥2) and hchild the child hypernode. P.N. Sawadogo et al. P.N. Sawadogo et al. Let us pursue the example of Section 5.1 by adding other hypernodes: tweets related to the company and a commercial video of the products. In a grouping on the origin of data, the tweet hypernode is alone in the “external source” col- lection, while the other two are in the “internal source” collection. In a second grouping on the format of the initial version, the video hypernode is alone in the “unstructured” collection, while the other two hypernodes are in the “semi- structured” collection. Collections are represented by dotted rectangles in Fig- ure 2 (attributes of the hypernodes are not represented for simplicity). 5.3 Global Metadata Global metadata are speciﬁc elements that are managed diﬀerently from other metadata. They “gravitate” around hypernodes and are exploited as needed, i.e., almost systematically, especially logs and indexes. We consider that seman- tic resources are stored in nodes, while indexes and event logs are rather physical structures and are highly dependent on the technology used to implement the data lake and the metadata system. 5.2 Inter-object Metadata An object grouping is modeled by a set of non-oriented hyperedges, i.e., edges that can link more than two (hyper)nodes. Each hyperedge corresponds to a collection of objects. If grouping is performed on a hypernode attribute, a hypernode belongs to the hyperarc that corresponds to its value for the attribute. Thus, there are as many hyperarcs as there are distinct values of the considered attribute. Note that not all attributes are necessarily used in groupings and that groupings can be made on other elements but attributes. A similarity link between two hypernodes is represented by a non-oriented edge with attributes: value of the similarity metric, type of metric used, date of the metric, etc. Two hypernodes connected by a similarity link must be compa- rable, i.e., they must each contain a representation that can be compared to the other with a similarity measure. A hypernode can be derived from other hypernodes through a parenthood link. To translate this relationship, we use an oriented hyperedge: all the “parent” hypernodes and the “child” hypernode are connected by this oriented hyperedge toward the child hypernode. This hyperedge also bears descriptive attributes. Deﬁnition 4. The set of inter-object metadata Minter is deﬁned by three pairs ⟨H, Eg⟩, ⟨H′, Es⟩and ⟨H′′, Ep⟩, where H ⊂Mintra, H′ ⊂Mintra and H′′ ⊂ Mintra are sets of hypernodes carrying attributes; Eg = {Eparam g \| Eparam g : H →P(H)} is the set of functions grouping hypernodes in collections w.r.t. a given parameter (often an attribute); Es = {s \| s ∈H′ × H′} is the set of edges (similarity links) carrying attributes; and Ep = {(h1, ..., hn, hchild) \| (h1, ..., hn, hchild) ∈ (H′′)n+1} is the set of parenthood relationships, with (h1, ..., hn) being the parent hypernodes (n ≥2) and hchild the child hypernode. 10 6 Conclusion After an overview of the deﬁnitions of a data lake from the literature, we propose in this paper our own deﬁnition of this concept. Then, we identify the six key features that the metadata system of a data lake must provide to be as robust as possible in addressing the big data issues and the schema-on-read approach. Comparing existing metadata systems, we show that some succeed in providing most features, but none oﬀers them all. Hence, we propose a new metadata model, MEDAL, based on the notion of object and a typology of metadata in three categories: intra-object, inter-object and global metadata. MEDAL adopts a graph-based organization. An object is represented by a hypernode containing nodes that correspond to the versions and representations of an object. Transformation and update operations are modeled by oriented edges linking the nodes. Hypernodes can be linked in several ways: edges to model similarity links and hyperarcs to translate parenthood relationships and object groupings. Finally, global resources are also present, in the form of knowledge bases, indexes or event logs. Thanks to all these elements, MEDAL supports all six key features we have identiﬁed, making it the most complete metadata model to the best of our knowl- edge. However, MEDAL is not implemented yet. It is the objective of future work in which we shall propose an application of our metadata model in a context of structured, semi-structured and unstructured data. This implementation will allow us to evaluate MEDAL in more detail, in particular by comparing it with other existing systems. 11 Metadata Systems for Data Lakes Metadata Systems for Data Lakes Acknowledgments Part of the research presented in this article is funded by the Auvergne- Rhône-Alpes Region, as part of the AURA-PMI project that ﬁnances Pegdwendé Nicolas Sawadogo’s PhD thesis. References 1. Alrehamy, H., Walker, C.: Personal Data Lake With Data Gravity Pull. In: BDCloud 2015, Dalian, china, IEEE Computer Society Washington, vol. 88, pp. 160-167 (2015). doi:10.1109/BDCloud.2015.62 2. 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https://openalex.org/W2267243353	https://europepmc.org/articles/pmc4716139?pdf=render	English	null	Antisaccadic Eye Movements Are Correlated with Corpus Callosum White Matter Mean Diffusivity, Stroop Performance, and Symptom Burden in Mild Traumatic Brain Injury and Concussion	Frontiers in neurology	2,016	cc-by	9,625	A Windsor Kwan-Chun Ting1,2 , Tom A. Schweizer2,3 , Jane Topolovec-Vranic2,4 and Michael D. Cusimano1,2,3* 1 Injury Prevention Research Office, Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada, 2 Neuroscience Research Program, St. Michael’s Hospital, Toronto, ON, Canada, 3 Department of Surgery, Division of Neurosurgery, St. Michael’s Hospital, Toronto, ON, Canada, 4 Department of Occupational Science and Occupational Therapy, University of Toronto, Toronto, ON, Canada Original Research published: 18 January 2016 doi: 10.3389/fneur.2015.00271 Keywords: antisaccade, concussion, mild traumatic brain injury, diffusion tensor imaging, executive function, corpus callosum, symptom burden Citation: Ting WK-C, Schweizer TA, Topolovec-Vranic J and Cusimano MD (2016) Antisaccadic Eye Movements Are Correlated with Corpus Callosum White Matter Mean Diffusivity, Stroop Performance, and Symptom Burden in Mild Traumatic Brain Injury and Concussion. Front. Neurol. 6:271. doi: 10.3389/fneur.2015.00271 Antisaccades are thought to involve higher level inputs from neural centers involved in rapid eye movement inhibition and control. Previous work has demonstrated that performance on the antisaccade task can help in the assessment of injury in acute and/ or chronic mild traumatic brain injury (mTBI). In this exploratory study, we performed cross-sectional and longitudinal comparisons of rapid eye movement, followed by cor- relations of antisaccade performance with assessments of symptom burden, diffusion tensor imaging, and a neuropsychological test of response inhibition. Significant deficits in antisaccade median latency, F(2, 31) = 3.65, p = 0.04 and prosaccade error mean duration, F(2, 31) = 3.63, p = 0.04 were found between patient groups and controls: the former was correlated with loss of white matter integrity in the splenium of the corpus callosum in acute mTBI, rho = 0.90, p = 0.0005. Furthermore, increased antisaccade median latency was also correlated with poor performance on an executive functioning task, r2 = 0.439, p = 0.03, and greater symptom burden, r2 = 0.480, p = 0.02 in the acute mTBI patients. Our preliminary research suggests that the antisaccade task could be useful as a neurological marker for mTBI and concussion, but more work is required. Antisaccades are thought to involve higher level inputs from neural centers involved in rapid eye movement inhibition and control. Previous work has demonstrated that performance on the antisaccade task can help in the assessment of injury in acute and/ or chronic mild traumatic brain injury (mTBI). In this exploratory study, we performed cross-sectional and longitudinal comparisons of rapid eye movement, followed by cor- relations of antisaccade performance with assessments of symptom burden, diffusion tensor imaging, and a neuropsychological test of response inhibition. Significant deficits in antisaccade median latency, F(2, 31) = 3.65, p = 0.04 and prosaccade error mean duration, F(2, 31) = 3.63, p = 0.04 were found between patient groups and controls: the former was correlated with loss of white matter integrity in the splenium of the corpus callosum in acute mTBI, rho = 0.90, p = 0.0005. Furthermore, increased antisaccade median latency was also correlated with poor performance on an executive functioning task, r2 = 0.439, p = 0.03, and greater symptom burden, r2 = 0.480, p = 0.02 in the acute mTBI patients. Reviewed by: Reviewed by: Eric Peter Thelin, Karolinska Institutet, Sweden Eugene Golanov, The Houston Methodist Hospital Research Institute, USA Reviewed by: Eric Peter Thelin, Karolinska Institutet, Sweden Eugene Golanov, The Houston Methodist Hospital Research Institute, USA *Correspondence: Michael D. Cusimano injuryprevention@smh.ca Specialty section: This article was submitted to Neurotrauma, a section of the journal Frontiers in Neurology Received: 09 October 2015 Accepted: 22 December 2015 Published: 18 January 2016 Specialty section: This article was submitted to Neurotrauma, a section of the journal Frontiers in Neurology Received: 09 October 2015 Accepted: 22 December 2015 Published: 18 January 2016 Our preliminary research suggests that the antisaccade task could be useful as a neurological marker for mTBI and concussion, but more work is required. Edited by: Firas H. Kobeissy, University of Florida, USA Edited by: Firas H. Kobeissy, University of Florida, USA INTRODUCTION The Stroop color and word test is a common measure of a specific type of executive functioning known as interference control. For mTBI patients, traumatic axonal injury is thought to be a result of damage to the neurofilament organization and axo- lemma (4). Diffusion tensor imaging (DTI) findings after mTBI (4) have found that the frontal association white matter tracts, namely, the anterior corpus callosum, superior longitudinal fasciculus, anterior corona radiata, and uncinate fasciculus were common areas of white matter damage in mTBI. Significant effect sizes of FA reductions and MD increases have been found across studies in a meta-analysis of the corpus callosum (5). The corpus callosum, in particular, is important for many cognitive functions; it is involved in bilateral information transfer and coordination between the prefrontal cortices (6). Croall et al. conducted a longitudinal study in mild/moderate TBI patients who were scanned acutely and returned for a chronic assessment, correlating structural integrity with neuropsychological perfor- mance, and found significant changes in fractional anisotropy and mean diffusivity in acute mTBI patients (on average 6 days post-mTBI) compared to controls in the ascending and posterior corpus callosum, respectively (7). However, this study did not investigate inhibitory control processes. Kinnunen et al. found significantly lower FA in the mTBI compared to control groups in the corpus callosum and several other areas (8). MD was elevated in the mTBI groups compared to the control groups in similar areas, including the internal and external capsule. The authors of this study did not find significant correlations between executive functioning and FA, but there was a correlation between higher mean diffusivity in the white matter of the superior temporal areas and worse performance on executive functioning tasks. To our knowledge, there does not seem to be any studies directly correlating a subset of executive functioning, namely interference control performance to FA/MD changes in both acute mTBI and persistent post-traumatic symptom (PTS) patients at the same time. l Research has suggested that mTBI patients exhibit deficits in saccadic generation and difficulty inhibiting a planned saccadic eye movement within 2 days of injury (18). Antisaccade latency returns to normal after 1 week post-mTBI (19, 20), but accuracy FIGURE 1 \| An illustrated schematic of the antisaccade task. Abbreviations: PS = prosaccade; AS = antisaccade. INTRODUCTION Few objective assessment tools exist to aid the clinician in the assessment of mild traumatic brain injury (mTBI) and concussion from both an emergency standpoint and in clinic follow-up. Furthermore, little is known about the relationship of symptom load to measures like white matter integrity and other objective measures of brain function. Comprehensive scales have been developed that sys- tematically seek to identify the collective cognitive, emotional, and somatic symptoms after mTBI January 2016 \| Volume 6 \| Article 271 Frontiers in Neurology \| www.frontiersin.org 1 Ting et al. Antisaccades in mTBI Recent work has advocated the use of rapid eye movements in the assessment of TBI (9). Generating a saccadic eye movement requires coordinated action between many control centers, and the neurophysiology involved has been well defined after dec- ades of study (10–12). Completing the antisaccade task requires making a rapid eye movement to the mirror-opposite location to where a saccade target appears, relative to a central fixation point as soon as possible (Figure 1). The cognitive demands of the color-word component of the Stroop color and word test are similar to those of the antisaccade. Participants must give a verbal response indicating the color ink in which the word is printed, but not what the word says. The word stimulus interferes with the correct verbal response, in much the same way that the prosaccade error target interferes with the correct antisaccade response. Performance on these two tasks should be correlated to some extent. Successful completion of interference tasks requires well-functioning response inhibition control systems from the higher cognitive systems in the frontal and parietal areas of the brain, which may be common to both neuropsychological and antisaccade performance. For the antisaccade task specifically (13), it is thought that the higher cortical regions in the frontal (14) and parietal lobes, namely the frontal eye fields (10, 15), supplementary eye fields, dorsolateral prefrontal cortex (10, 15, 16), posterior parietal cortex, and parietal eye field (10) are responsible for inhibiting the reflexive prosaccade error (17).i of all etiologies, but an objective test would be an advantage for assessment. Executive functioning deficits after TBI encompass domains such as attention (1), working memory (2, 3), response inhibition, and other cognitive subsystems involved in the control and regulation of behavior, due to frontal lobe susceptibility to injury. Inclusion and Exclusion Criteriai We used the World Health Organization definition of mTBI (27) (see Supplementary Methods in Supplementary Material) with some qualifications. PTS patients sustained mTBI at least 3 months prior to testing and reported PTS 3 months or longer after their injury. We included concussions within our group of mTBI patients. We chose to focus on mTBI and not purely concussion, as positive neuroimaging should not preclude antisaccade measurement if this tool were to be eventually used in the clinic environment. Inclusion criteria for the study also included age greater than or equal to 16 years, able to provide informed consent and adequate verbal English language skills. All participants required a structured history to rule out horizontal diplopia, severe loss of visual acuity and exclusion conditions noted below. The control group was matched to the patient groups based on age (±5 years), sex, and years of educa- tion (±5 years). Previous work has correlated antisaccade performance with a cluster of executive functioning tests and found a significant negative correlation between antisaccade latency and a pooled executive functioning score for a sample of TBI subjects of all severities – only considering the mTBI patients, they found trends (23). Also important is their use of pooled scores for their cor- relational analyses, which may have masked direct correlations between specific executive functioning tests and antisaccade performance. Symptom assessment, neuroimaging, and neuropsychologi- cal assessment are helpful but insufficient for diagnosing mTBI. Rapid eye movements can be measured after injury, either alone or as an adjunct to the measures previously discussed. Although we have reviewed several eye movements that may be useful for more severe disorders of consciousness inflicted by moderate to severe TBI (26), more information is needed to determine whether antisaccades are related to common assessment metrics in mTBI, and this is a gap in the current literature.i Acute mTBI, PTS, and healthy control participants were excluded if they were medically unstable or intoxicated (by drugs or alcohol) at the time of recruitment, had medical comorbidities of multiple sclerosis, prior incisional brain surgery, prior brain irradiation, prior hydrocephalus, a history of stroke, early/ alcohol-related dementia, neurodegenerative disease including Alzheimer’s disease, and uncontrolled diabetes. In terms of optic pathology, participants were excluded if they had a his- tory of eye disease (amblyopia or glaucoma) or orbital trauma due to the present injury. INTRODUCTION Functional hyperactivation was observed in the cerebellum and V5/V1 cortical areas, but not the frontal areas during the antisaccade task, contrary to their initial predictions. Antisaccade performance in TBI patients with chronic symptoms several years after injury has been investigated (23). Kraus et al. found significant increases in the number of prosaccade errors (saccade toward stimulus) and latency (in the overlap condition) in the antisaccade task in TBI patients compared with healthy controls but no differences between TBI patients of different severities (mTBI versus moderate/severe TBI). This suggested that there was low specificity of the antisaccade task in differenti- ating between mTBI and more severe head injuries in the chronic stages, but their use of electrooculography limits the precision of antisaccade measurement compared to the chip-based measure- ment methods. Frontiers in Neurology \| www.frontiersin.org INTRODUCTION It is important to understand the relationships of symptom burden and executive functioning to antisaccade performance in acute mTBI and PTS, but antisaccades’ relationship to DTI has not been investigated. Specifically, we do not know whether the major white matter pathways damaged in mTBI (corpus callosum, superior longitudinal fasciculus, uncinate fasciculus, and anterior corona radiata) are necessarily com- mon to the white matter pathways necessary for antisaccade performance (projections from the dorsolateral prefrontal cortex, anterior cingulate cortex to the superior colliculus) in mTBI or are related to their function in some way. One focus of this study will be identifying correlations with significant dif- ferences in saccade performance variables. There is no literature investigating whether there is any correlation between acute structural integrity (measured by DTI) and performance on the antisaccade. FIGURE 1 \| An illustrated schematic of the antisaccade task. Abbreviations: PS = prosaccade; AS = antisaccade. January 2016 \| Volume 6 \| Article 271 Frontiers in Neurology \| www.frontiersin.org 2 Ting et al. Antisaccades in mTBI mTBI, it should improve over time, and it should be worse in PTS individuals. Our secondary aim is to examine neuroimaging of these patients, hypothesizing there would be lower fractional anisotropy and increased mean diffusivity in acute and PTS patients’ scans compared to control patients’ scans in white mat- ter regions of interest (ROIs) due to diffuse axonal injury after mTBI. Specific areas of interest included the corpus callosum and all its component areas (genu, body, and splenium), uncinate fasciculus, superior longitudinal fasciculus, internal capsule (part of the anterior corona radiata). Importantly, we hypothesized that antisaccade performance would be correlated with FA and MD in these white matter areas. In our final aim, we hypothesized that patients with poor antisaccade performance would also show poor performance in neuropsychological tests of response inhi- bition since antisaccades involve inhibitory processes mediated by the frontal and prefrontal cortices. Further, we hypothesized that measures of symptom burden improve after mTBI, and the former would be correlated to antisaccade performance. differences have been documented up to few months postinjury (20). Some studies have shown that antisaccade accuracy appears to be impaired in the acute stages, with patients making more errors than controls (19–21); others indicate better performance (22). Only one study was found to relate acute antisaccade perfor- mance to neuroimaging, and the focus was mainly on functional performance (21). MATERIALS AND METHODS All participants provided written informed consent before being prospectively enrolled in the study. We screened patients who registered in the Emergency Department and Outpatient Head Injury Clinic at St. Michael’s Hospital (SMH). Healthy controls were recruited from word of mouth to the friends and relatives of the participants. This work was part of a larger study, which was approved by the Research Ethics Board at SMH. Studies to date in PTS patients have not correlated antisac- cade performance with gold standard structural neuroimaging. A useful antisaccade marker should be furthermore related to the symptoms that patients are experiencing. Heitger et al. correlated saccade deficiencies in the acute phase with quality of life in the PTS phase (24). Importantly, oculomotor performance (including antisaccade tasks as well as other saccade test results) 1 week after injury was significantly predictive of greater symptom burden on the Rivermead Post-Concussive Symptoms Questionnaire (RPQ) and certain domain scores of the Short-Form-36 questionnaire at 3 and 6 months following mTBI. This study expanded upon Heitger et al.’s earlier results but performed correlations within the same visit, similar to their later work (25) but with a greater number of antisaccade trials per participant. Rapid Eye Movement Measurement Rapid Eye Movement Measurement We used a portable research device, the Saccadometer Advanced from Ober Consulting. The saccade and fixation targets were projected onto the wall with three red low-power laser lights. The lights were projected onto a matte white wall 1.5–3.5 m away from the participant’s eyes. All saccadometry was conducted under darkened conditions as per a published pilot study by our research group (28). The saccadometer automatically adjusted for minor deviations in distance between the participant’s forehead and the distance to the wall so long as it was within the range defined above. It also automatically marked and excluded inter- vening blinks. Diffusion MR images were preprocessed and analyzed using the Functional Magnetic Resonance of the Brain (FMRIB) FSL Toolbox (FMRIB Software Library, Release 5.0, 2012, University of Oxford, England on OS X 10.9) (29). Raw NIfTI images were inspected manually for any gross acquisition abnormalities. Scans that developed excessive gross distortions due to severe head motion or dental braces during acquisition, or incomplete scans were excluded from further analyses. Images were eddy-corrected using the EDDY program in the FSL toolbox to reduce distortion effects and skull-stripped using the Brain Extraction Tool (BET) from the FSL toolbox to exclude the non-brain voxels. Using FSLVIEW, each scan was manually checked for eddy correction and brain extraction errors. Binary masks were manually drawn for each scan using FSLVIEW and used for diffusion tensor fitting using DTIFIT. Parameters included the original eddy-corrected diffusion weighted image, the manually drawn binary mask, and the eigenvalue/eigenvector files, respectively. All participants were guided through calibration and tutorial sessions prior to the antisaccade task. Participants who were myopic or hyperopic were asked to wear contacts to ensure corrected visual acuity for the saccade tests or were examined to ensure they could see all three saccadic targets clearly and with- out horizontal diplopia before proceeding. The antisaccade task was preprogramed into the saccadometer by the manufacturer. Participants were shown a red center light, after which it jumped either to the left or to the right (fixed probability 50%) with 10° horizontal displacement. There was a fixed foreperiod of 1000 ms and a random foreperiod with a maximum of 1000 ms. They were instructed to look at the center light. When it jumped to the left or to the right, participants were instructed to look in the mirror-opposite location as accurately and quickly as they could. Inclusion and Exclusion Criteriai Participants were excluded from the saccadometry analysis if they were taking medications known to Our primary aim is to confirm whether components of the antisaccade performance are affected by acute mTBI and those with PTS when compared to controls, hypothesizing that anti- saccade performance should be most affected early after acute January 2016 \| Volume 6 \| Article 271 3 Ting et al. Antisaccades in mTBI interfere with saccadic eye movement function (benzodiazepines and antipsychotics). interfere with saccadic eye movement function (benzodiazepines and antipsychotics). Raw saccadometry data were transferred to a computer with LatencyMeter Software version 5.2. The software and integrated hardware automatically accounted for eye blinks and head move- ment during its preprocessing. Saccades with latencies <50 ms (anticipatory saccades) and >600 ms (increased latency due to inattention/possible attempt at malingering) were excluded from the analysis, forming the lower and upper bounds of antisaccade latency, respectively. Saccade trials falling within these bounds were defined as accepted. We attempted to make a more homoge- neous and reliable subset of trials using this method.hi Participant Assessments Acute mTBI patients were asked to come in for two visits to SMH: the first visit within a week of injury, and the second visit 2–4 weeks after the injury. Patients with PTS recruited from the head injury clinic were invited to SMH once to participate in all study activi- ties. Healthy control participants who had no history of TBI were recruited and invited to come into SMH twice, 2–4 weeks apart. We attempted to minimize the latency between injury and the first visit. During the first visit, the participant completed an antisaccade task for 100 trials, a series of neuropsychological tests and the MRI scan if scheduling allowed. Prior to this, we obtained demographics information and injury characteristics through taking a brief history. A second assessment was carried out between 2 and 4 weeks after the participant’s mTBI. During the second assessment, we carried out the same tests as we did in the first assessment to assess recovery of function after mTBI on both neuropsychological testing and antisaccade performance. PTS participants conducted all neurological and neuropsycho- logical tests, as well as the MRI on one visit. The antisaccade directional accuracy was operationally defined by the number of accepted antisaccades in the correct (anti-) direction for each session. After individual saccadic trials were screened based on the criteria above, intraparticipant median latencies were calculated across trials for each participant at each visit. The interparticipant means of these median values were used for statistical comparisons between groups. An analogous set of dependent variables were calculated for incorrect prosac- cade errors, which fit within the defined latency range for all the three groups across all the visits. January 2016 \| Volume 6 \| Article 271 Neuroimage Acquisition and Processing Neuroimage Acquisition and Processing We used a 2.4 mm × 2.4 mm × 3.0 mm voxel interleaved echo- planar acquisition, on a 3.0-T Siemens Skyra Magnetom (204 coil elements), with 12 non-collinear diffusion directions and one b1 = 0 reference scan at 3.0 mm slice thickness. The b2 fac- tor = 1000 s/mm2. For acute patients, the majority of the MRI scans were conducted on the second visit (n = 9) so the neuroim- age correlational analyses were conducted using data from this visit. For control participants, the majority of MRI scans were also conducted on the second visit (n = 7) to match the acute patients.f Frontiers in Neurology \| www.frontiersin.org Statistical Analyses Cross-sectional antisaccade and neuropsychological testing data were analyzed using analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) in post hoc comparisons. Longitudinal antisaccade data were analyzed using paired t-tests. Neuroimaging data were analyzed using Wilcoxon rank-sum and Welch’s t-tests depending on the data distribution. Pearson corre- lations were used for parametric data and Spearman correlations for non-parametric data. Statistical analyses were conducted in SAS 9.4 (SAS Institute, Inc., Cary, NC, USA) and GNU R 3.0.3 for Windows (R-Core-Team, 2014). Antisaccade Latency and Error Duration Is Detrimentally Affected after Injury y j y Saccadometry data are in Table 2, and more details are in Supplementary Tables S2 and S3 in Supplementary Material. There was a significant omnibus difference on median antisaccade latency, F(2, 31) = 3.65, p = 0.04. Post hoc LSD comparisons sug- gested that acute mTBI participants [mean = 278.36 (SD = 28.8)] and PTS participants [272.86 (26.1)] had greater latency than the control group [241.17 (45.2)]. Figure 2A illustrates this result. There was a significant overall difference in mean error duration, F(2, 31) = 3.63, p = 0.038. Post hoc LSD comparisons suggested that acute mTBI participants [54.07 (11.3)] had greater error duration than the PTS group [45.96 (3.99)] and the control group [45.73 (9.10)]. Figure 2B illustrates this finding. Cross-sectional comparisons on the first visit between acute mTBI, PTS, and control groups were not significant for number of accepted antisaccades, antisaccade latency, mean duration, mean amplitude, and mean peak velocity. There were no sig- nificant differences within the acute participants between the first and second visits on all antisaccade performance metrics. In healthy controls, the error median latency was significantly greater in the first visit [189.31 (40.18)] compared to the second visit [168.38 (24.59)] [t(7) = 3.155, p = 0.016]. Other antisaccade performance metrics were not significant between the two visits of control participants. Symptom Burden and Executive Function Assessment Participants completed the RPQ and the symptom assessment scale of the Sport Concussion Assessment Tool Version 3 (SCAT3). Participants also completed the Center for Epidemiological Studies – Depression (CES-D) scale to assess affective state. To measure executive function, a core set of tests within a larger bat- tery was used, including the Stroop color and word test, the trail making test, and the phonemic fluency score from the Montreal Cognitive Assessment (MoCA), English Version 3. The Stroop color and word test was administered with modified instructions from Golden (1945), reviewed by Dr. Grant Killian (31). Rapid Eye Movement Measurement The waiting and trial break time for each saccade was 2000 and 1000 ms, respectively. We performed the gap antisaccade task, in which the center light turned off before presentation of the antisaccade target. Each participant demonstrated understand- ing of the expected antisaccade task before recording of the data started. Nine acute mTBI participants returned for their second visit, but antisaccade data could not be collected for one partici- pant (n = 8). i Four scans could not be included in the DTI analysis. Two PTS participants were excluded due to severe distortion during acquisition of the magnetic resonance image. Two control par- ticipants were not included in the analysis because the accom- panying eigenvector and eigenvalue files could not be generated from the accompanying DTI scan and because of an adverse event, respectively. We used tract-based spatial statistics (TBSS) to generate the fractional anisotropy and mean diffusivity images necessary for further analysis and calculation of the ROI-based January 2016 \| Volume 6 \| Article 271 Frontiers in Neurology \| www.frontiersin.org 4 Ting et al. Antisaccades in mTBI values. We defined white matter tracts from the Johns Hopkins University (JHU) DTI Atlas (30). ROI-based mean FA and MD values were then used in the further statistical comparisons. Mean diffusivity maps were generated in a similar fashion to fractional anisotropy, but the final processing step was completed using a custom TBSS script, which projected the mean diffusiv- ity maps onto the original fractional anisotropy data in order to generate a skeletonized mean diffusivity map. MD ROI values were calculated in a similar fashion using the binarized templates from the JHU Atlas. This process was completed separately for an acute versus control comparison and a PTS versus control comparison. head injury, and about half had comorbid medical conditions, such as bone, cardiovascular, respiratory, or renal disease. About 64% (n = 7) of the acute group needed corrected vision due to myopia or hyperopia. The PTS group had their single visit a median of 8.2 months after their mTBI. A third of the PTS patients endorsed a history of previous concussion or head injury; about half (n = 8) had comorbid medical conditions. Diffusion Tensor Imaging Identified Correlations with Antisaccadesii A significant positive relationship was identified between the mean diffusivity in the splenium of the corpus callosum and the median antisaccade latency at the second visit for acute mTBI patients [Rho (8) = 0.9048, p = 0.00045] (Figure 3A). There was a significantly greater mean diffusivity in the acute (median, Mdn = 0.000805) group compared to the control group (Mdn = 0.000766) in the splenium of the corpus callosum (p = 0.03112). There was a significantly lower mean diffusivity in the PTS (M = 0.000743) group compared to the control group RESULTS Participant Characteristics Frontiers in Neurology \| www.frontiersin.org Participant Characteristics Mean (SD) Acute mTBI visit 1 PTS visit 1 Healthy control visit 1 Results N 11 14 9 Antisaccade number accepted 51.9 (25.3) 55 (21.6) 72.2 (26.6) F(2, 31) = 2.01, p = 0.15 Antisaccade median latency (ms) 278.36 (28.8) 272.86 (26.1) 241.17 (45.2) F(2, 31) = 3.65, p = 0.04 Post hoc: acute > control (confidence limits 7.05, 37.20), PTS > control (−60.34, −3.04) Antisaccade mean duration (ms) 85.12 (36.0) 68.33 (32.5) 63.45 (17.2) F(2, 31) = 1.46, p = 0.25 Antisaccade mean amplitude (°) 15.48 (5.98) 13.04 (6.14) 12.58 (5.36) F(2, 31) = 0.75, p = 0.48 Antisaccade mean peak velocity (°/s) 387.46 (154.38) 440.92 (259.76) 437.61 (143.88) F(2, 31) = 0.25, p = 0.78 Error median latency (ms) 178.64 (1.36) 182.29 (28.9) 193.17 (39.3) F(2, 31) = 0.69, p = 0.51 Error mean duration (ms) 54.07 (11.3) 45.96 (3.99) 45.73 (9.10) F(2, 31) = 3.63, p = 0.038 Post hoc: acute > PTS (1.29, 14.9), acute > control (0.73, 15.6) Error mean amplitude (°) 9.92 (1.47) 9.36 (2.09) 8.41 (2.05) F(2, 31) = 1.58, p = 0.2214 Error mean peak velocity (°/s) 432.53 (75.95) 470.90 (249.19) 406.85 (101.53) F(2, 31) = 0.39, p = 0.6808 FIGURE 2 \| Antisaccade differences between study groups. Graphs of (A) antisaccade latency performance and (B) error duration across participant groups. Acute mTBI and PTS groups had significantly greater antisaccade median latency than the control group. The acute mTBI group had greater error duration than the PTS and control groups. Error bars are ±SEM. TABLE 2 \| Cross-sectional comparisons of antisaccade performance in the first visit. FIGURE 2 \| Antisaccade differences between study groups. Graphs of (A) antisaccade latency performance and (B) error duration across participant groups. Acute mTBI and PTS groups had significantly greater antisaccade median latency than the control group. The acute mTBI group had greater error duration than the PTS and control groups. Error bars are ±SEM. (M = 0.000761) in the corticospinal tract (p = 0.04917). There were no other significant differences between groups in fractional anisotropy and in mean diffusivity. There were no significant cor- relations between right corticospinal tract mean diffusivity and antisaccade median latency/error duration for PTS patients at their only visit. SCAT3 total score F(2, 33) = 30.8, p < 0.0001 was significantly greater in the acute mTBI group [13.3 (4.76)] and PTS group [16.3 (5.91)] compared to controls [1.3 (2.26)]. Participant Characteristics Basic demographic characteristics of our participant groups are in Table 1, and more information is in Supplementary Table S1 in Supplementary Material. A significant proportion (64%; n = 7) of the acute mTBI group had a history of previous concussion or TABLE 1 \| Study participant demographic characteristics of acute mTBI, PTS, and healthy control groups. Participant group N (first visit) Mean age (SD) Sex (M:F) Education (full years from Grade 1) mean (SD) GCS score Acute mTBI V1 11 36.5 (17) years 7:4 15.8 (5) years All 15 in Emergency Department (ED) All 15 at recruitment PTS V1 15 42.5 (15) years 5:10 14.9 (4) years 30 min post injury or later upon reassessment in ED (based on ED chart or clinical note describing injury): GCS 13: 1; GCS 14: 1; GCS 15: 13. All 15 at recruitment Healthy control V1 10 35.5 (21) years 5:5 15.4 (1) years All 15 January 2016 \| Volume 6 \| Article 271 5 Frontiers in Neurology \| www.frontiersin.org TABLE 1 \| Study participant demographic characteristics of acute mTBI, PTS, and healthy control groups. January 2016 \| Volume 6 \| Article 271 5 Ting et al. Antisaccades in mTBI Ting et al. TABLE 2 \| Cross-sectional comparisons of antisaccade performance in the first visit. Participant Characteristics SCAT3 symptom severity score F(2, 33) = 13.9, p < 0.0001 was also significantly different between all the three groups in ascending order, start- ing with controls [2.3 (3.95)], then to acute mTBI patients [31.18 (21.6)], and then to PTS patients [52.87 (31.0)]. Analysis of RPQ total score indicated significant differences in symptom reporting cross-sectionally between participant groups, F(2, 33) = 40.5, p < 0.0001. All the three groups were significantly different from one another in ascending order, starting with controls [1.3 (3.47)], then acute mTBI [18 (10.3)], and finally PTS [40.53 (14.0)]. Stroop color performance was significantly impaired between groups F(2, 33) = 4.67, p = 0.0164. Post hoc comparisons indicated that acute mTBI participants [75.8 (17.3)] scored worse compared to controls [89 (13.6)] and PTS participants [72.4 (10.2)] also scored worse than controls. Main Findinghi The main finding of this study is that there is a distinguishing char- acteristic of the antisaccade, which is significantly affected after injury, and that antisaccade performance is correlated with white matter integrity, response interference, and symptom burden. Antisaccade Latency and Error Duration Is Detrimentally Affected after Injuryhii y j y The median antisaccade latency at the first visit was significantly greater in the patient groups compared to controls, but we found no significant difference between acute mTBI and PTS groups at the first visit. Our analyses suggested that antisaccade latency may be a good metric for distinguishing injury from non-injury, although the heterogeneity of mTBI and persistence of symptoms remain a problem. To some extent, this supports what has been found in the literature in cross-sectional comparisons between injury and control groups. Heitger et al. identified significant increases in antisaccade latency in acute mTBI groups within 1 week of injury compared to control groups (20), and Williams et al. also identified similar greater antisaccade latency in severely injured TBI patients compared to controls (32). There was also a greater spread in latency scores within the control group compared to the injury groups in our results. This could mean that there is a natural variation or normal range of antisaccade latency in our control sample (similar to a normal range of blood pressure or body weight in humans). There are also numerous other factors like attention and alertness level, which may affect performance on the antisaccade task. This is important to investigate further in future studies. Overall, as the antisaccade task involves response inhibition, increased latency in the antisaccade task may reflect greater cortical processing time needed to generate the correct oculomotor command. This finding was in line with our initial hypotheses. Control participants took less time to make an error prosaccade toward the stimulus during the second visit compared to the first visit (Supplementary Material). The results suggest that some anti- saccade variables might not be stable over several weeks, even in healthy controls. Further studies are needed to identify the factors influencing antisaccade performance. The difference in antisac- cade median latency in the pairwise control comparisons was not significant, suggesting stable measurement over time on this spe- cific antisaccade variable. The error mean duration was also similar between both visits in each group. Hence, not all measurements taken acutely are reliable indicators of change in clinical state. DISCUSSION The duration of a saccade is related to its amplitude and veloc- ity. We found that the average amplitude for all three participant groups were larger than the target amplitude of 10° on the correct antisaccade responses, but overall were smaller than the target amplitude on the prosaccade error responses. It was interesting that a deficit was observed in the error mean duration, but not anti- saccade duration. It was noted by an early reviewer that our results in the patient group do not conform to the typical main sequence relationships between amplitude and velocity. Heitger et al. (25) attributed their observed deficits in PTS patients in prosaccade and antisaccade duration to damage in subcortical processing (25). Their results suggest that prosaccade mean duration can potentially be used to distinguish between acute mTBI and PTS. The duration of a saccade is related to its amplitude and veloc- ity. We found that the average amplitude for all three participant groups were larger than the target amplitude of 10° on the correct antisaccade responses, but overall were smaller than the target amplitude on the prosaccade error responses. It was interesting that a deficit was observed in the error mean duration, but not anti- saccade duration. It was noted by an early reviewer that our results in the patient group do not conform to the typical main sequence relationships between amplitude and velocity. Heitger et al. (25) attributed their observed deficits in PTS patients in prosaccade and antisaccade duration to damage in subcortical processing (25). Their results suggest that prosaccade mean duration can potentially be used to distinguish between acute mTBI and PTS. Frontiers in Neurology \| www.frontiersin.org Symptom Burden and Executive Functioning Correlated with Antisaccadeshi There was a significant positive correlation between antisaccade median latency and SCAT3 symptom severity score, r2 = 0.480, p = 0.02 in the acute mTBI patients at their first visit (Figure 3B). There was a significant negative correlation between antisaccade median latency and stroop color-word score, r2 = 0.439, p = 0.03 in the acute mTBI patients at the first visit (Figure 3C). There was a significant difference between groups on the Stroop color-word score, F(2, 33) = 6.59, p = 0.0039. Post hoc comparisons indicated that acute mTBI participants [39.7 (9.7)] scored worse compared to controls [52.1 (12.0)] and PTS participants [39.1 (7.1)] also scored worse than controls. January 2016 \| Volume 6 \| Article 271 Frontiers in Neurology \| www.frontiersin.org 6 Ting et al. Antisaccades in mTBI FIGURE 3 \| Antisaccade correlations with study measures. (A) Significant positive correlation at second visit between acutely injured white matter integrity in the corpus callosum splenium and antisaccade latency performance; (B) significant positive correlation at first visit between SCAT3 symptom severity score and antisaccade median latency performance in acute mTBI patients; (C) significant negative correlation at first visit between Stroop color-word score and antisaccade latency in acute mTBI patients. FIGURE 3 \| Antisaccade correlations with study measures. (A) Significant positive correlation at second visit between acutely injured white matter integrity in the corpus callosum splenium and antisaccade latency performance; (B) significant positive correlation at first visit between SCAT3 symptom severity score and antisaccade median latency performance in acute mTBI patients; (C) significant negative correlation at first visit between Stroop color-word score and antisaccade latency in acute mTBI patients. Diffusion Tensor Imaging Identified Correlations with Antisaccadesii Further analysis stratifying the impact location for acute mTBI and PTS patients (i.e., frontal, temporal, parietal, or occipital) may provide some mechanistic explanations for the negative findings in these areas, and the DTI analysis as a whole. We did not find significant dif- ferences elsewhere. However, traumatic brain injury is a complex biophysical process often characterized by rotational injury as well as blunt trauma (37). There are likely variations in the severity and nature of white matter injury in the mTBI patients that were scanned, which could be further characterized by localizing the area of injury and a review of the structural images for distribution of focal white matter hyperintensities and/or infarcts. Contrary to our hypothesis, there were no significant differences in fractional anisotropy between the acute and control groups in all the ROIs studied. However, FA measures a different aspect of diffusion flow and white matter integrity compared to mean diffusivity and fur- ther studies are needed to examine why some differences were seen in MD, and why FA was left unaltered postinjury. Longitudinal studies of DTI have found fluctuations in mean diffusivity differ- ences from the days to a year following injury (7). It is understood that FA and MD will rise and fall over time from the acute stages to chronic stages, and our findings may reflect these natural fluctua- tions in the corticospinal tract. Even in the acute stages, there is little consensus as to whether FA and MD will increase or decrease, as different findings have been identified between studies (38). Connections have been found in past research in PTS patients, between antisaccade performance and executive functioning (39). We could not confirm this relationship in our results. This was sur- prising, given that the level of executive functioning impairment in PTS (measured by the Stroop color-word score) was similar to the acute group. One possible explanation is that in PTS patients, the changes in microstructure leading to sustained impairment in antisaccade performance were different compared to acute patients. This explanation is supported by our finding that the dif- ferences in mean diffusivity were found in the corpus callosum in the acute patients, but not in the PTS patients; likewise, significant MD differences were identified in the right corticospinal tract in the PTS patients, but not the acute patients. Thus, a difference in the neuroanatomy of PTS may be an explanation for the negative findings here. Diffusion Tensor Imaging Identified Correlations with Antisaccadesii It is possible that diffuse axonal injury of the many connections serviced by the corpus callosum splenium plays a significant role in the execution of an efficient antisaccade.hf ifi The difference we found in MD of the acute group’s corpus cal- losum splenium compared to the control group partially confirmed our hypothesis that there would be decreased white matter integrity postinjury in the corpus callosum. Significant microstructural dam- age and change in the corpus callosum in TBI patients over time have been identified (35, 36). As discussed, a recent meta-analysis specifically focusing on the corpus callosum found that DTI analy- sis of this area was a sensitive marker for microstructural damage after mild TBI (5). Our findings suggest that corpus callosum microstructural alterations are present several weeks after injury in acute mTBI patients compared to healthy controls. The finding that MD was significantly elevated compared to controls supports the hypothesis that there might be increased edema and inflammation in the splenium component of this critical white matter bundle.hif y q y p g y Since there was a significant negative association between antisaccade performance and Stroop color-word score, a poten- tial mechanistic explanation is that antisaccade performance reflected this executive neurocognitive impairment, which in turn was associated with increased symptom burden on the SCAT3. Currently, there is no validated subscoring system for the symp- tom assessment scale of the SCAT3. Future work could do factor analyses on the SCAT3 symptom scale to identify a stable cogni- tive performance subscale on the SCAT3 (similar to the RPQ) and use this subscore to correlate with antisaccade performance and executive functioning. We found that task interference inhibi- tion was impaired in acute mTBI patients with the Stroop test. A similar cognitive process is needed in the brain to generate an effective antisaccade: the participant must inhibit the easiest rapid eye movement, an interfering one moving toward the stimulus (committing a prosaccade error), and cognitively program an anti- saccade, instead rapidly moving the eyes to the mirror-opposite location. The interference deficit between the acute mTBI patients and controls suggest the antisaccade task could potentially be a marker for this specific neurocognitive domain and provides a neuropsychological link to the larger implications of this work. There were no significant differences in the genu and body of the corpus callosum, which was surprising as previous studies have identified vulnerability of these areas (5). Diffusion Tensor Imaging Identified Correlations with Antisaccadesii In line with our hypotheses, we identified a significant positive cor- relation between median latency of antisaccades at the acute mTBI second visit and MD in the splenium of the corpus callosum. There appeared to be a connection between the time necessary for inhibi- tory processes to take place, generating a successful antisaccade, and integrity of this white matter bundle. This correlation did not hold for PTS patients. Some have attributed PTS to psychosocial aspects of the injury, whereas the acute manifestations are a product of neurobiological changes in reaction to the impact (33). The lack of effective correlations in PTS patients may reflect this underlying There was also a significant omnibus group-wise difference in mean duration of their error trials, with the acute mTBI participants exhibiting greater duration than the PTS and control participants. January 2016 \| Volume 6 \| Article 271 7 Ting et al. Antisaccades in mTBI between antisaccade median latency and SCAT3 symptom load, and a negative correlation between antisaccade median latency and Stroop interference performance at the first visit. The first finding made sense in that poorer antisaccade performance was correlated to increased symptom burden on the SCAT3. Increased antisaccade latency is a disorder of saccadic initiation. The time required for the integration and calculation of the antisaccade signal reflects the time needed to process this task involving higher brain structures and interhemispheric connectivity (10), in part mediated by the corpus callosum. Intact white matter architecture and functioning of the higher cerebral centers are necessary for effective saccadic initiation and normal cognitive functioning. Impaired cognitive functioning and specifically an effective way to cope with response interference may push symp- tom burden higher in acute mTBI and PTS patients. It remains to be determined why this relationship was only identified for the acute mTBI group and not the PTS group, even though the PTS group reported significantly greater symptom burden on the RPQ and the SCAT3 on almost every question and symptom category.i disconnect between neurological functioning and the structural neuroanatomy. Similar relationships between antisaccade per- formance and corpus callosum integrity have been documented in patients with fetal alcohol spectrum disorder in studies where the patients completed antisaccade tasks as well as diffusion MRI using voxel-based DTI analysis methods (34). The antisaccade task requires much greater recruitment of cerebral resources compared to a simple prosaccade task. Diffusion Tensor Imaging Identified Correlations with Antisaccadesii As discussed in the introduction, the most drastic effects of the neurometabolic cascade happen acutely after injury, but they can ease into permanent damage in the chronic phases. Symptom Burden and Executive Symptom Burden and Executive Functioning Correlated with Antisaccades Correlational analyses identified several associations between antisaccade performance and symptom load/executive perfor- mance that were meaningful. We found a positive correlation Functioning Correlated with Antisaccades Correlational analyses identified several associations between antisaccade performance and symptom load/executive perfor- mance that were meaningful. We found a positive correlation January 2016 \| Volume 6 \| Article 271 Frontiers in Neurology \| www.frontiersin.org 8 Ting et al. Antisaccades in mTBI Perhaps, these changes are reflected in our neuroimaging results, but the study’s small sample size limits our conclusions. Perhaps, these changes are reflected in our neuroimaging results, but the study’s small sample size limits our conclusions. adequately power a ROI-based inquiry of DTI changes after mTBI. As reviewers noted, since computerized tomography results are more likely to be used for mTBI, future work may consider any correlations between tomography findings and PTS. Furthermore, this population is inherently heterogeneous and future work may need to account for this variability, and a sensitivity/specificity analysis would be very informative with a larger sample size. Finally, although our experimental design and timing of visits was adjusted to overall clinical course for recovery in mTBI patients and the expected course of recovery in saccadic eye movements, it is possible that this timeframe was not long enough for significant resolution of symptoms in the acute mTBI patients. We also did not correct for multiple comparisons due to the exploratory nature and small sample size of this study. Nevertheless, we think this work is an important step toward identifying the relationship of the antisaccade to other assessment methods used for these patients. In terms of executive functioning, in line with our hypotheses, there was a significant difference between the injured and control groups on the Stroop color-word score, with injured participants covering fewer items in the same amount of time as controls (hence scoring more poorly). The results suggested that the injured groups had significantly impaired interference control as tested by the Stroop interference trial compared to healthy control groups. We did not see significant differences between the acute and PTS group, suggesting similar levels of response inhibition impairment. FUNDING We would like to thank the Canadian Institutes of Health Research MOP 123371 for study funding. WT was financially supported by the Institute of Medical Science Open Fellowship Award at the University of Toronto. CONCLUSION In conclusion, there were differences in antisaccade median latency and error mean duration between mTBI patient groups and controls. These measures were associated with loss of white matter integrity in the splenium of the corpus callosum; increased antisaccade median latency was also associated with poor per- formance on executive functioning tasks and greater symptom load. This study adds to the existing literature suggesting that the antisaccade task may have potential to be useful as a neurological marker for acute mTBI, but with our low sample sizes and the exploratory nature of this study further investigation is required. Our work supports that antisaccade latency is related to impaired inhibitory functioning, diffuse axonal injury in the corpus callo- sum, and symptom load. We confirmed significant characteristics of antisaccade functioning, which were affected in injury patients. We also identified deficits in specific types of executive function- ing, and identified relationships between antisaccade performance and symptom load. Our findings indicate that it may be possible to distinguish between mTBI participant groups and controls using antisaccades, both in the acute and chronic phases. However, any antisaccade metric that will be used for clinical purposes needs to be selected carefully and must be robust. ACKNOWLEDGMENTS We did not include a task to account for malingering on the neuropsychological testing and inflated symptom reporting in the acutely or chronically injured patients. We did not account for depression score and other factors that may affect the relationships studied (age and time since injury, for example). Furthermore, we did not exclude patients who had a comorbid history of post- traumatic stress disorder (PTSD), which affects the psychological aspect of neurocognitive functioning and symptom burden. We also did not account for history of previous concussion due to low sample sizes. We used the latency range of 50–600 ms for our initial screening and identification of “accepted” saccades based on previous work on the step saccade task (28). However, close to half of the antisaccade trial responses ended up falling out of these boundaries, limiting the validity of our conclusions. A new upper and lower bound for antisaccade latencies used for this task may be needed. We averaged the FA and MD values over the whole white matter region of interest (ROI). Thus, it is possible that any individual differences at specific points along the tracts were masked. This limitation was touched upon by Niogi et al. in their review (4) noting the large number of participants needed to Special thanks to Dr. Rowan Jing for assistance with the statistical analysis. We would like to thank Stanley Zhang, Anthony Wong, Iryna Pshonyak, Jocelyn Lee, and Harrish Nithianandan for their assistance with this study. We would also like to thank Dr. Glen Bandiera for helpful comments on WT’s M.Sc. work upon which this manuscript is based. AUTHOR CONTRIBUTIONS Conception of the work: MC, TS, JT-V, and WT; collection of data: WT and MC; data analysis: WT; data interpretation: MC, TS, JT-V, and WT; wrote initial draft of manuscript: WT; and revisions to the manuscript for significant intellectual content: MC, TS, JT-V, and WT. Symptom Burden and Executive We found significant differences in the Stroop color score with poorer performance in the injury groups; there have been reports of impaired color score differences in patients with postconcussive syndrome compared to controls (40).hifl There were no significant differences in phonemic word flu- ency on the MoCA, and also no significant differences on the Trail Making Test B score although TMT-A performance was altered. The finding that differences in TMT-A were observed suggested there are differences in psychomotor speed between groups. 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https://openalex.org/W4388187439	https://journalofmedula.com/index.php/medula/article/download/610/427	Indonesian	null	Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung	Medula/MEDULA (Medical Profession Journal of Universitas Lampung)	2,023	cc-by-sa	3,938	Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Abstrak Volume oksigen maksimal (VO2max) merupakan ambilan oksigen maksimal selama latihan. Orang dengan kebugaran yang baik memiliki nilai VO2max lebih tinggi dan dapat melakukan aktivitas lebih kuat daripada mereka yang kebugarannya tidak baik. Indeks massa tubuh (IMT) merupakan salah satu faktor komposisi tubuh yang mempengaruhi VO2max. VO2max dapat diukur melalui berbagai macam metode, salah satunya adalah dengan metode Balke test. Penelitian ini bertujuan untuk mengetahui hubungan indeks massa tubuh terhadap VO2max mahasiswa tingkat pertama Fakultas Kedokteran Universitas Lampung. Penelitian ini merupakan penelitian yang bersifat analitik korelatif dengan pendekatan cross sectional menggunakan data primer yang dilakukan dalam satu kali pertemuan pada bulan November 2022. Data diperoleh dengan pengukuran indeks massa tubuh (IMT) terlebih dahulu, dan dilanjutkan dengan pengukuran VO2max menggunakan metode Balke test. Penelitian ini menggunakan sampel sebanyak 39 mahasiswa laki-laki Fakultas Kedokteran Universitas Lampung Angkatan 2022 yang telah melewati seleksi kriteria inklusi dan eksklusi sebelumnya. Hasil pada penelitian ini menunjukkan adanya hubungan yang signifikan antara indeks masa tubuh dengan VO2max pada mahasiswa tingkat pertama Fakultas Kedokteran Universitas Lampung. Pada penelitian didapatkan nilai p<0,001 dan koefisien korelasi sebesar -0,812 yang berarti terdapat hubungan korelasi negatif yang sangat kuat antarvariabel. Hubungan korelasi negatif tersebut menunjukkan bahwa semakin tingginya indeks massa tubuh (IMT) akan berakibat pada semakin rendahnya nilai VO2max individu, begitu pula sebaliknya. Kata kunci: Balke test, indeks massa tubuh, VO2max Kata kunci: Balke test, indeks massa tubuh, VO2max Abstract Maximum oxygen volume (VO2max) is the maximum oxygen uptake during exercise. People with good fitness have higher VO2max value and can perform activities more vigorously than those with poor fitness. Body mass index (BMI) is one of the body composition factors that affect VO2max. VO2max can be measured through various methods, one of which is the Balke test. This study aims to determine the relationship of body mass index to VO2max in the first grade students of the Faculty of Medicine, University of Lampung. This research is a correlative analytic study with a cross-sectional approach using primary data which was conducted in one meeting in November 2022. The data is obtained using measuring body mass index (BMI) first, and followed by measuring VO2max using the Balke test method. This study uses sample of 39 batch 2022 male students of the Faculty of Medicine, University of Lampung who have previously passed the inclusion and exclusion criterias. The result in this study indicates that there is a significant relationship between body mass index and VO2max in the first grade students of the Faculty of Medicine, University of Lampung. In this study, the p value is <0.001 and the correlation coefficient is -0.812, which means that there is a very strong negative correlation between variables. This negative correlation relationship indicates that the higher the body mass index (BMI) will result in lower individual VO2max values, and vice versa. Keywords: Balke test, body mass index, VO2max Korespondensi: Salma Khairunnisa \| Jl. Dewi Sartika No.123, Metro Utara, Metro \| HP 082372510998 e-mail: salmakhrnnsa09@gmail.com akibat kegiatan sedentari yang tinggi. Mahasiswa Fakultas Kedokteran tahun pertama cenderung memiliki kegiatan akibat kegiatan sedentari yang tinggi. Mahasiswa Fakultas Kedokteran tahun pertama cenderung memiliki kegiatan Kata kunci: Balke test, indeks massa tubuh, VO2max Pendahuluan Sebagian besar mahasiswa kedokteran memiliki tingkat aktivitas fisik yang rendah Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 28 Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung sedentari yang lebih tinggi dibanding mahasiswa tahun kedua. Hal ini mungkin terjadi karena penyesuaian kebiasaan belajar dan beraktivitas pada mahasiswa tahun pertama yang cenderung menghabiskan sebagian besar waktunya untuk duduk dan belajar atau mengerjakan tugasnya. Selain itu, tingkat stress akibat penyesuaian pada mahasiswa tahun pertama memberikan pengaruh terhadap kebiasaan melakukan kegiatan sedentary 1,2. merupakan salah satu metode pengukuran VO2max yang pelaksanaannya cukup mudah, memerlukan peralatan yang sederhana, dan dapat digunakan untuk mengukur kebugaran banyak orang sekaligus dengan hasil yang cukup akurat dengan cara berlari sejauh- jauhnya jarak selama 15 menit 14,15. VO2max pada dasarnya dipengaruhi oleh beberapa faktor, di antaranya faktor genetik, latihan fisik, jenis kelamin, usia, berat badan, tinggi badan, komposisi tubuh, tekanan darah, denyut nadi, dan suhu tubuh. Indeks massa tubuh (IMT) merupakan salah satu faktor komposisi tubuh yang mempengaruhi VO2max. Seseorang dengan indeks massa tubuh yang tinggi (overweight) cenderung memiliki nilai VO2max yang rendah. Begitu pula sebaliknya, seseorang dengan indeks massa tubuh normal akan memiliki nilai VO2max yang tinggi. Suatu penelitian mendapatkan bahwa setiap penambahan 1 kg/m2 indeks massa tubuh akan menurunkan volume oksigen maksimal (VO2max) sebesar 1,349 mlO2/kg/menit 4,8,10,11,13,16–20. Mahasiswa yang menghabiskan waktunya untuk kegiatan sedentari yang tinggi tersebut mayoritas memiliki indeks massa tubuh yang tergolong obesitas, sedangkan mahasiswa dengan waktu kegiatan sedentari yang rendah memiliki indeks massa tubuh normal. Hal ini diperkuat dengan ditemukannya sebanyak 30% mahasiswa Fakultas Kedokteran Universitas Kristen Indonesia mengalami obesitas dengan tingkat aktivitas fisik yang rendah. Selain itu, pada Fakultas Kedokteran Universitas YARSI ditemukan sebanyak 54,5% mahasiswa kedokteran tahun pertama dengan jenis kelamin laki-laki memiliki indeks massa tubuh dengan kategori obesitas. Pendahuluan Tingkat aktivitas fisik yang rendah dan indeks massa tubuh yang tinggi tersebut berakibat pada rendahnya VO2max 1–5. Metode Penelitian ini merupakan penelitian yang bersifat analitik korelatif dengan pendekatan cross sectional, dimana penelitian ini akan mencari hubungan indeks massa tubuh terhadap VO2max mahasiswa tingkat pertama Fakultas Kedokteran Universitas Lampung. Penelitian ini dilakukan di Stadion Pahoman Bandar Lampung pada bulan November 2022. Pengambilan sampel penelitian ini menggunakan teknik total sampling dengan syarat sampel yang memenuhi kriteria inklusi dan eksklusi, yakni didapatkan sebanyak 39 orang mahasiswa laki-laki Fakultas Kedokteran Universitas Lampung angkatan 2022. Penelitian hanya diadakan dalam satu kali pertemuan, dimana subjek yang telah memenuhi kriteria inklusi dan eksklusi diminta persetujuannya untuk menjadi sampel penelitian dengan menandatangani lembar informed consent. Penelitian ini telah telah mendapatkan persetujuan etik dari Komisi Etik Penelitian Kesehatan Fakultas Kedokteran Universitas Lampung dengan nomor surat VO2max merupakan ambilan oksigen maksimal selama latihan. Orang dengan kebugaran yang baik memiliki nilai VO2max lebih tinggi dan dapat melakukan aktivitas lebih kuat daripada mereka yang kebugarannya tidak baik. VO2max dibatasi oleh kemampuan dari sistem kardiorespirasi dalam menghantar oksigen menuju otot. VO2max dapat digunakan sebagai penanda kesehatan saat ini dan prediktor kesehatan masa depan terhadap berbagai faktor risiko penyakit tidak menular. Nilai VO2max yang rendah menjadi salah satu faktor predisposisi yang penting dalam peningkatan angka kematian dini akibat gangguan kardiovaskular 6–13. VO2max dapat dinilai dengan berbagai uji yang mengaktifkan sekelompok otot-otot besar selama uji tersebut memiliki intensitas dan durasi yang memadai untuk menibulkan transfer energi aerobik yang maksimal. Salah satu metodenya adalah Balke test. Balke test 4048/UN26.18/PP.05.02.00/2022. Penelitian 4048/UN26.18/PP.05.02.00/2022. Penelitian Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 29 Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Tabel 2. Analisis univariat indeks massa tubuh Karakteristik Subjek Min. Maks. Rerata Indeks Massa Tubuh (kg/m2) 16,275 41,461 24,96 ± 6,058 ini menggunakan data primer yang diperoleh dengan pengukuran secara langsung indeks massa tubuh (IMT) dan VO2max masing-masing sampel penelitian. Pengukuran indeks massa tubuh dilakukan dengan cara melakukan pengukuran berat badan menggunakan timbangan injak digital dan pengukuran tinggi badan dengan menggunakan microtoise, dimana nantinya hasil berat badan dan tinggi badan dikonversikan ke dalam rumus indeks massa tubuh. Pengukuran VO2max dilakukan dengan menghitung jarak tempuh yang kemudian dikonversikan menggunakan rumus VO2max metode Balke. Metode Setelah itu, data yang diperoleh dilakukan pengolahan dan analisis data menggunakan uji Spearman dengan bantuan software SPSS. Berdasarkan tabel 2 di atas, didapatkan indeks massa tubuh sampel memiliki nilai minimum sebesar 16,275, nilai maksimum sebesar 41,461, nilai median sebesar 24,048, nilai rerata sebesar 24,96 ± 6,058. Tabel 3. Persebaran kategori indeks massa tubuh responden Kategori IMT Frekuensi (n) Persentase (%) Kurus 4 10,3 Normal 20 51,3 Gemuk 15 38,5 Total 39 100 Tabel 3. Persebaran kategori indeks massa tubuh responden Hasil Penelitian ini telah dilaksanakan pada bulan November tahun 2022 di Stadion Pahoman Bandar Lampung. Sampel yang digunakan pada penelitian ini sebanyak 39 orang sampel. Setelah dilakukan analisis data diperoleh hasil sebagai berikut. Data tersebut menunjukkan bahwa sebagian besar sampel memiliki indeks massa tubuh dengan kategori normal, yaitu sebanyak 20 orang, kemudian diikuti dengan sampel yang memiliki indeks massa tubuh dengan kategori gemuk (overweight) sebanyak 15 orang, dan kategori kurus (underweight) sebanyak 4 orang. Tabel 1. Karakteristik subjek penelitian Karakteristik Subjek Min. Maks. Rerata Usia (tahun) Berat Badan (kg) Tinggi Badan (cm) 18 41,3 152,9 20 120,9 185,1 18,26 ± 0,498 70,64 ± 19,468 167,7 ± 6,224 Tabel 1. Karakteristik subjek penelitian Tabel 4. Analisis univariat VO2max Karakteristik Subjek Min. Maks. Rerata VO2max (ml/kg/menit) 26,82 38,25 32,546 ± 3,07 Berdasarkan tabel 1 di atas, didapatkan rentang usia sampel adalah 18 tahun sampai dengan 20 tahun dengan dominasi dari usia 18 tahun. Seluruh sampel berjenis kelamin laki- laki. Berdasarkan berat badan, didapatkan berat badan minimum sampel sebesar 41,3 kg dan berat badan maksimum sebesar 120,9 kg. Sedangkan tinggi badan minimum sampel sebesar 152,9 cm dan tinggi badan maksimum sebesar 185,1 cm. Berdasarkan tabel 3 di atas, didapatkan nilai VO2max memiliki nilai minimum sebesar 26,82, nilai maksimum sebesar 38,25, nilai median sebesar 33,13, nilai rerata sebesar 32,546 ± 3,07. Tabel 5. Persebaran kategori VO2max responden Kategori VO2max Frekuensi (n) Persentase (%) Sangat rendah 12 30,8 Rendah 27 69,2 Total 39 100 Tabel 5. Persebaran kategori VO2max responden Data tersebut menunjukkan bahwa sebagian besar responden memiliki nilai VO2max dengan kategori rendah, yaitu Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 30 Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung sebanyak 27 orang, kemudian diikuti dengan responden yang memiliki VO2max dengan kategori sangat rendah sebanyak 12 orang. Hasil penelitian ini sejalan dengan penelitian yang dilakukan oleh Gantarialdha (2021) tentang hubungan profil antropometri dengan VO2max pada mahasiswa Fakultas Kedokteran angkatan 2018 yang menyatakan bahwa terdapat hubungan antara profil antropometri berupa indeks massa tubuh dan VO2max dengan nilai koefisien korelasi sebesar -0,513 yang berarti tingkat hubungan kuat. Hasil Hal ini juga tidak bertolak belakang dengan hasil penelitian oleh Irianto (2016) yang juga mendapatkan bahwa semakin tinggi IMT siswa maka semakin rendah kebugaran kardiorespirasinya dengan korelasi yang memiliki kekuatan hubungan kuat (r = -0,509) dan nilai signifikansi <0,001. Tabel 6. Gambaran hubungan indeks massa tubuh terhadap vo2max dengan uji korelasi spearman Indeks Massa Tubuh VO2max Indeks Massa Tubuh Koefisien Korelasi Sig. (2- tailed) N 1,000 39 -0,812 <0,001 39 VO2max Koefisien Korelasi Sig. (2- tailed) N -0,812 <0,001 39 1,000 39 Tabel 6. Gambaran hubungan indeks massa tubuh terhadap vo2max dengan uji korelasi spearman Indeks massa tubuh (IMT) merupakan antropometri untuk menilai massa tubuh yang terdiri dari tulang, otot, dan lemak. IMT adalah cara yang praktis untuk memantau status gizi orang dewasa (usia ≥18 tahun), khususnya yang berhubungan dengan kekurangan dan kelebihan berat badan. IMT tidak dapat diterapkan pada kelompok umur yang masih tumbuh seperti bayi, anak, remaja, dan kelompok khusus seperti ibu hamil yang mengalami peningkatan berat badan ketika hamil dan olahragawan atau atlet yang sebagian besar massa tubuhnya terdiri dari otot. Selain itu, IMT pun tidak dapat diterapkan pada keadaan khusus dimana orang mengidap penyakit seperti edema, asites, dan hepatomegali 21. Berdasarkan hasil uji korelasi Spearman dalam tabel 4 di atas, didapatkan nilai koefisien korelasi -0,812 dengan nilai signifikansi <0,001. Nilai signifikansi dianggap berarti apabila p <0,05. Oleh karena itu, nilai signifikansi penelitian ini menunjukkan bahwa H0 ditolak, artinya terdapat korelasi yang signifikan antara indeks massa tubuh dengan VO2max. Koefisien korelasi yang terbentuk yaitu -0,812 yang menunjukkan korelasi negatif sangat kuat antarvariabel, artinya semakin rendah indeks massa tubuh maka semakin besar nilai VO2max. Namun, hasil penelitian menunjukkan nilai VO2max yang lebih buruk pada indeks massa tubuh di bawah normal (kurus/underweight) jika dibandingkan dengan nilai VO2max pada indeks massa tubuh normal. Terdapat beberapa faktor yang dapat menentukan nilai VO2max seseorang, diantaranya yaitu usia, jenis kelamin, aktivitas fisik, dan komposisi tubuh. Faktor yang berperan cukup besar adalah komposisi tubuh yang dapat diukur dengan profil antropometri berupa indeks massa tubuh. Seseorang dengan status indeks massa tubuh yang normal akan memiliki tubuh yang lebih bugar dibanding seseorang dengan status indeks massa tubuh yang tinggi terutama obesitas. Indeks massa tubuh tinggi terutama obesitas pada seseorang akan membatasi fleksibilitasnya dalam berbagai aktivitas. Hal ini akan membuat orang dengan indeks massa tubuh yang tinggi tersebut seringkali menjalani gaya hidup yang tidak banyak bergerak, Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 31 Pembahasan Berdasarkan hasil penelitian, dapat diketahui bahwa hasil nilai p <0.001 (<0.05) yang berarti terdapat korelasi bermakna antara indeks massa tubuh dengan VO2max pada mahasiswa Fakultas Kedokteran Universitas Lampung angkatan 2022. Nilai koefisien korelasi pada hasil penelitian didapatkan sebesar -0,812 yang berarti bahwa indeks massa tubuh dan VO2max memiliki korelasi negatif sangat kuat, semakin tinggi indeks massa tubuh maka semakin rendah nilai VO2max individu. Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 31 Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung sehingga kadar kebugaran jasmaninya lebih rendah 13,17,22,23. membutuhkan lebih banyak lagi ATP sehingga otot akan berkontraksi lebih kuat. Hal itu akan menyebabkan peningkatan konsumsi oksigen oleh otot untuk melangsungkan respirasi aerobik. Akan tetapi, peningkatan konsumsi oksigen oleh otot tersebut bersamaan dengan kurangnya suplai oksigen dari darah ke otot. Hal ini mengakibatkan persediaan oksigen semakin menipis dan akhirnya tidak cukup berlangsungnya respirasi aerobik oleh otot. Oleh karena itu, otot mengalihkan respirasi aerobik ke respirasi anaerobik yang tidak melibatkan oksigen dalam prosesnya, yaitu glikolisis anaerobik. Glikolisis anaerobik sangat bermafaat dalam penyediaan ATP dalam waktu yang relatif cepat dan tanpa menggunakan oksigen. Namun, glikolisis anaerobik menghasilkan suatu produk akhir berupa asam laktat yang sangat merugikan karena dapat menyebabkan kelelahan otot 24. Indeks massa tubuh yang tinggi akan menyebabkan peningkatan low density lipoprotein (LDL) dan penurunan high density lipoprotein (HDL). Beban yang terlalu berat akan mengganggu fungsi jantung. Tingginya lemak dalam tubuh terutama pada penderita obesitas akan menjadi kendala dan menimbulkan beban tambahan pada fungsi jantung. Penurunan fungsi ini akan berdampak pada turunnya curah jantung. Hal ini menyebabkan lebih sedikit darah yang dipompa sehingga oksigen yang beredar ke otot juga lebih sedikit. Hal ini pun menyebabkan penurunan ambilan oksigen untuk metabolisme intraseluler, terutama sel muskuloskeletal. Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 32 Daftar Pustaka 1. Faiq AR, Zulhamidah Y, Widayanti E. Gambaran Sedentary Behaviour dan Indeks Massa Tubuh Mahasiswa Fakultas Kedokteran Universitas YARSI di Masa Pendidikan Tahun Pertama dan Kedua. MAJALAH SAINSTEKES. 2018;5(2):66–73. 1. Faiq AR, Zulhamidah Y, Widayanti E. Gambaran Sedentary Behaviour dan Indeks Massa Tubuh Mahasiswa Fakultas Kedokteran Universitas YARSI di Masa Pendidikan Tahun Pertama dan Kedua. MAJALAH SAINSTEKES. 2018;5(2):66–73. 11. Teresa S, Widodo S, Winarni TI. Hubungan Body Mass Index dan Persentase Lemak Tubuh dengan Volume Oksigen Maksimal pada Dewasa Muda. Jurnal Kedokteran Diponegoro. 2018;7(2):840–53. 2. Riskawati YK, Prabowo ED, Rasyid HA. Tingkat Aktivitas Fisik Mahasiswa Program Studi Pendidikan Dokter Tahun Kedua, Ketiga, Keempat. Majalah Kesehatan. 2018;5(1):26–32. 12. Saniyyah A. Hubungan Status Gizi dengan Tingkat Kebugaran Kardiorespirasi pada Siswa Kelas IV dan V SDN 13 Sungai Pisang Padang [skripsi]. Padang: Universitas Andalas; 2020. 3. Utami TWW, Bebasari E, Ernalia Y. Relationship of Physical Activity Levels with Genesis Obesity in Faculty of Medicine University of Riau Generation 2012 & 2013. JOM FK. 2015;2(2):1–12. 3. Utami TWW, Bebasari E, Ernalia Y. Relationship of Physical Activity Levels with Genesis Obesity in Faculty of Medicine University of Riau Generation 2012 & 2013. JOM FK. 2015;2(2):1–12. 13. Gantarialdha N. Hubungan Indeks Massa Tubuh Terhadap Ketahanan Kardiorespirasi Dinyatakan dalam VO2max. Jurnal Medika Hutama. 2021;2(4):1162–8. 4. Adidharma NC. Karakteristik Tingkat Kebugaran Kardiorespirasi Siswa Kelas 6 SD di Desa Mengwitani Tahun 2014. E- Jurnal Medika. 2016 May;5(5):1–7. 14. Mitchell RD, Crandall C. Validation of the 15 Minute Balke Field Test for Competitive, Adult 5K Runners: From Treadmill VO2max Testing to Enhancing Performance. American Journal of Sports Science and Medicine. 2017;5(3):44–7. 5. Diani YH. Faktor-Faktor yang Berhubungan dengan Obesitas pada Mahasiswa Fakultas Kedokteran Universitas Kristen Indonesia. Jurnal Ilmiah WIDYA. 2018;5(1):1–5. 5. Diani YH. Faktor-Faktor yang Berhubungan dengan Obesitas pada Mahasiswa Fakultas Kedokteran Universitas Kristen Indonesia. Jurnal Ilmiah WIDYA. 2018;5(1):1–5. 15. Mubarak R. Efektivitas Latihan Berjalan Terhadap Kapasitas Fungsional dan Fungsi Keseimbangan pada Calon Jemaah Haji Dewasa Sehat [tesis]. Jakarta: Universitas Indonesia; 2018. 6. Tomkinson GR, Annandale M, Ferrar K. Global Changes in Cardiovascular Endurance of Children and Youth Since 1964: Systematic Analysis of 25 Million Fitness Test Results from 28 Countries. Circulation. 2013;128(22). 6. Tomkinson GR, Annandale M, Ferrar K. Global Changes in Cardiovascular Endurance of Children and Youth Since 1964: Systematic Analysis of 25 Million Fitness Test Results from 28 Countries. Circulation. 2013;128(22). 16. World Health Organization. A healthy lifestyle [Internet]. Simpulan 9. Nugraha AR, Berawi KN. Pengaruh High Intensity Interval Training (HIIT) terhadap Kebugaran Kardiorespirasi. Majority. 2017;6(1):1–5. p Terdapat hubungan antara indeks massa tubuh dengan VO2max pada mahasiswa tingkat pertama Fakultas Kedokteran Universitas Lampung. 10. Teresa S. Hubungan Body Mass Index dan Persentase Lemak Tubuh dengan Volume Oksigen Maksimal pada Dewasa Muda [skripsi]. Semarang: Universitas Diponegoro; 2017. Pembahasan Dengan berat badan yang berlebih, pada saat beraktivitas fisik tubuh Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 32 Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Kedokteran Dan Kesehatan. 2014;1(1):54–62. kapasitas residu fungsional dan volume cadangan ekspirasi sehingga mengharuskan bernapas menjadi lebih datar, dangkal, cepat, dan kurang efisien. Kurva tekanan-volume paru pada obesitas juga dapat meningkatkan kerja otot pernapasan 25,26. 8. Kalyanshetti SB, Veluru S. A cross- sectional Study of Association of Body Mass Index and VO2 Max by Nonexercise Test in Medical Students. Natl J Physiol Pharm Pharmacol. 2017;7(2):228–31. Pembahasan Karena timbunan lemak yang tidak proporsional, sistem muskuloskeletal tidak bisa memperoleh jumlah oksigen yang optimal selama latihan 13. Indeks massa tubuh yang tinggi terutama overweight dan obesitas menyebabkan terjadinya peningkatan lemak di dinding dada dan abdomen. Hal ini menyebabkan peregangan yang berlebihan pada dinding thorax. Peregangan dinding thorax yang berlebihan ini akan mengakibatkan penurunan compliance dinding dada, membatasi gerakan tulang-tulang kosta atau penurunan mobilitas thorax, serta otot pernapasan harus bekerja lebih keras untuk menghasilkan tekanan yang tinggi pada rongga pleura untuk memungkinkan aliran udara masuk saat inspirasi. Otot pernapasan yang harus bekerja lebih keras tersebut meningkatkan konsumsi O2 dan produksi CO2 yang akan berakibat pada peningkatan ventilasi. Kemudian, lama- kelamaan terjadi hipoventilasi karena bertambahnya retensi CO2 25,26. Apabila nilai indeks massa tubuh seseorang tinggi dan termasuk dalam kategori overweight sampai obesitas, maka akan terjadi peningkatan jaringan lemak dalam tubuh. Seseorang yang memiliki indeks massa tubuh tinggi memiliki jaringan lemak yang lebih banyak dibanding jaringan otot. Jaringan adiposa adalah jaringan tubuh yang tidak terlibat langsung dalam proses pembentukan energi, sedangkan jaringan otot merupakan jaringan yang berperan aktif dalam pembentukan energi. Seseorang yang lebih banyak memiliki jaringan lemak dalam tubuh akan memiliki kemampuan menghasilkan energi yang rendah. Artinya, berat badan berlebih memiliki lebih banyak massa jaringan yang tidak aktif dan akan meningkatkan beban jaringan aktif sehingga otot akan berkontraksi lebih kuat untuk menopang kelebihan berat badan. Hal itu akan menyebabkan peningkatan konsumsi O2 oleh otot untuk memperoleh ATP 13,24 Deposisi jaringan adiposa subkutan dan visceral dapat menyebabkan keterbatasan ekspansi paru-paru selama inspirasi dan mengurangi kaliber saluran napas perifer sehingga saluran napas akan menyempit secara luas dan menjadi pemicu berkembangnya hipoksemia. Deposisi lemak subkutan di dada bagian atas yang paling berpengaruh pada fungsi pernapasan. Orang yang obesitas akan mengalami transmisi tekanan yang tinggi di kompartemen intraabdominal menuju ke toraks yang secara langsung dapat mengurangi Tubuh membutuhkan ATP sebagai energi selama beraktivitas fisik. ATP diperoleh dari respirasi yang terjadi di otot, khususnya respirasi aerobik yang menghasilkan ATP paling banyak dibanding respirasi anaerobik, dimana respirasi aerobik membutuhkan oksigen untuk membentuk ATP. Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 34 Daftar Pustaka Jenewa: World Health Organization; 2010 [diperbarui tanggal 6 Mei 2010; disitasi tanggal 4 Oktober 2022]. Tersedia dari: https://www.who.int/europe/news- room/fact-sheets/item/a-healthy- lifestyle---who-recommendations 7. Alfarisi R. Pengaruh Kebugaran Jasmani, Aktifitas Fisik, dan Indeks Massa Tubuh Terhadap Indeks Prestasi Kumulatif (IPK) Mahasiswa Kedokteran Universitas Malahayati Bandar Lampung. Jurnal Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 33 Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung Salma Khairunnisa, Dewi Nur Fiana, Helmi Ismunandar, Khairun Nisa Berawi \| Hubungan Indeks Massa Tubuh terhadap Volume Oksigen Maksimal (VO2max) Mahasiswa Tingkat Pertama Fakultas Kedokteran Universitas Lampung 17. Jayusfani R, Afriwardi, Yerizel E. Hubungan IMT (Indeks Massa Tubuh) dengan Ketahanan (Endurance) Kardiorespirasi pada Mahasiswa Pendidikan Dokter Unand 2009-2012. Jurnal Kesehatan Andalas. 2015;4(2):409– 14. 18. Irianto. Korelasi Indeks Massa Tubuh dengan Kebugaran Kardiorespirasi pada Siswa SMA Sutomo 2 Medan [skripsi]. Medan: Universitas Sumatera Utara; 2016. 19. Novitasari A, Setiarini A. Hubungan Komposisi Tubuh Dengan Nilai Volume Oksigen Maksimal (VO2maks) pada Atlet Pria Kelompok Usia Remaja dan Dewasa Awal (10-30 Tahun): Systematic Review. Jurnal Kesehatan Komunitas. 2020;6(1):7–12. 20. Weir CB, Jan A. BMI Classification Percentile and Cut Off Points. Treasure Island (FL): StatPearls Publishing; 2022. 21. Par’i HM, Wiyono S, Harjatmo TP. Penilaian Status Gizi. Jakarta: Kementerian Kesehatan Republik Indonesia; 2017. 22. Alfarisi R, Rivai PP. Hubungan Indeks Massa Tubuh Terhadap Ketahanan Kardiorespirasi Diukur dari VO2max pada Mahasiswa Fakultas Kedokteran Universitas Malahayati. Jurnal Ilmu Kedokteran dan Kesehatan. 2017;4(2):67–73. 23. Andriani R. Hubungan Antara Indeks Massa Tubuh dan Aktivitas Fisik dengan Volume Oksigen Maksimum [skripsi]. Surakarta: Universitas Muhammadiyah Surakarta; 2016. 24. Hidayah I. Peningkatan Kadar Asam Laktat dalam Darah Sesudah Bekerja. The Indonesian Journal of Occupational Safety and Health. 2018;7(2):141–131. 25. Saminan. Efek Kelebihan Berat Badan terhadap Pernafasan. Jurnal Kedokteran Nanggroe Medika. 2019;2(4):27–33. 26. Gili SS, Pramita I, Permadi AW. Hubungan Obesitas Pada Anak Terhadap Mobilitas Thorax Saat Inspirasi Di Desa Tegal Kertha, Kecamatan Denpasar Barat, Kota Denpasar, Bali. Physiotherapy Heath Science. 2021;3(2):58–66. Medula \| Volume 13 \| Nomor 1 \| April 2023 \| 34
https://openalex.org/W3214267374	https://figshare.com/articles/journal_contribution/Plastics_in_Australian_Archives_An_Industry_Survey_Regarding_Prevalence_Condition_and_Preservation_Strategies/16929143/1/files/31322255.pdf	English	null	Plastics in Australian Archives: An Industry Survey Regarding Prevalence, Condition, and Preservation Strategies	Studies in conservation	2,021	cc-by	2,137	SUPPLEMENTARY MATERIAL SUPPLEMENTARY MATERIAL Questionnaire Questionnaire Part I: Participant information (1) Your role at your institution is best described as: • Archivist, records manager or archive specialist • Conservator or preservation specialist • Curator or museum specialist • Librarian or library specialist • Other: (2) At your institution, what is the approximate number of staff members (individuals financially compensated for ongoing full-time or part-time work)? • 100 or more • 50 to 99 • 20 to 49 • 10 to 19 • 5 to 9 • 1 to 4 • 0 (volunteers only) (3) Are archive-based activities the primary function of your institution (e.g. independent archive), or is archiving part of a larger organisation’s activities (e.g. archive in a museum or school)? • Archiving is the primary function • Archive is part of a larger organisation (3) Are archive-based activities the primary function of your institution (e.g. independent archive), or is archiving part of a larger organisation’s activities (e.g. archive in a museum or school)? • Archiving is the primary function • Archive is part of a larger organisation 1 (4) Which of the following material types is most representative of the composition of your archive-based collection? • Paper-based records • Analogue audio-visual records (e.g. film) • Digital records • Mixture of above • Other: (5) According to map provided below, which Group best describes the climate at your institution’s geographic location? [Image not reproduced due to copyright. Köppen-Geiger climate map of Australia’ available from https://people.eng.unimelb.edu.au/mpeel/koppen.html, from the article Peel MC, Finlayson BL & McMahon TA 2007, Updated world map of the Köppen-Geiger climate classification, Hydrol. Earth Syst. Sci., no. 11, 1633-1644.] • Group A: Af, Am, Aw (tropical) • Group B: BWh, BWk, BSh, BSk (dry) • Group C: Csa, Csb, Cwa, Cfa, Cfb (temperate) (4) Which of the following material types is most representative of the composition of your archive-based collection? (5) According to map provided below, which Group best describes the climate at your institution’s geographic location? [Image not reproduced due to copyright. Köppen-Geiger climate map of Australia’ available from https://people.eng.unimelb.edu.au/mpeel/koppen.html, from the article Peel MC, Finlayson BL & McMahon TA 2007, Updated world map of the Köppen-Geiger climate classification, Hydrol. Earth Syst. Sci., no. 11, 1633-1644.] • Group A: Af, Am, Aw (tropical) • Group B: BWh, BWk, BSh, BSk (dry) • Group C: Csa, Csb, Cwa, Cfa, Cfb (temperate) • Group C: Csa, Csb, Cwa, Cfa, Cfb (temperate) Part II: Collection plastic types (6) To the best of your knowledge, does your archive-based collection hold any of the following plastic-containing objects (select all that apply)? (6) To the best of your knowledge, does your archive-based collection hold any of the following plastic-containing objects (select all that apply)? • Photographic bases (e.g. negatives, microfilms, animation cels) • Mechanical sound carriers (e.g. gramophone records, cylinders) • Magnetic tapes (e.g. audio cassettes, video tapes, floppy discs) • Optical discs (e.g. CDs, DVDs) • Digital storage drives (e.g. computer hard disks) • Plastic book bindings (e.g. comb bindings, spiral bindings, faux leather covers) • Plastic document organisers (e.g. binders, albums, boxes) • Plastic document protection (e.g. laminated papers, sleeves, envelopes) • Coated papers or synthetic papers (e.g. Polyart, Yupo, resin-coated papers) • Photographic bases (e.g. negatives, microfilms, animation cels) • Mechanical sound carriers (e.g. gramophone records, cylinders) • Magnetic tapes (e.g. audio cassettes, video tapes, floppy discs) • Plastic document protection (e.g. laminated papers, sleeves, envelopes) • Coated papers or synthetic papers (e.g. Polyart, Yupo, resin-coated papers) 2 2 : For Question 7, only object types selected in Question 6 were shown to the participant. (7) Which of the following plastic-containing archive-based objects have you observed to be in poor condition over the course of your work (select all that apply)? Note: ‘Poor condition’ is here used to describe an object that you would consider warrants immediate or prompt intervention from a conservator. • Photographic bases (e.g. negatives, microfilms, animation cels) • Photographic bases (e.g. negatives, microfilms, animation cels) • Mechanical sound carriers (e.g. gramophone records, cylinders) • Magnetic tapes (e.g. audio cassettes, video tapes, floppy discs) • Optical discs (e.g. CDs, DVDs) • Digital storage drives (e.g. computer hard disks) • Plastic book bindings (e.g. comb bindings, spiral bindings, faux leather covers) • Plastic document organisers (e.g. binders, albums, boxes) • Plastic document protection (e.g. laminated papers, sleeves, envelopes) • Coated papers or synthetic papers (e.g. Polyart, Yupo, resin-coated papers) • Coated papers or synthetic papers (e.g. Polyart, Yupo, resin-coated papers) Note: For Question 8, object types selected in Question 7 were shown to the participant as the first column of a table, with each degradation type listed as the subsequent column headings. (8) For the archive-based object types selected in the previous question, please select the type of degradation change you observed: • Colour (e.g. yellowing) • Structure (e.g. cracking) • Shape (e.g. Part II: Collection plastic types warping) • Odour (e.g. acidic smell) • Hardness (e.g. embrittlement) • Texture (e.g. sticky) • Colour (e.g. yellowing) • Structure (e.g. cracking) • Shape (e.g. warping) • Odour (e.g. acidic smell) • Hardness (e.g. embrittlement) • Texture (e.g. sticky) If there are other types of degradation you have observed, please briefly describe them here: If there are other types of degradation you have observed, please briefly describe them here: (9) To the best of your knowledge, are any of the following malignant polymer types present in your archive-based collection (select all that apply)? • Cellulose nitrate (e.g. analogue film, book coatings) • Cellulose acetate (e.g. analogue film, laminated paper) • Poly(vinyl chloride) (e.g. gramophone records, binder covers, document sleeves) • Polyurethane (e.g. faux leather covers, foams, magnetic tapes) • Rubber (e.g. elastic bands) • None of the above • Not sure/unknown 3 3 Part III: Current strategies Part III: Current strategies (10) Does your institution employ material compatibility or safety tests for new materials when they are first introduced for use in storage or display (e.g. VOC, Oddy, PAT, pH testing)? (10) Does your institution employ material compatibility or safety tests for new materials when they are first introduced for use in storage or display (e.g. VOC, Oddy, PAT, pH testing)? • Yes, always (11) Does your institution employ any of the following strategies for plastics identified to be in poor condition, excluding film collections (select all that apply)? Note: ‘Poor condition’ is here used to describe an object that you would consider warrants immediate or prompt intervention from a conservator. • Cold or cool storage (e.g. cold vaults, freezers) • Desiccants (e.g. molecular sieves, charcoal) • Ventilation (e.g. ventilated boxes) • Separation of plastics by polymer type (e.g. separation of cellulose acetate, cellulose nitrate and polyester) • Monitoring techniques (e.g. A-D strips) • Replacement of aged plastic storage materials with new ones (e.g. enclosures, sleeves or bags) • Removal of plastic components (e.g. disbinding or delaminating) • Digitisation • None of the above are employed for plastics • Other: Note: Questions 12 to 14 were only shown to the participant if ‘photographic bases’ was selected in Question 6. (12) Does your institution employ any of the following strategies for film preservation (select all that apply)? • Cold or cool storage (e.g. cold vaults, freezers) • Desiccants (e.g. molecular sieves, charcoal) • Ventilation (e.g. Part II: Collection plastic types ventilated film cannisters) • Separation of films by polymer type (e.g. separation of cellulose acetate, cellulose nitrate and polyester) • Monitoring techniques (e.g. A-D strips) • Plastic storage materials (e.g. enclosures, sleeves or bags) • Duplication • Digitisation • None of the above are employed for film • Other: 4 (13) To the best of your knowledge, the storage environment for your institution’s film collection is (round values to the closest integer): • Storage area 1: o Temperature (°C): _________ ± __________ o Humidity (%): _________ ± __________ • Storage area 2 (if any): o Temperature (°C): _________ ± __________ o Humidity (%): _________ ± __________ (14) If your institution does not use cold or cool storage for film collections, why not (select all that apply)? (14) If your institution does not use cold or cool storage for film collections, why not (select all that apply)? (14) If your institution does not use cold or cool storage for film collections, why not (select all that apply)? • Collection is stored below 0 degrees Celsius using other means Part IV: Useful resources (15) On a scale of 1 to 5, how would you rate the usefulness of the following proposed resources at your institution (1 = not at all useful, 5 = very useful)? • Digital reference database of plastic materials • Workshop for plastics identification • Standards for plastics preservation (16) On a scale of 1 to 5, how would you rate the need for the following areas of knowledge at your institution (1 = not at all needed, 5 = very needed)? • Identification techniques for plastics • Condition analysis techniques for plastics • Degradation patterns of different types of plastics • Treatment techniques for plastics • Storage techniques for plastics (17) If you have any further comments you would like to express concerning plastic types, deterioration, preservation strategies and resources, please enter them here. If there are any (17) If you have any further comments you would like to express concerning plastic types, deterioration, preservation strategies and resources, please enter them here. If there are any other institutions you think we should contact, please also enter their details here: (17) If you have any further comments you would like to express concerning plastic types, deterioration, preservation strategies and resources, please enter them here. If there are any other institutions you think we should contact, please also enter their details here: End of survey 5 5 SM Figure 1. Employment roles of respondents (n = 46). Only 3 were conservators. SM Figure 2. Approximate number of staff in responding institutions (n = 46). 3 volunteer-only archives with no staff members are shown. SM Figure 1. Employment roles of respondents (n = 46). Only 3 were conservators. SM Figure 1. Employment roles of respondents (n = 46). Only 3 were conservators. SM Figure 1. Employment roles of respondents (n = 46). Only 3 were conservators. SM Figure 2. Approximate number of staff in responding institutions (n = 46). 3 volunteer-only archives with no staff members are shown. SM Figure 2. Approximate number of staff in responding institutions (n = 46). 3 volunteer-only archives with no staff members are shown. 6 SM Table 1. Fisher’s exact test on the physical formats collected by paper and mixed collections. No significant difference was found between the two collection types (p < 0.05). SM Table 1. Fisher’s exact test on the physical formats collected by paper and mixed collections. an = sample size, y = number of ‘yes’ responses, m = y/n, p = two-sided significance value. Part IV: Useful resources No significant difference was found between the two collection types (p < 0.05). mixed (n = 22)a paper (n = 22) Physical format ymixed mmixed ypaper mpaper mmixed - mpaper p Photographic 21 .95 21 .95 .00 1.000 Mechanical 13 .59 8 .36 .23 .227 Magnetic 21 .95 21 .95 .00 1.000 Optical 22 1.00 20 .91 .09 .488 Electronic 15 .68 18 .82 -.14 .488 Visual 13 .59 7 .32 .27 .129 Bindings 22 1.00 21 .95 .05 1.000 Organisers 20 .91 21 .95 -.05 1.000 Protectors 20 .91 22 1.00 -.09 .488 an = sample size, y = number of ‘yes’ responses, m = y/n, p = two-sided significance value. SM Table 2. Fisher’s exact test on the implementation of preservation techniques for non-film plastics comparing paper and mixed collections. No significant difference was found between the two collection types (p < 0.05). mixed (n = 22)a paper (n = 22) Preservation technique ymixed mmixed ypaper mpaper mmixed - mpaper p Cold/cool 6 .27 8 .36 -.09 .747 Desiccants 0 .00 2 .09 -.09 .488 Ventilation 5 .23 5 .23 .00 1.000 Separation 10 .45 6 .27 .18 .347 Monitoring 3 .14 4 .18 -.05 1.000 Replacement 19 .86 19 .86 .00 1.000 Removal 14 .64 15 .68 -.05 1.000 Digitisation 14 .64 17 .77 -.14 .510 None 2 .09 3 .14 -.05 1.000 an = sample size, y = number of ‘yes’ responses, m = y/n, p = two-sided significance value. SM Table 2. Fisher’s exact test on the implementation of preservation techniques for non-film plastics comparing paper and mixed collections. No significant difference was found between the two collection types (p < 0 05) SM Table 2. Fisher’s exact test on the implementation of preservation techniques for non-film plastics comparing paper and mixed collections. No significant difference was found between the two collection types (p < 0.05). an = sample size, y = number of ‘yes’ responses, m = y/n, p = two-sided significance value. 7
https://openalex.org/W2993338317	https://europepmc.org/articles/pmc6943541?pdf=render	English	null	Review of Ginseng Anti-Diabetic Studies	Molecules/Molecules online/Molecules annual	2,019	cc-by	11,400	Received: 19 September 2019; Accepted: 6 December 2019; Published: 9 December 2019 Received: 19 September 2019; Accepted: 6 December 2019; Published: 9 December 2019 Abstract: Ginseng is one of the most valuable and commonly used Chinese medicines not only in ancient China but also worldwide. Ginsenosides, also known as saponins or triterpenoids, are thought to be responsible for the beneficial effects of ginseng. In this review, we summarize recent publications on anti-diabetic studies of ginseng extracts and ginsenosides in cells, animals, and humans. It seems that the anti-diabetic eﬀect of ginseng is positive for type 2 diabetic patients but has no signiﬁcant impact on prediabetes or healthy adults. Regulation of insulin secretion, glucose uptake, anti-oxidative stress, and anti-inﬂammatory pathways may be the mechanisms involved with ginseng’s anti-diabetic eﬀects. Taken together, this summary provides evidence for the anti-diabetes eﬀects of ginseng extracts and ginsenosides as well as the underlying mechanisms of their impact on diabetes. Keywords: ginseng; ginsenosides; anti-diabetes; insulin; blood glucose molecules molecules molecules Review www.mdpi.com/journal/molecules Molecules 2019, 24, 4501; doi:10.3390/molecules24244501 Wei Chen 1,2,3 , Prabhu Balan 2,3 and David G. Popovich 1,* 2 Riddet Institute, Massey University, Palmerston North 4442, New Zealand; P.Balan@massey.ac.nz 3 Alpha-Massey Natural Nutraceutical Research Centre, Massey University, Palmerston North 4442, New Zealand * Correspondence: D.G.Popovich@massey.ac.nz; Tel.: +64-6356-9099 1. Introduction As a medicinal intervention management to diabetes, the Asian (Panax ginseng) and American (Panax quinquefolius L.) ginsengs are the two most widely used varieties and have recently attracted a lot of attention from those trying to understand the molecular mechanisms of ginseng’s anti-diabetic eﬀects. Bureau of People’s Welfare Pharmacies) recorded that ginseng was employed to cure Xiaoke disease, which is nowadays known as diabetes [10]. As a medicinal intervention management to diabetes, the Asian (Panax ginseng) and American (Panax quinquefolius L.) ginsengs are the two most widely used varieties and have recently attracted a lot of attention from those trying to understand the molecular mechanisms of ginseng’s anti-diabetic eﬀects. Ginseng contains diversiﬁed components, including saponins, polysaccharides, polyacetylenes, phenols, and alkaloids. Ginseng saponins, known as ginsenosides, are an important class of natural triterpene saponins, which are thought to be responsible for the anti-diabetic eﬀect in ginseng. So far, nearly two hundred ginsenosides have been detected from ginseng plants and heat-processed ginseng products [11]. Most known ginsenosides are classiﬁed as members of the dammarane family, and consist of a four-ring, steroid-like structure, such as ginsenosides Rb1, Rg3, Re, and Rg1 (structures shown in Figure 1). Increasing studies suggest that ginseng extracts, processed ginseng extracts, and ginsenosides show anti-diabetic eﬀects, although the mechanism of action is still not clear. A systematic summary of anti-diabetic studies of ginseng will be helpful to explore its anti-diabetic mechanism. Molecules 2019, 24, x FOR PEER REVIEW 3 of 19 Figure 1. Chemical structures of triterpenoid saponins mentioned in this study. Glc, Rha, Arap, Araf, and Xyl refer to β-D-glucopyranosyl, α-L-rhamnopranosyl, α-L-arabinopyranosyl, α-L- arabinofuranosyl, and β-D-xylopyranosyl, respectively. Figure 1. Chemical structures of triterpenoid saponins mentioned in this study. Glc, Rha, Arap, Araf, and Xyl refer to β-d-glucopyranosyl, α-l-rhamnopranosyl, α-l-arabinopyranosyl, α-l-arabinofuranosyl, and β-d-xylopyranosyl, respectively. Figure 1. Chemical structures of triterpenoid saponins mentioned in this study. Glc, Rha, Arap, Araf, and Xyl refer to β-D-glucopyranosyl, α-L-rhamnopranosyl, α-L-arabinopyranosyl, α-L- arabinofuranosyl, and β-D-xylopyranosyl, respectively. Figure 1. Chemical structures of triterpenoid saponins mentioned in this study. Glc, Rha, Arap, Araf, and Xyl refer to β-d-glucopyranosyl, α-l-rhamnopranosyl, α-l-arabinopyranosyl, α-l-arabinofuranosyl, and β-d-xylopyranosyl, respectively. 2. Anti-Diabetic Effects of Ginseng in Human Trials Although ginseng has been used as a natural herb medicine for a long time in East Asia, interest in the anti-diabetic efficacy of ginseng has drawn increased attention from modern medicine. 1. Introduction The main type of diabetes is type 1 diabetes, which is caused by insulin deﬁciency, and type 2 diabetes, which is characterized by insulin resistance [1]. The majority of diabetes is type 2 diabetes, and it is a formidable challenge for public health [2]. According to the International Diabetes Federation statistics, 463 million people aged 20–79 years worldwide had type 2 diabetes in 2019, and this number is estimated to reach 700 million by 2045 [3]. There is also a huge population with prediabetes, which is very likely to develop into type 2 diabetes [4]. Without proper management, diabetes can cause serious health problems, especially diabetic complications. For example, it can damage eyes, kidneys, and nerves, and it also causes heart disease, stroke, and can even necessitate limb removal [5]. Thus, proper treatment of diabetes is important. Although many researchers are looking at ways to reduce insulin resistance to treat type 2 diabetes, type 2 diabetes is still a severe disease and cannot be cured [6]. Currently, management of diabetes mainly focuses on insulin or its peptide derivatives, anti-diabetic oral drugs, and diet control [7]. Insulin and its peptide derivative treatments need to be injected via vein every day, leading to inconvenience and burdens for patients [8]. Long-term oral administration of chemical drugs can be harmful for many diabetic patients because of drug toxicities [8]. Researchers are exploring the possibilities oﬀered by complementary and alternative medicine, such as traditional herbal medicine. There has been increasing popularity in Chinese medicine, especially since the 2015 Nobel Prize was awarded to Tu Youyou, who discovered that artemisinin from traditional Chinese medicine can treat malaria [9]. This popularity indicates that even in modern times, ancient Chinese herbal remedies can be valuable as an alternative therapy for relieving serious diseases. One of the Chinese medicinal herbs with promising anti-diabetic eﬀects is ginseng [10]. In fact, ginseng has long been used to treat diabetes in China. In the Song Dynasty (1078 A.D.), oﬃcial documentation (Formularies of the Molecules 2019, 24, 4501; doi:10.3390/molecules24244501 www.mdpi.com/journal/molecules 2 of 16 Molecules 2019, 24, 4501 Bureau of People’s Welfare Pharmacies) recorded that ginseng was employed to cure Xiaoke disease, which is nowadays known as diabetes [10]. 2. Anti-Diabetic Eﬀects of Ginseng in Human Trials Although ginseng has been used as a natural herb medicine for a long time in East Asia, interest in the anti-diabetic efficacy of ginseng has drawn increased attention from modern medicine. An increasing number of human trials are exploring the efficacy of ginseng intervention in diabetes. Shishtar et al. found ginseng signiﬁcantly reduced fasting blood glucose compared to the control (−0.31 mmol/L [95% CI: −0.59 to −0.03], p = 0.03) based on a meta-analysis of sixteen randomized controlled clinical trials; there was no signiﬁcant eﬀect on fasting plasma insulin and glycated hemoglobin [13]. Another meta-analysis included eight trials that suggested ginseng supplementation showed signiﬁcant diﬀerences on fasting glucose (−0.306 mmol/L [95% CI: −0.539 to −0.074], p = 0.01), postprandial insulin (−2.132 mmol/L [95% CI: −3.706 to −0.558], p = 0.008), and HOMA-IR (−0.397 mmol/L [95% CI: −0.679 to −0.115], p = 0.006) compared to the control group; there was no signiﬁcant diﬀerence on postprandial glucose and fasting insulin between ginseng treatment and control groups [14]. Since 2012, there have been 13 human trials published that have focused on the anti-diabetic eﬀect of ginseng (Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius L.)). Diﬀerent ginseng extracts or diﬀerently processed ginseng products exhibited varied impact on diabetic patients. The information of ginseng extracts mentioned in this study is shown in Table 1. Ten clinical trials on Asian ginseng have been reported in recent years. In one trial, thirty-six diabetic patients were randomized to receive 1.5 g/day of ginsam, which is enriched ginsenoside Rg3, though a vinegar extraction from P. ginseng, or a daily placebo for eight weeks. HbA1c levels and fasting blood glucose in the ginsam-treated group signiﬁcantly reduced by 0.56% and 21.40 mg/dL compared to the placebo group [15]. Park et al. reported that 23 impaired fasting glucose participants were randomly administrated with 960 mg/day of hydrolyzed Asian ginseng extract or a placebo for eight weeks. The hydrolyzed ginseng extract, containing 7.54 mg/g of Rg1, 6.30 mg/g of compound K, 5.42 mg/g of Rb1, 1.87 mg/g of Re, 0.70 mg/g of Rd, 0.36 mg/g of Rb2, and 0.29 mg/g of Rc, signiﬁcantly decreased fasting plasma glucose (p = 0.017) and postprandial glucose (p = 0.01) compared to the placebo but there were no signiﬁcant diﬀerences in fasting plasma insulin and postprandial insulin between the groups [16]. 1. Introduction An Until 2011, there were eleven publications (twelve human trials) about ginseng extracts being used for diabetes intervention. Four of them studied American ginseng root extracts, ﬁve of them studied Asian ginseng root extracts, two used ginsenosides (Rb1 and Rg1, Re), and one tested eight types of 3 of 16 Molecules 2019, 24, 4501 ginseng (American, American-wild, Asian, Asian-red, Vietnamese-wild, Siberian, Japanese-rhizome, and Sanchi ginseng) on acute postprandial glycemic indices in healthy humans. Through summarizing these twelve human trials, along with in vitro cell studies and in vivo animal studies, the authors deduced that ginseng may modulate insulin production/secretion, glucose metabolism and uptake, or inﬂammatory pathway to exert anti-diabetic eﬀects [12]. Recently, many studies, including human studies, in vitro and in vivo studies have been conducted on diabetes intervention with ginseng extracts or ginsenoside. In order to better know the progress and basis for the evaluation of the anti-diabetic eﬀects of ginseng, this current review summarizes the research into type 2 diabetes intervention with ginseng extracts or ginsenosides conducted in human trials, in vivo animal studies, and in vitro cell studies since 2012. 2. Anti-Diabetic Eﬀects of Ginseng in Human Trials p In a randomized, double-blind, placebo-controlled trial involving forty-two subjects, 2.7 g/day of fermented red ginseng given for four weeks signiﬁcantly increased postprandial insulin of subjects with impaired fasting glucose or type 2 diabetes (35.5 µU/mL in the placebo group vs. 56.3 µU/mL in the ginseng group, p = 0.040). Moreover, in the ginseng treatment group, their postprandial glucose and glucose area under curves (AUC) signiﬁcantly reduced by 17.2% (p = 0.0001) and 27.4% (p = 0.002) compared to the baseline values. However, the ginseng treated group did not show a remarkable diﬀerence in fasting blood glucose and insulin relative to the placebo [21]. It is known that fermentation can increase the bioavailability (absorption and bioactivity) of ginseng due to small ginsenosides (ginsenoside Rg3, compound K, etc.) produced through the fermentation process. As reported by Bang et al. in the clinical trial [20], 5 g/day of Korean red ginseng can reduce fast blood glucose and glucose AUC. However, in this study [21], the fermented red ginseng only modulated postprandial glucose and insulin, not fasting glucose and insulin. The short period of time (four weeks) may be responsible for the lack of signiﬁcant diﬀerences in fasting glucose and insulin because the improvement of fasting glucose and insulin is usually expected over a long-term treatment, while postprandial glucose and insulin are more sensitive to slight interventions in glycemic control. Long-term clinical trials for fermented red ginseng are needed. These studies suggest that diﬀerent Asian ginseng extracts consistently reduce the fasting blood glucose or postprandial glucose in type 2 diabetic patients and could be considered as an optional therapy for managing type 2 diabetes. However, other results from human studies cast doubts about this view. Sixty-eight obese subjects without diabetes were administrated 6 g/day of KRG (orally) or a placebo for 12 weeks in a randomized, double-blind, and placebo-controlled trial. This study showed that there was no signiﬁcant eﬀect on the insulin level and the insulin sensitivity index between the ginseng treatment group and placebo group [22]. Another randomized, double-blind, placebo-controlled 8-week trial conducted among ﬁfty obese women found that there were signiﬁcant improvements in the obesity index between before and after treatment with 6 g/day KRG, but no signiﬁcant diﬀerence between the KRG treatment group and the placebo group [23]. Both of these studies indicate that KRG does not enhance insulin sensitivity in obese people without diabetes. 2. Anti-Diabetic Eﬀects of Ginseng in Human Trials These results showed that hydrolyzed ginseng extract cut the absorption of glucose in the intestinal lumen. Further large-scale and long-term studies are needed to fully evaluate whether it can decrease insulin secretion. The ginseng berry has a diﬀerent ginsenoside composition and contains more plentiful ginsenosides than its root [17]. An in vivo study using ob/ob diabetic mice reported that after being treated with 150 mg/kg ginseng berry extract, fasting blood glucose signiﬁcantly decreased to 150 mg/dL on day 5 and 129 mg/dL on day 12. The same experiment was carried out with ginseng root extract. Although the same dose of ginseng root extract signiﬁcantly reduced the fasting blood glucose to 143 mg/dL on day 5, the fasting blood glucose had not decreased by day 12 (155 mg/dL) [18]. This study showed that ginseng berry extract exhibited more potent anti-hyperglycemic eﬀects compared to ginseng root extract administrated at the same concentrations using ob/ob diabetic mice. Recently, a 12-week, randomized, double-blind, placebo-controlled clinical trial was completed with 72 participants with a fasting glucose level ranging from 100–140 mg/dL. The study showed that ginseng berry extract 4 of 16 Molecules 2019, 24, 4501 signiﬁcantly reduced fasting blood glucose and postprandial glucose at 60 min in an oral glucose tolerance test after a 12-week treatment, but it did not regulate serum glucose to normal levels [19], indicating that the ginseng berry extract failed to show anti-hyperglycemic eﬀects in this human study. It should be noted that the participants in this study were prediabetes rather than type 2 diabetic patients. The anti-hyperglycemic eﬀect was observed in the ob/ob diabetic mice model [18]. Maybe the anti-hyperglycemic eﬀect of ginseng berry extract is positive for type 2 diabetes rather than prediabetes. Further investigation of people with type 2 diabetes is needed to test this hypothesis. Participants with impaired fasting glucose, impaired glucose tolerance, or type 2 diabetes consuming 5 g/day Korean red ginseng (KRG) extract were found to have signiﬁcantly reduced blood glucose. Speciﬁcally, −1.23 ± 0.59 mmol/L of serum glucose and –0.97 ± 0.30 mmol/L of whole blood glucose were decreased at a 30 min oral glucose tolerance test after a 12-week intervention [20]. The plasma insulin sensitivity index increased by 33% compared to the placebo group in this 12-week randomized, double-blind and placebo-controlled trial [20]. 2. Anti-Diabetic Eﬀects of Ginseng in Human Trials In both trials, the regulation of glucose homeostasis was not observed in the prediabetes situation for subjects who were obese without diabetes, impaired glucose tolerance or mild type 2 diabetes. Recently, a multicenter, double-blind, randomized, and placebo-controlled trial was conducted on 1000 healthy adults. The test group consumed 2 g/day of KRG for 24 weeks, which was found to be safe and well-tolerated in healthy adults, and there were no signiﬁcant abnormal changes from anthropometric, laboratory, and vital sign measurements between the KRG group and the placebo group [24]. Therefore, it seems that the anti-diabetic eﬀect of Asian ginseng is positive for type 2 diabetic patients, but there has no signiﬁcant eﬀect on prediabetes or healthy adults. 5 of 16 Molecules 2019, 24, 4501 Besides the human trials using Asian ginseng, there have been three trials using American ginseng on type 2 diabetic patients. In one study, thirty type-2 patients were involved in a randomized, placebo-controlled crossover trial. HbA1c levels were 0.31% lower (p = 0.011) after treatment with 6 g konjac-glucomannan-based ﬁber blended together with 3 g American ginseng per day for 12 weeks. Plasma lipids of the ginseng intervention group also signiﬁcantly decreased compared to the placebo control group [25]. This study did not consider the eﬀect of konjac-glucomannan-based ﬁber on diabetes. The study did not report whether the konjac-glucomannan-based ﬁber blend or the American ginseng was responsible for reducing the glucose levels in the trial subjects. The study could be improved by planning another group administrated with konjac-glucomannan-based ﬁber, or adding it to the placebo. Vuksan et al. [26] conducted a randomized, double-blind, and placebo-controlled crossover clinical study to assess the anti-diabetic eﬃcacy and safety of American ginseng. Twenty-four individuals with well-controlled type 2 diabetes completed the study. Using a double-blind, crossover design, the participants were randomized to receive either 3 g/day of American ginseng extract or a placebo for eight weeks together with their original treatment. The eﬃcacy was assessed by HbA1c, and safety was assessed by liver and kidney function testing. The researchers found that American ginseng signiﬁcantly reduced HbA1c levels (−0.29%, p = 0.041), fasting blood glucose (−0.71 mmol/L, p = 0.008), and systolic blood pressure (−5.6 mmHg, p < 0.001) compared to the placebo. Furthermore, the beneﬁcial changes did not aﬀect the safety proﬁles. 2. Anti-Diabetic Eﬀects of Ginseng in Human Trials Another human study speciﬁcally tested the safety of American ginseng as an adjunct to conventional therapy in type 2 diabetes [27]. Seventy-four type 2 diabetic patients were given 3 g/day American ginseng extract or a placebo for 12 weeks. The investigators found that there was no signiﬁcant diﬀerence between the ginseng treatment and the placebo in the safety parameters, such as kidney function (urates and creatinine), liver function (AST and ALT), and hemostatic function (PT and INR). These studies suggest that American ginseng extract is eﬀective and safe as an additional treatment in the management of type 2 diabetes. Together, these human studies show that both Asian ginseng and American ginseng do indeed decrease blood glucose in type 2 diabetics. At the same time, it should be noted that this efficacy was observed in several limited ginsenosides (6–7 ginsenosides analyzed), small sample sizes (23–94 subjects), and short-term studies (4–12 weeks). Larger scale clinical trials are needed to completely illuminate the long-term beneﬁts of this herbal supplement in the management of type 2 diabetes. The studies are summarized in Table 2. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng Apart from the clinical evidence for the anti-diabetic eﬀect of ginseng, there have also been animal studies conducted to explore the underlying mechanisms of the anti-diabetic eﬀect of ginseng, along with some cell experiments in vitro. Diﬀerent processed ginseng extracts and some individual ginsenosides (structures shown in Figure 1) have been reported to exhibit an anti-diabetic eﬀect in cell and animal experiments (Tables 3 and 4). Although the anti-diabetic mechanism of ginseng has not been entirely clariﬁed, the available data indicate that the regulation of blood glucose by ginseng is possibly related to the following four aspects. First, ginseng modulates blood glucose levels by improving β-cell function and enhancing insulin sensitivity. P. ginseng berry extract increased β-cell proliferation and insulin secretion to improve glycemic control in streptozotocin (STZ) -induced diabetic mice [28]. Ginseng berry extract could also improve insulin sensitivity in C57BL/6 mice over 15 months old, likely by increasing the activation of IRS-1 and AKT [29]. Kim et al. found that 200 mg/kg of black ginseng extract inhibited β-cell apoptosis and improved islet architecture, which led to the enhancement of β-cell function and reduction of hyperglycemia in STZ-treated mice [30]. While exploring the anti-diabetic component of ginseng extracts, both PPD-type ginsenosides and PPT-type ginsenosides were investigated in animal studies. Ginsenoside Rb1, one of the main PPD-type ginsenosides, was found to reduce symptoms of decreased insulin sensitivity and elevated blood glucose caused by the high-fat diet induction of type 2 diabetic mice [31]. Using the STZ-induced diabetic rat model, researchers found that a main PPT-type 6 of 16 Molecules 2019, 24, 4501 ginsenoside, Rg1, can lower insulin resistance and blood glucose, and also improve the blood lipid proﬁle and liver function [32], suggesting that Rg1 may be a potential adjuvant therapy for type 2 diabetic patients with fatty liver disease. As well as the main components in fresh ginseng extracts, heat-treated ginsenoside Rg3 has also been tested. Ginsenoside Rg3 is the main metabolite degraded from other abundant ginsenosides during heat-processing fresh ginseng to manufacture red ginseng and black ginseng. Kim et al. screened the GLP-1 release ability of 15 ginsenosides and found that Rg3 showed the strongest GLP-1 secretion (about 27 pM/mg) eﬀect in NCI-H716 cells. In this in vivo trial, 10 µM Rg3 signiﬁcantly raised production of GLP-1 and insulin to reduce blood glucose in db/db mice [33]. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng In mouse islet cells, insulin secretion was signiﬁcant: 2.3 times higher in the 4 µM Rg3 treatment group compared to control group [34]. Taken together, Rg3 is presumably the main active anti-diabetic ingredient of ginseng, although administration with other ginsenosides has also shown hypoglycemic eﬀects; these ginsenosides can be hydrolysed to Rg3 in the gastrointestinal tract [35] to further exert pharmacological eﬀects. Table 1. Ginseng materials mentioned in this study. Types Samples Processing Main Components (mg/g Dry Weight) Ref. White ginseng WKGE Shade dried, Soxhlet extracted by water 1.93 Re, 1.91 Rc, 1.81 Rb2, 1.56 Rb1, 1.24 Rg1, 0.6 Rf, (×10−3) [36] KWG Air dried 9.1 Re, 3.0 Rg1, 2.4 Rb1, 1.3 Rc, 0.7 Rb2 [37] TCMGAR Air dried 11.2 Rg3, 4.2 Rd, 4.1 Rb1, 3.8 Rh2, 2.3 Rb2, 2.0 Rc [38] AG Air dried 96.7 total ginsenosides (PPD:PPT = 3.03:1) [26] Red ginseng KRG Steamed and air dried 1.93 Rb1, 1.3 Rg3, 1.0 Rd, 0.93 Rb2 [39] KRG Steamed at 90–100 ◦C; for 3 h and air dried 51.6 Rb1, 28.9 Rg1, 22.2 Rc, 21.6 Re, 18.2 Rb2 [22] KRG Steamed and air dried 2.43 Rb1, 1.58 Rg1, 0.95 Rc, 0.89 Rb1, 0.62 Re [40] KRG Steamed and air dried 4.6 Rb1, 2.8 Rc, 2.3 Rb2, 1.4 Rg2, 1.2 Rg3, 1.2 Re [41] KRG Steamed and air dried 8.03 Rb1, 3.29 Rc, 2.80 Rb2, 2.50 Rg3, 1.47 Rf, 1.29 Re, 1.18 Rg1, 1.0 Rd [24] KRG Steamed and air dried 16.58 total ginsenosides (PPD:PPT = 1.65:1) [20] ARG Steamed in autoclave and dried Related fatty acids, 58.1 cinnamic acid, 50.1 ferulic acid [42] Black ginseng BGE Steamed and dried, repeat several cycles, extracted by 70% ethanol at 70 ◦C for 12 h 5.6 C-K, 4.7 Rg5, 1.7 Rg3, 1.5 Rb1, 0.8 Rg2, 0.7 Rc [30] GBG05-FF Repeated steaming at 95 ◦C for 6 h and drying at 60 ◦C, extracted by 70% ethanol at 80 ◦C for 8 h 11.7 Rg5, 6.9 Rk1, 5.2 Rg3, 1.9 Rh4 [43] Fermented ginseng FRG RGE incubated with yeast at 40 ◦C for 12 h 4.9 Rg3, 4.8 Rb1, 3.4 Rb2, 2.9 Rg2, 1.8 Re, 1.4 C-K, 0.8 Rg1 [44] FRG RGE incubated with L. plantarum at 35–40 ◦C for 15 d 4.9 C-K, 3.5 Re, 3.3 Rb1, 3 Rb2, 2.4 Rd, 2 Rc [21] FSGB SGB incubated with L. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng plantarum at 30 ◦C for 72 h Quinic acid, linoleic acid, palmitic acid [45] FGE GE incubated with microorganism 61.0 C-K, 27.7 Rg3, 12.1 Rh1, 9.5 Rd, 8.2 Rg2, 3.1 Rh2, 2.3 Rb2 [46] GS-E3D RGE incubated with 10% pectin lyase at 50 ◦C for 5 d 30.2 Rb1, 17.6 Rb2, 14.0 Rc, 12.6 Re, 5.9 Rg1, 4.7 Rf, 2.7 Rg3, 1.5 Rg5 [47] VEG GE incubated with vinegar (pH 2.3) at 90 ◦C for 6 h 40.5 Rg3, 4.1 Rc, 4.0 Rd, 1.9 Rb2, 1.1 Rf [15] HGE GE incubated with an enzyme solution 7.54 Rg1, 6.3 C-K, 5.42 Rb1, 1.87 Re, 0.70 Rd [16] Fractioned ginseng Rg3-RGE RG multiple extracted by 55% ethanol 51.7 Rg3, 3.86 Rb1, 3.71 Rh1, 3.55 Rg2, 1.6 Rd, 1.53 Rb2 [48] GB Removed seeds and air dried, reﬂuxed with 70% ethanol for 10 h 110.6 Re, 21.1 Rc, 19.0 Rb2, 16.6 Rg1, 16.5 Rd, 8.4 Rg2, 7.7 Rb1 [19] Table 1. Ginseng materials mentioned in this study. Table 1. Ginseng materials mentioned in this study. 7 of 16 Molecules 2019, 24, 4501 Table 2. Eﬀects of ginseng on diabetes-related parameters in human studies. Material Design (Sample Size and Subjects) Drug Treatment and Duration Results Ref. BW HbA1c FBG FI PG PI HOMA-IR Safety Vinegar extract of ginseng (VEG) RCT (72 type 2 diabetic patients) Four group (n = 18/group): 1500, 2000, 3000 mg of VEG, or placebo daily; 8 weeks # + + + + # # * [15] Korean red ginseng (KRG) RCT (50 obese women) 6 g/d of KRG n = 24, or placebo n = 26; 8 weeks + # - # # # # # [23] Korean red ginseng (KRG) Multicenter, RCT (1000 healthy adults) 2 g/d of KRG n = 495, or placebo n = 505; 24 weeks. y weight; FBG: fasting blood glucose; FI: fasting insulin; HbA1c: hemoglobin A1c; HOMA-IR: homeostasis model assessment-insulin resistance; PG: postprandial glucose; PI: dial insulin; RCT: randomized controlled trial. +: indicates the positive outcome with signiﬁcant diﬀerence compared to the control (p < 0.05); -: indicates no signiﬁcant diﬀerences d to the control; #: indicates not tested in the literature; : indicates the safety proﬁles were unaﬀected. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng Eﬀects of ginseng on diabetes-related param Material Dose [Route of Administration] Duration Animal Ginseng extracts Fermented steam-dried ginseng berry (FSGB) 0.5 g/kg [ig] 7 wk db/db mice D Ginseng berry (GB) 100, 200 mg/kg [ig] 10 wk STZ-induced mice E gluc Tissue culture raised mountain ginseng adventitious root (TCMGARs) 125, 250, 500 mg/kg [diet] 4 wk STZ-induced rats Sign Ginseng berry (GB) 0.05% [diet] 6 m C57BL/6 mice (15 months old mice) Wild Korean ginseng extract (WKGE) 100, 200, 300 mg/kg [ig] 8 wk STZ-induced rats Sig Black ginseng extract (BGE) 50, 100, 200 mg/kg [ig] 5 wk STZ-induced mice R in Black ginseng ethanol extract (GBG05-FF) 300, 900 mg/kg [diet] 4 wk db/db mice Re glu lev and Red ginseng (RG) 100, 200 mg/kg [ig] 8 wk ICR mice for type 1 (STZ induced) db/db mice for type 2 Im d de with Korean red ginseng (KRG) 200 mg/kg [ig] 10 wk STZ-induced rats 3 tw Pectin lyase-modiﬁed red ginseng extract (GS-E3D) 25, 50, 100 mg/kg [ig] 6 wk STZ-induced rats De Rg3-enriched red ginseng extract (Rg3-RGE) 2.5, 5 mg/kg [ig] 12 wk LDL-/- mice Re Korean red ginseng (KRG) 200 mg/kg [diet] 12 wk Goto Kakizaki rats Re an Fermented red ginseng (FRG) 0.5%, 1% [diet] 16 wk ob/ob mice D ex Red ginseng extract (RGE) 2 g/kg [ig] 5 wk STZ-induced rats Met re re Fermented ginseng extract (FGE) 0.1% w/w [diet] 8 wk db/db mice De an se Ginseng polysaccharides 0, 12.5, 25, 50, 100, 200 mg/kg [ig] 10 d STZ-induced ICR mice Incr a Ginseng powder 150 mg/kg [ig] 7 d STZ-induced rats Enh American red ginseng (ARG) 150 mg/kg [ip] 30 d db/db mice Red an American ginseng 200 mg/kg [ig] 2 m (type 1), 4 m (type2) C57BL/6 mice for type 1 (STZ- induced), db/db mice for type 2 Inc car American ginseng saponin (PQS) of stem and leaf 30, 60 mg/kg [ig] 8 wk STZ-induced rats Up Table 3. Eﬀects of ginseng on diabetes-related parameters in animal studies. Material Dose [Route of Administration] Duration Animal Molecular Mechanism Ref. Ginseng extracts Fermented steam-dried ginseng berry (FSGB) 0.5 g/kg [ig] 7 wk db/db mice Decreased the blood glucose and body weight; increased the immune cell population and GLUT1 expression. [45] Ginseng berry (GB) 100, 200 mg/kg [ig] 10 wk STZ-induced mice Enhanced beta-cell proliferation and glucose tolerance, decreased blood glucose. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng [28] Tissue culture raised mountain ginseng adventitious root (TCMGARs) 125, 250, 500 mg/kg [diet] 4 wk STZ-induced rats Signiﬁcantly reduced the blood glucose, TC, and TG levels. [38] Ginseng berry (GB) 0.05% [diet] 6 m C57BL/6 mice (15 months old mice) Increased the parameters of insulin sensitivity, IRS, AKT, and FOXO1; decreased PPAR-γ. [29] Wild Korean ginseng extract (WKGE) 100, 200, 300 mg/kg [ig] 8 wk STZ-induced rats Signiﬁcantly reduced blood glucose, ALT and alkaline phosphatase levels. [36] Black ginseng extract (BGE) 50, 100, 200 mg/kg [ig] 5 wk STZ-induced mice Reduced hyperglycemia and NF-κB; increased the insulin/glucose ratio and β-cell function. [30] Black ginseng ethanol extract (GBG05-FF) 300, 900 mg/kg [diet] 4 wk db/db mice Reduced the parameters of fasting blood glucose, glucose tolerance, HbA1c, TG, TC levels, and lipid accumulation; enhanced the phosphorylation of the AMPK, and up-regulated the expressions of GLUT2 and GLUT4. [43] Red ginseng (RG) 100, 200 mg/kg [ig] 8 wk ICR mice for type 1 (STZ induced) db/db mice for type 2 Improved the threshold shift of hearing, delayed latencies, and signal intensity decrease in type 2 diabetic mice; changes with no signiﬁcance in type 1 diabetic mice. [41] Korean red ginseng (KRG) 200 mg/kg [ig] 10 wk STZ-induced rats 39 genes were upregulated more than two-fold; 84 genes were down-regulated. [49] Pectin lyase-modiﬁed red ginseng extract (GS-E3D) 25, 50, 100 mg/kg [ig] 6 wk STZ-induced rats Decreased the urinary levels of albumin, 8-OHdG, and AGEs; suppressed oxidative stress. [47] Rg3-enriched red ginseng extract (Rg3-RGE) 2.5, 5 mg/kg [ig] 12 wk LDL-/- mice Reduced the levels of glucose, TG, LDL, AST, and AST; inhibited atheroma formation. [48] Korean red ginseng (KRG) 200 mg/kg [diet] 12 wk Goto Kakizaki rats Reduced blood glucose, PTP-1B, UCP-2, and PARP; enhanced the production of GLUT-4 and insulin. [40] Fermented red ginseng (FRG) 0.5%, 1% [diet] 16 wk ob/ob mice Decreased body weight, blood glucose, and hyperlipidemia; increased the expressions of IR, LPL, GLUT1, GLUT4, PPAR-γ, and PEPCK. [44] Red ginseng extract (RGE) 2 g/kg [ig] 5 wk STZ-induced rats Metformin or RGE administered alone can reduce the FBG, but co-administration recovered the FBG to the control level. [39] Fermented ginseng extract (FGE) 0.1% w/w [diet] 8 wk db/db mice Decreased blood glucose, HbA1c, TNF-α, and lymphocytes; increased adiponectin, serum insulin, PPAR-γ2, and GLUT-2. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng - # # # # # # [24] Korean red ginseng (KRG) RCT (68 obese participants without diabetes) 6 g/d of KRG n = 34, or placebo n = 34; 12 weeks - # - - # # - # [22] Korean red ginseng (KRG) RCT (41 type 2 diabetic patients) 5 g/d of KRG n = 21, placebo n = 20; 12 weeks - - + + + + + # [20] Fermented red ginseng (FRG) RCT (42 impaired fasting glucose or type 2 diabetic patients) 2.7 g/d of FRG n = 21, placebo n = 21; 4 weeks # # - - + + # * [21] Hydrolyzed ginseng extract (HGE) RCT (23 impaired fasting glucose participants) 960 mg/d of HGE n = 12, placebo n = 11; 8 weeks - # + - + - - * [16] Korean white ginseng (KWG) RCT crossover trial (25 type 2 diabetic patients) 1 g, 3 g, 6 g KWG, or 3 g placebo together with 50 g glucose-load, acute test # # - # - # # * [37] Ginseng berry extract (GBE) RCT (72 participants) 1 g/d GBE n = 34, placebo n = 38; 12 weeks - - + - + - - * [19] American ginseng (AG) RCT parallel trial (74 type 2 diabetic patients) 3 g/d of AG n = 35, placebo n = 39; 12 weeks # # # # # # # * [27] American ginseng (AG) RCT crossover trial (39 type 2 diabetic patients) 6 g/d of ﬁber from KGB together with 3 g AG; 12 weeks - + - - # # # * [25] American ginseng (AG) RCT crossover trial (24 type 2 diabetic patients) 3 g/d AG or placebo with original treatment; 8 weeks - + + - # # # * [26] BW: body weight; FBG: fasting blood glucose; FI: fasting insulin; HbA1c: hemoglobin A1c; HOMA-IR: homeostasis model assessment-insulin resistance; PG: postprandial glucose; PI: postprandial insulin; RCT: randomized controlled trial. +: indicates the positive outcome with signiﬁcant diﬀerence compared to the control (p < 0.05); -: indicates no signiﬁcant diﬀerences compared to the control; #: indicates not tested in the literature; *: indicates the safety proﬁles were unaﬀected. Table 2. Eﬀects of ginseng on diabetes-related parameters in human studies. 8 of 16 Molecules 2019, 24, 4501 Table 3. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng [46] Ginseng polysaccharides 0, 12.5, 25, 50, 100, 200 mg/kg [ig] 10 d STZ-induced ICR mice Increased the concentration of insulin, SOD, and glycogen; decreased the content of MDA. [50] Ginseng powder 150 mg/kg [ig] 7 d STZ-induced rats Enhanced the expression of PPAR-δ and the phosphorylation of troponin1. [51] American red ginseng (ARG) 150 mg/kg [ip] 30 d db/db mice Reduced blood glucose, plasma cholesterol and LDL; increased glycogen and HDL. [42] American ginseng 200 mg/kg [ig] 2 m (type 1), 4 m (type2) C57BL/6 mice for type 1 (STZ- induced), db/db mice for type 2 Increased stroke volume, ejection fraction, cardiac output, and left ventricle pressure; reduced oxidative stress. [52] American ginseng saponin (PQS) of stem and leaf 30, 60 mg/kg [ig] 8 wk STZ-induced rats Up-regulated the expressions of HGF, NO, ET-1, TNF-α, and slCAM-1. [53] Table 3. Eﬀects of ginseng on diabetes-related parameters in animal studies. 9 of 16 Molecules 2019, 24, 4501 Table 3. Cont. Table 3. Cont. Material Dose [Route of Administration] Duration Animal Molecular Mechanism Ref. Ginsenosides Panaxatriol 11 mg/d + 45 min/d (15 m/min) exercise [diet] 6 wk KK-Ay/Ta Jcl (KKAy) mice Signiﬁcantly lowered blood glucose; no signiﬁcant diﬀerences in body weight; signiﬁcantly improved insulin resistance. [54] Rb1 10 mg/kg [ip] 1 wk C57BL/6 mice (high fat) Decreased fasting blood glucose, glucose tolerance, and 11β-HSD1; increased insulin sensitivity. [31] Re 5, 10, 20 mg/kg [ig] 4 wk C57BL/6 mice (high fat) Decreased the parameters of TG, TC, LDL-C, GOT, and GPT; increased HDLC, regulating ACh, AChE, MDA, SOD, and GSH via JNK pathway. [55] Rg1 10 mg/kg [ip] 4 wk STZ-induced rats Improved angiogenesis, increased eNOS activation, up-regulated VEGF expression, and inhibited apoptosis. [56] Rg1 10,15, 20 mg/kg [ip] 12 wk STZ-induced rats Decreased the parameters of cTnI, CK-MB, MDA, apoptosis, and CASP3; increased expressions of SOD, CAT, GSH, and Bcl-xL. [57] Rg1 10,15, 20 mg/kg [iv] 12 wk STZ-induced rats Reduced the expressions of cTnI, GRP78, and CHOP; inhibited endoplasmic reticulum stress-induced apoptosis. [58] Rg1 56.25 µM/kg [iv] 10 d STZ-induced rats Reduced the cerebral infarction volume; promoted neuronal recovery. [59] Rg1 25, 50 mg/kg [ig] 8 wk STZ-induced rats Reduced blood glucose levels and insulin resistance index; increased the parameters of TC, TG, LDL-C, AST, and ALT. [32] Rg3 0.5 mg/kg [ig] Once db/db mice Increased the production of GLP-1 and insulin; decreased blood glucose. 11β-HSD1: 11β-hydroxysteroid dehydrogenase type I; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; Ach: acetylcholine; AChE: acetylcholinesterase; AGEs: advanced glycation end-products; AKT: protein kinase B; ALT: alanine aminotransferase; AMPK: 5’ adenosine monophosphate-activated protein kinase; AST: aspartate aminotransferase; Bcl-xL: B-cell lymphoma-extra-large; BUN: blood urea nitrogen; CASP3: caspase-3; CAT: catalase; CCN2: connective tissue growth factor; CHOP: C/EBP homologous protein; CK-MB: creatinine kinase MB; CTGF: connective tissue growth factor; cTnI: cardiac troponin I; eNOS: endothelial nitric oxide synthase; ET-1: endothelin-1; FBG: fasting blood glucose; FOXO1: forkhead box protein O1; GLP-1: glucagon-like peptide-1; GLUT1: glucose transporter 1; GLUT-2: glucose transporter protein 2; GLUT-4: glucose transporter protein 4; GOT: glutamic-oxaloacetic transaminase; GPT: glutamic-pyruvic transaminase; GRP78: glucose-regulated protein 78; GSH (-PX): glutathione peroxidase; HbA1c: hemoglobin A1c; HDL: high- density lipoprotein; HDL-C: high-density lipoprotein cholesterol; HGF: hepatocyte growth factor; IL: interleukin; IR: insulin receptor; IRS: insulin receptor substrate; JNK: c-Jun N-terminal protein kinase; LDL: low-density lipoprotein; LDL-C: low-density lipoprotein cholesterol; LPL: lipoprotein lipase; β2-MG: β2-microglobulin; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; NF-κB: nuclear factor–κB; NO: nitric oxide; PEPCK: phosphoenolpyruvate carboxykinase; PI3K: phosphoinositide 3 kinase; PPAR-γ2: peroxisome proliferator-activated receptor gamma 2; PPARδ: peroxisome proliferator-activated receptors δ; PTP-1B: protein tyrosine phosphatases-1B; SCr: serum creatinine; Sicam-1: soluble intercellular adhesion molecule 1; SOD: superoxide dismutase; STAT3: signal transducer and activator of transcription 3; STZ: streptozotocin; T-AOC: total anti-oxidative capacity; TC: total cholesterol; TG: triglycerides; TGF-β: transforming growth factor beta; TNF-α: tumor necrosis factor alpha; UCP-2: uncoupling protein 2; UCr: urinary creatinine; VEGF: vascular endothelial growth factor. ip: intraperitoneal injection; iv: intravenous injection; ig: intragastric injection. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng [33] Rh2 5 mg/kg [iv] 4 wk STZ-induced rats Decreased fasting blood glucose, ratio of heart weight/body weight, and PPAR-δ; increased STAT3, CCN2, and ﬁbronectin. [60] Malonyl ginsenosides (MGR) 50, 100 mg/kg [ip] 3 wk STZ-induced rats (high fat) Signiﬁcantly lowered FBG levels; reduced TG and TC; increased glucose disposal; no eﬀect on body weight. [61] Ginsenoside Rg1 + Astragaloside IV (50 Rg1 + 16 IV) mg/kg [ig] 8 wk STZ-induced rats Lowered MDA level and increased levels of CAT, GSH-PX, and T-AOC. Signiﬁcantly reduced the levels of BUN, SCr, β2-MG, and UCr. Diminished the mRNA overexpression of TGF-β1 and CTGF; increased Smad7 expression. [62] Rg3 10 mg/kg/2 days [ip] 4 wk STZ-induced ApoE-/- mice 20(S)-Rg3 showed stronger anti-proliferative and anti-migratory eﬀects than 20(R)-Rg3; (S)-isomer signiﬁcantly reduced the plaque size of diabetic atherosclerosis. [63] Rg3 0.5 mg/kg [ig] 3 m STZ-induced rats (high fat) Inhibited inﬂammatory response (inhibited the expression of TGF-β1, IL-1, IL-6, IL-12) and promoted the activation of PI3K and MAPK signaling pathways to prevent lung tissue damage induced by hyperglycemia. [64] Ref. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng 11β-HSD1: 11β-hydroxysteroid dehydrogenase type I; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; Ach: acetylcholine; AChE: acetylcholinesterase; AGEs: advanced glycation end-products; AKT: protein kinase B; ALT: alanine aminotransferase; AMPK: 5’ adenosine monophosphate-activated protein kinase; AST: aspartate aminotransferase; Bcl-xL: B-cell lymphoma-extra-large; BUN: blood urea nitrogen; CASP3: caspase-3; CAT: catalase; CCN2: connective tissue growth factor; CHOP: C/EBP homologous protein; CK-MB: creatinine kinase MB; CTGF: connective tissue growth factor; cTnI: cardiac troponin I; eNOS: endothelial nitric oxide synthase; ET-1: endothelin-1; FBG: fasting blood glucose; FOXO1: forkhead box protein O1; GLP-1: glucagon-like peptide-1; GLUT1: glucose transporter 1; GLUT-2: glucose transporter protein 2; GLUT-4: glucose transporter protein 4; GOT: glutamic-oxaloacetic transaminase; GPT: glutamic-pyruvic transaminase; GRP78: glucose-regulated protein 78; GSH (-PX): glutathione peroxidase; HbA1c: hemoglobin A1c; HDL: high- density lipoprotein; HDL-C: high-density lipoprotein cholesterol; HGF: hepatocyte growth factor; IL: interleukin; IR: insulin receptor; IRS: insulin receptor substrate; JNK: c-Jun N-terminal protein kinase; LDL: low-density lipoprotein; LDL-C: low-density lipoprotein cholesterol; LPL: lipoprotein lipase; β2-MG: β2-microglobulin; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; NF-κB: nuclear factor–κB; NO: nitric oxide; PEPCK: phosphoenolpyruvate carboxykinase; PI3K: phosphoinositide 3 kinase; PPAR-γ2: peroxisome proliferator-activated receptor gamma 2; PPARδ: peroxisome proliferator-activated receptors δ; PTP-1B: protein tyrosine phosphatases-1B; SCr: serum creatinine; Sicam-1: soluble intercellular adhesion molecule 1; SOD: superoxide dismutase; STAT3: signal transducer and activator of transcription 3; STZ: streptozotocin; T-AOC: total anti-oxidative capacity; TC: total cholesterol; TG: triglycerides; TGF-β: transforming growth factor beta; TNF-α: tumor necrosis factor alpha; UCP-2: uncoupling protein 2; UCr: urinary creatinine; VEGF: vascular endothelial growth factor. ip: intraperitoneal injection; iv: intravenous injection; ig: intragastric injection. 10 of 16 Molecules 2019, 24, 4501 Table 4. Eﬀects of ginseng on diabetes-related molecular targets in cell line studies. Material Cell Line Drug Dose Molecular Mechanism Ref. Ginseng total saponin Mouse podocytes 1 µg/mL for 6, 24, and 48 h Ginseng total saponins improved p130Cas protein of podocytes. [65] Ginsenoside Rg3 Human NCI-H716 10 µM Enhanced secreting of GLP-1. [33] Ginsenoside Rg3 Mouse islet cell 4 µM Increased glucose-induced insulin secretion (2.3-fold higher); enhanced islet function and attenuated apoptosis. [34] Ginsenoside Rg3 Human pulmonary cell BEAS-2B 25, 50, 75, 100 µg/mL No eﬀect on the viability of BEAS-2B cells; high concentration could induce apoptosis compared with the control group. [64] Ginsenoside Rg1 C2C12 muscle cell 10, 20, 40 µM Enhanced glucose uptake and GLUT4 by AMPK pathways. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng [66] Ginsenoside Re 3T3-L1 cell 3, 10, 30, 60 µM Increased glucose uptake and the expressions of PPAR-γ2, IRS-1, ap2, and adiponectin genes expressions; helped the translocation of GLUT4 to the membranes; inhibited the expression and production of TNF-α. [67] Ginsenoside F2 3T3-L1 cell 10, 50, 100 µM Reduced the content of lipid accumulation; down-regulated expression of PPAR-γ and perilipin. [68] Ginsenoside Rg5:Rk1 mixture 3T3-L1 cell 10, 100 µg/mL Inhibited lipid accumulation and reduced TG level. Decreased the mRNA level of STAT3, PPAR-γ, CEBPα, and ap2; reduced protein expression of PPAR-γ and CEBPα. [69] AMPK: adenosine 5’-monophosphate (AMP)-activated protein kinase; ap2: activating protein; CEBPα: CCAAT/enhancer binding protein alpha; GLP-1: glucagon-like peptide-1; GLUT4: glucose transporter protein 4; IRS-1: insulin receptor substrate 1; PPAR: peroxisome proliferators-activated receptors; STAT3: signal transducer and activator of transcription 3; TNF: tumor necrosis factor. Table 4. Eﬀects of ginseng on diabetes-related molecular targets in cell line studies. AMPK: adenosine 5’-monophosphate (AMP)-activated protein kinase; ap2: activating protein; CEBPα: CCAAT/enhancer binding protein alpha; GLP-1: glucagon-like peptide-1; GLUT4: glucose transporter protein 4; IRS-1: insulin receptor substrate 1; PPAR: peroxisome proliferators-activated receptors; STAT3: signal transducer and activator of transcription 3; TNF: tumor necrosis factor. AMPK: adenosine 5’-monophosphate (AMP)-activated protein kinase; ap2: activating protein; CEBPα: CCAAT/enhancer binding protein alpha; GLP-1: glucagon-like peptide-1; GLUT4: glucose transporter protein 4; IRS-1: insulin receptor substrate 1; PPAR: peroxisome proliferators-activated receptors; STAT3: signal transducer and activator of transcription 3; TNF: tumor necrosis factor. Second, ginseng can enhance glucose uptake by up-regulating the expression of glucose transporters (GLUT). One percent Fermented red ginseng (FRG) signiﬁcantly decreased the body weight of ob/ob mice compared with the controls after 4 weeks of administration during the 16-week treatment period; blood glucose was also signiﬁcantly reduced after 16-weeks treatment with FRG [44]. Furthermore, the study found that the expressions of GLUT1 and GLUT4 were signiﬁcantly up-regulated by FRG in liver and muscle. This suggests that FRG may decrease blood glucose through enhancing glucose uptake in skeletal muscle. Similar results were also observed in db/db mice treated with ginseng extracts. 0.5 g/kg fermented steam-dried ginseng berry (FSGB) [45] signiﬁcantly decreased body weight and blood glucose in db/db mice during a 35-day treatment; furthermore, the plasma insulin in the FSGB treated group (17.05 ± 0.49 ng/mL) signiﬁcantly increased almost double compared to the untreated db/db group (8.21 ± 0.60 ng/mL). GLUT1 mRNA expression was up-regulated by FSGB in the L6 skeletal muscle cells. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng During an 8-week treatment with 0.1% fermented ginseng extract (FGE) in db/db mice, the body weight, fasting blood glucose, and HbA1c level signiﬁcantly (p < 0.05) decreased compared to control groups [46]. FGE intervention signiﬁcantly enhanced the mRNA expression of GLUT2 and lowered the glucose-6-phosphotase (G6Pase) expression in liver tissues [46]. G6Pase plays an important role in glucose homeostasis because it is a main enzyme of gluconeogenesis and glycogenolysis in the liver. The reduction of G6Pase and increase of GLUT2 expressions mean FGE may improve the transport of blood glucose into the liver, which leads to decreasing the blood glucose level in db/db mice. Moreover, the FGE group exhibited a higher level of insulin secretion than the control group in the islet primary cell culture; the FGE treated group had weaker damage and inﬂammation than the control group in vivo hematoxylin–eosin staining assay [46]. These suggest that FGE has a protective eﬀect on the pancreas and improves the production/secretion of insulin. Kang et al. found that 900 mg/kg black ginseng ethanol extract (GBG05-FF) signiﬁcantly reduced fasting blood glucose, glucose tolerance, and plasma HbA1c after four weeks of treatment. Furthermore, GBG05-FF up-regulated the expressions 11 of 16 11 of 16 Molecules 2019, 24, 4501 of GLUT2 in the liver and GLUT4 in the muscles [43], indicating that black ginseng can enhance glucose uptake in surrounding tissues to regulate blood glucose levels. In addition, other beneﬁcial physiological changes, such as a reduction of total cholesterol and triglycerides, and increased PPAR-γ and PEPCK were also observed after administration with fermented ginseng berry [46] or black ginseng [43]. Ginsenosides Rg1 [66] and Re [67] increased expressions of GLUT4 through AMPK pathways in C2CI2 muscle cells and via increasing PPAR-γ activity in 3T3-L1 cells, respectively. Furthermore, KRG at a dose of 0.2 g/kg/d for a 12-week treatment period enhanced insulin action and secretion in diabetic Goto-Kakizaki rats by up-regulating the GLUT4 expression in adipose tissue and down-regulating the expressions of UCP2 and PARP in the pancreas, and PTP-1B in adipose tissue and skeletal muscle [40]. Third, suppression of oxidative stress through increasing SOD activity and decreasing MDA production was reported to occur in treatment with either ginseng polysaccharides in STZ-induced ICR diabetic mice [50], or ginsenoside Rg1 in STZ-induced diabetic rats [57]. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng Administration of GS-E3D (pectin lyase-modiﬁed red ginseng extract) for six weeks signiﬁcantly decreased urinary levels of albumin, 8-hydroxy-2′-deoxyguanosine (8-OHdG), and advanced glycation end-products (AGEs) in STZ-induced diabetic rats. Moreover, all symptoms of diabetic nephropathy were improved by GS-E3D treatment via suppressing renal accumulation of AGEs and oxidative stress [47]. Tissue culture raised mountain ginseng adventitious root (TCMGARs) extracts at dosage levels of 250, and 500 mg/kg signiﬁcantly reduced the blood glucose, total cholesterol, and triglyceride content in STZ-induced diabetic rats [38]. In an LDL-/- mouse model, Rg3-enriched red ginseng extract (44.91 mg/g, 66.6% occupy for total ginsenosides) signiﬁcantly decreased the levels of glucose, triglyceride, low-density lipoprotein, alanine aminotransferase and aspartate aminotransferase compared to the control [48]. In a high-fat induced C57BL/6 mouse model, ginsenoside Re signiﬁcantly reduced fasting blood glucose levels and related biochemical parameters, including cholesterol, low-density lipoprotein cholesterol, total triglyceride, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase. Re also regulated the level of ACh, AChE, and oxidative stress-related parameters (MDA, SOD, GSH) via the JNK pathway [55]. In addition to the above three potential anti-diabetic mechanisms, ginseng can also show anti-diabetic activity through other pathways. Several animal studies have found that ginseng processed products or individual ginsenosides regulate blood glucose in diabetic mouse or rat models, accompanied by regulating the expression of TNF-α, eNOS [46,53,56], suggesting that diabetes is associated with inﬂammation, and eﬀective modulation by ginseng on inﬂammation may be able to prevent the development of insulin resistance. Recently, Shen et al. reported that Rg1 released in brain tissue using nano-drug delivery systems reduced the volume of cerebral infarction and improved neural recovery in diabetic rats with cerebral infarction [59], indicating that the hypoglycemic eﬀect of Rg1 may be regulated through the central nervous system. Additionally, in an STZ-induced type 1 diabetic rat model, ginsenoside Rh2 reduced fasting blood glucose and improved cardiac function via enhancing PPAR-δ signaling in diabetic rats with cardiac ﬁbrosis [60]. In the 3T3-L1 cells, researchers that found ginsenoside F2 [68], and a ginsenosides Rg5 + Rk1 mixture [69] could reduce lipid accumulation and down-regulate expression of PPAR-γ. Based on studies on diabetes intervention with ginseng in vivo and in vitro, the potential mechanism of ginseng extracts (ginsenosides) on type 2 diabetes is summarized in Figure 2. 12 of 16 ial Molecules 2019, 24, 4501 p Based on studies 12 of 16 ial Figure 2. 3. Potential Mechanisms of the Anti-Diabetic Eﬀect of Ginseng The potential mechanism of ginseng extracts (ginsenosides) on type 2 diabetes. Bcl-xL: B- cell lymphoma-extra-large; CASP3: caspase-3; CAT: catalase; eNOS: endothelial nitric oxide synthase; G6Pase: glucose-6-phosphotase; GLP-1: glucagon-like peptide-1; GLUT: glucose transporter protein; GSH (-PX): glutathione peroxidase; IL: interleukin; MAPK: mitogen-activated protein kinase; MDA: Figure 2. The potential mechanism of ginseng extracts (ginsenosides) on type 2 diabetes. Bcl-xL: B-cell lymphoma-extra-large; CASP3: caspase-3; CAT: catalase; eNOS: endothelial nitric oxide synthase; G6Pase: glucose-6-phosphotase; GLP-1: glucagon-like peptide-1; GLUT: glucose transporter protein; GSH (-PX): glutathione peroxidase; IL: interleukin; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; PEPCK: phosphoenolpyruvate carboxykinase; SOD: superoxide dismutase; T-AOC: total anti-oxidative capacity; TC: total cholesterol; TG: triglycerides. gure 2. The potential mechanism of ginseng extracts (ginsenosides) on type 2 diabetes. Bcl-xL: B- ll lymphoma-extra-large; CASP3: caspase-3; CAT: catalase; eNOS: endothelial nitric oxide synthase; 6Pase: glucose-6-phosphotase; GLP-1: glucagon-like peptide-1; GLUT: glucose transporter protein; SH (-PX): glutathione peroxidase; IL: interleukin; MAPK: mitogen-activated protein kinase; MDA: Figure 2. The potential mechanism of ginseng extracts (ginsenosides) on type 2 diabetes. Bcl-xL: B-cell lymphoma-extra-large; CASP3: caspase-3; CAT: catalase; eNOS: endothelial nitric oxide synthase; G6Pase: glucose-6-phosphotase; GLP-1: glucagon-like peptide-1; GLUT: glucose transporter protein; GSH (-PX): glutathione peroxidase; IL: interleukin; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; PEPCK: phosphoenolpyruvate carboxykinase; SOD: superoxide dismutase; T-AOC: total anti-oxidative capacity; TC: total cholesterol; TG: triglycerides. 4. Conclusions Acknowledgments: We would like to acknowledge Ceire Hopley (librarian) for her help in literature searching and analysis. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Kim, S.; Shin, B.C.; Lee, M.S.; Lee, H.; Ernst, E. Red ginseng for type 2 diabetes mellitus: A systematic review of randomized controlled trials. Chin. J. Integr. Med. 2011, 17, 937–944. [CrossRef] [PubMed] 8. Che, J.Y.; Lu, D. Rethink Of Diabetes Treatment and Drug Development. Cell Dev. Biol. 2014, 3, e12 . Tu, Y. Artemisinin-A Gift from Traditional Chinese Medicine to the World (Nobel Lecture). Angew. Chem Ed. Engl. 2016, 55, 10210–10226. [CrossRef] 0. Bai, L.; Gao, J.; Wei, F.; Zhao, J.; Wang, D.; Wei, J. Therapeutic Potential of Ginsenosides as an Adjuv Treatment for Diabetes. Front. Pharm. 2018, 9, 423. [CrossRef] 11. Chen, W.; Balan, P.; Popovich, D.G. Chapter 6-Comparison of the ginsenoside composition of Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius L.) and their transformation pathways. Stud. Nat. Prod. Chem. 2019, 63, 161–195. 12. Yuan, H.D.; Kim, J.T.; Kim, S.H.; Chung, S.H. Ginseng and diabetes: The evidences from in vitro, animal and human studies. J. Ginseng Res. 2012, 36, 27–39. [CrossRef] [PubMed] 13. Shishtar, E.; Sievenpiper, J.L.; Djedovic, V.; Cozma, A.I.; Ha, V.; Jayalath, V.H.; Jenkins, D.J.; Meija, S.B.; de Souza, R.J.; Jovanovski, E.; et al. The eﬀect of ginseng (the genus panax) on glycemic control: A systematic review and meta-analysis of randomized controlled clinical trials. PLoS ONE 2014, 9, e107391. [CrossRef] [PubMed] 14. Gui, Q.F.; Xu, Z.R.; Xu, K.Y.; Yang, Y.M. The Eﬃcacy of Ginseng-Related Therapies in Type 2 Diabetes Mellitus: An Updated Systematic Review and Meta-analysis. Medicine (Baltim.) 2016, 95, e2584. [CrossRef] [PubMed] 15. Yoon, J.W.; Kang, S.M.; Vassy, J.L.; Shin, H.; Lee, Y.H.; Ahn, H.Y.; Choi, S.H.; Park, K.S.; Jang, H.C.; Lim, S. Eﬃcacy and safety of ginsam, a vinegar extract from Panax ginseng, in type 2 diabetic patients: Results of a double-blind, placebo-controlled study. J. Diabetes Investig. 2012, 3, 309–317. [CrossRef] 16. Park, S.H.; Oh, M.R.; Choi, E.K.; Kim, M.G.; Ha, K.C.; Lee, S.K.; Kim, Y.G.; Park, B.H.; Kim, D.S.; Chae, S.W. An 8-wk, randomized, double-blind, placebo-controlled clinical trial for the antidiabetic eﬀects of hydrolyzed ginseng extract. J. Ginseng Res. 2014, 38, 239–243. [CrossRef] 17. Kim, Y.K.; Yoo, D.S.; Xu, H.; Park, N.I.; Kim, H.H.; Choi, J.E.; Park, S.U. Ginsenoside content of berries and roots of three typical Korean ginseng (Panax ginseng) cultivars. Nat. Prod. Commun. 2009, 4, 903–906. [CrossRef] 18. Dey, L.; Xie, J.T.; Wang, A.; Wu, J.; Maleckar, S.A.; Yuan, C.S. Anti-hyperglycemic eﬀects of ginseng: Comparison between root and berry. 4. Conclusions Human, animal, and cell studies have shown that diﬀerent processed ginseng extracts and speciﬁc ginsenosides possess beneﬁcial eﬀects on diabetes, especially type 2 diabetes. Most studies of individual ginsenosides have focused on Rb1, Re, or Rg1, which are the main components of ginseng and are easily obtained. However, these ginsenosides have a large molecule structure, resulting in poor systemic bioavailability. Reeds et al. reported on overweight and obese participants with impaired glucose tolerance or newly diagnosed type 2 diabetes who consumed ginseng root extract (8 g/day), ginsenoside Re (250–500 mg/day), or placebo for 30 days; ginsenosides Rb1, Rb2, and Re were not detected in plasma after treatment with ginseng root extract or ginsenoside Re [70]. However, another study conducted in healthy volunteers showed that ginsenoside Re and its potential metabolites (including Rg2, F1, Rh1, and PPT) were detected in plasma after oral administration of ginsenoside Re [71]. It seems that the large molecule ginsenosides (Rb1, Rc, Re) may be a form of storage for saponins in ginseng plants rather than the active form in vivo. The related but smaller molecule ginsenosides (Rg3, Rh1) may be the ingredient that exerts therapeutic eﬀects. This is also supported by the evidence found in experiments with red ginseng, fermented ginseng, and black ginseng. Therefore, the metabolic ginsenosides (Rg3, Rh1) need to be investigated to determine the active form of ginsenosides in vivo. Another concern is that the results from clinical data for diﬀerent processed ginseng extracts are inconsistent; some studies showed that the ginseng extracts possessed anti-hyperglycemic or diabetes-related eﬀects, while others did not. These results could be caused by subject factors and/or drug reasons. The physical activity, body weight, diabetic degree, and sample size of volunteers may aﬀect the outcomes of clinical trials. Another inﬂuencing factor is the variability of ginseng extracts. Diﬀerent sources, species, or extraction processes lead to diﬀerent ginseng ingredients. Even when using the same species and the same extraction process, diﬀerent batches of ginseng can have various components, which inﬂuence the curative eﬀect. Thus, characteristic detection and quality control are needed for ginseng products in clinical applications. 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https://openalex.org/W2345773639	https://link.springer.com/content/pdf/10.1007%2FJHEP08%282016%29120.pdf	English	null	The holographic dual of a Riemann problem in a large number of dimensions	The Journal of high energy physics/The journal of high energy physics	2,016	cc-by	17,564	Published for SISSA by Springer Received: June 14, 2016 Accepted: August 12, 2016 Published: August 22, 2016 Received: June 14, 2016 Accepted: August 12, 2016 Published: August 22, 2016 Open Access, c⃝The Authors. Article funded by SCOAP3. Published for SISSA by Springer The holographic dual of a Riemann problem in a large number of dimensions shock waves 11 4.2 Solving the Riemann problem using ideal hydrodynamics 16 4.3 A numerical solution to the Riemann problem 19 4.4 Restoring gradient corrections 20 5 Discussion 25 A Comment about entropy production across a shock 26 B A bestiary of plots 27 B.1 RR conﬁgurations 27 B.2 SS conﬁgurations 28 B.3 RS conﬁgurations 28 B.4 Error analysis 29 Contents 1 Introduction 1 2 The holographic dual of the Riemann problem for large d 4 3 Comparison with hydrodynamics 7 3.1 Stress tensor from ﬂuid-gravity correspondence 7 3.2 Entropy from gravity 9 4 Near equilibrium steady states 11 4.1 Rarefaction waves vs. shock waves 11 4.2 Solving the Riemann problem using ideal hydrodynamics 16 4.3 A numerical solution to the Riemann problem 19 4.4 Restoring gradient corrections 20 5 Discussion 25 A Comment about entropy production across a shock 26 B A bestiary of plots 27 B.1 RR conﬁgurations 27 B.2 SS conﬁgurations 28 B.3 RS conﬁgurations 28 B.4 Error analysis 29 1 Introduction 2 The holographic dual of the Riemann problem for large d JHEP08(2016)120 The holographic dual of a Riemann problem in a large number of dimensions JHEP08(2016)120 Christopher P. Herzog,a Michael Spillanea and Amos Yaromb aC.N. Yang Institute for Theoretical Physics, Department of Physics and Astronomy, Stony Brook University, Stony Brook, NY 11794, U.S.A. bDepartment of Physics, Technion, Haifa 32000, Israel E-mail: christopher.herzog@stonybrook.edu, Christopher P. Herzog,a Michael Spillanea and Amos Yaromb aC.N. Yang Institute for Theoretical Physics, Department of Physics and Astronomy, Stony Brook University, Stony Brook, NY 11794, U.S.A. bDepartment of Physics, Technion, Haifa 32000, Israel E-mail: christopher.herzog@stonybrook.edu, michael.spillane@stonybrook.edu, ayarom@physics.technion.ac.il Abstract: We study properties of a non equilibrium steady state generated when two heat baths are initially in contact with one another. The dynamics of the system we study are governed by holographic duality in a large number of dimensions. We discuss the “phase diagram” associated with the steady state, the dual, dynamical, black hole description of this problem, and its relation to the ﬂuid/gravity correspondence. Keywords: AdS-CFT Correspondence, Black Holes, Holography and condensed matter physics (AdS/CMT) ArXiv ePrint: 1605.01404 Open Access, c⃝The Authors. Article funded by SCOAP3. doi:10.1007/JHEP08(2016)120 doi:10.1007/JHEP08(2016)120 Contents 1 Introduction 1 2 The holographic dual of the Riemann problem for large d 4 3 Comparison with hydrodynamics 7 3.1 Stress tensor from ﬂuid-gravity correspondence 7 3.2 Entropy from gravity 9 4 Near equilibrium steady states 11 4.1 Rarefaction waves vs. shock waves 11 4.2 Solving the Riemann problem using ideal hydrodynamics 16 4.3 A numerical solution to the Riemann problem 19 4.4 Restoring gradient corrections 20 5 Discussion 25 A Comment about entropy production across a shock 26 B A bestiary of plots 27 B.1 RR conﬁgurations 27 B.2 SS conﬁgurations 28 B.3 RS conﬁgurations 28 B.4 Error analysis 29 Contents 1 Introduction 1 2 The holographic dual of the Riemann problem for large d 4 3 Comparison with hydrodynamics 7 3.1 Stress tensor from ﬂuid-gravity correspondence 7 3.2 Entropy from gravity 9 4 Near equilibrium steady states 11 4.1 Rarefaction waves vs. 1 Introduction The Riemann problem may provide a relatively simple setting in which to study the non- equilibrium physics of quantum ﬁeld theory. The problem asks for the time evolution of piece wise constant initial conditions with a single discontinuity in the presence of some number of conservation laws, for example of energy, momentum, mass, or charge. In our case, we consider a ﬂuid phase of a conformal ﬁeld theory (CFT) with an initial planar interface, where the energy density jumps from eL on the left of the interface to eR on its right. We also allow for a discontinuity in the center of mass velocity of the ﬂuid across the interface. For simplicity, we will make a number of further restrictions. We assume a conformal ﬁeld theory that has a dual gravity description via the AdS/CFT correspondence. A priori, this will allow us to study the system beyond the hydrodynamic limit. We also take the – 1 – Figure 1. A phase diagram for the solution to the Riemann problem in a large d limit. Given a pair (eL, 0) and (eR, jR), the selection of shock and rarefaction waves is determined by the value of eR/eL and jR/eL. The dashed and solid lines are “critical”: the dashed line indicates the values of (eR, jR) connected to (eL, 0) by a single rarefaction wave while the solid line indicates the values of (eR, jR) connected to (eL, 0) by a single shock wave. JHEP08(2016)120 Figure 1. A phase diagram for the solution to the Riemann problem in a large d limit. Given a pair (eL, 0) and (eR, jR), the selection of shock and rarefaction waves is determined by the value of eR/eL and jR/eL. The dashed and solid lines are “critical”: the dashed line indicates the values of (eR, jR) connected to (eL, 0) by a single rarefaction wave while the solid line indicates the values of (eR, jR) connected to (eL, 0) by a single shock wave. limit that the number of spatial dimensions d is very large. In this limit, we ﬁnd that the system is described by two conservation equations ∂te −∂2 ζe = −∂ζj , ∂tj −∂2 ζj = −∂ζ j2 e + e . (1.1) (1.1) where e is, up to gradient corrections, the energy density and j the energy current. These equations are a special case of equations derived in ref. [1]. 1 Introduction An analysis of shock waves and their relation to two dimensional turbulence was carried out in [6].) In more than two space-time dimensions, conformal symmetry alone is not enough to specify the evolution completely and one needs additional assumptions about the structure of the conserved currents. Recent work appealed to the gauge/gravity duality [7–10], an analogy with 1 + 1 dimensions [5], and hydrodynamics [7, 11–13]. These papers focused on the case jR = jL = 0 and eL > eR such that from a hydrodynamic perspective a left moving rarefaction wave and a right moving shock wave are expected to emerge. JHEP08(2016)120 The distinction between rarefaction and shock waves was ignored in some of these papers [5, 7, 11]. Indeed, when working with 2 + 1 or 3 + 1 dimensional conformal ﬁeld theories, the diﬀerence between, say, an SS solution to the Riemann problem and an RS solution to the Riemann problem is very small for all but extreme initial energy diﬀerences. As the spacetime dimension d increases however, the diﬀerence between a rarefaction wave type of solution and a shock wave solution becomes signiﬁcant [13]. This ampliﬁcation of the diﬀerence between the two solutions serves as a motivator for studying this Riemann problem in a large number of dimensions. Interestingly, a large d limit has independently been a topic of recent interest [1, 14– 25] in the study of black hole solutions to Einstein’s equations. Of particular relevance to our work is the connection between black holes in asymptotically AdS spaces and hy- drodynamics [26]. Certain strongly interacting conformal ﬁeld theories are known to have dual classical gravitational descriptions. In the limit where these conformal ﬁeld theories admit a hydrodynamic description, a solution to the relevant hydrodynamic equations can be mapped to a solution of Einstein’s equations, in a gradient expansion where physical quantities change slowly in space and time. Transport coeﬃcients such as shear viscosity are ﬁxed by the form of Einstein’s equations. Thus, one may study the Riemann problem in conformal ﬁeld theories with a large number of dimensions by studying an equivalent Riemann-like problem involving an initially discontinuous metric of a black hole in an asymptotically AdS background. Given that extensive analyses of conservation equations like (1.1) can be found in many hydrodynamics textbooks and papers, one can legitimately ask why we bother to redo the analysis here. 1 Introduction In these variables the Riemann problem amounts to a determination of e and j given an initial conﬁguration of the form (e, j) = ( (eL, jL) z < 0 (eR, jR) z > 0 . (1.2) (1.2) y choosing an appropriate reference frame, we may set jL = 0 without loss of generality. By choosing an appropriate reference frame, we may set jL = 0 without loss of generality. A i h h i f hi f Ri bl i h As it happens, there are extensive treatments of this type of Riemann problem in hy- drodynamics textbooks. See for example ref. [2]. Typically, a pair of rarefaction and/or shock waves form and move away from each other, creating in their wake a region with almost constant e and j. In recent literature, this intermediate region has been called a non- equilibrium steady state (NESS) [3, 4]. One of the main results of this paper is a “phase” diagram valid in a large d limit (see ﬁgure 1) that describes, given the conservation equa- tions (1.1) and initial conditions (1.2), which pair of waves are formed: rarefaction-shock (RS), shock-shock (SS), shock-rarefaction (SR), or rarefaction-rarefaction (RR). A physical reason for the preference of a rarefaction wave to a shock wave is entropy production. Recent interest in this type of Riemann problem was spurred by a study of the problem in 1+1 dimensional conformal ﬁeld theory [3] where the evolution is completely determined by the conformal symmetry and a hydrodynamic limit need not be taken. Conservation – 2 – and tracelessness of the stress tensor imply that the stress tensor is a sum of right moving and left moving parts. When jR = jL = 0 one ﬁnds a NESS in between the two asymptotic regions, characterized by an energy density (eR +eL)/2 and an energy current proportional to eR −eL. The NESS is separated from the asymptotic regions by outward moving shock waves traveling at the speed of light. (An extension of the analysis of [3] which includes a discontinuity in the center of mass velocity, holomorphic currents and chiral anomalies can be found in [5]. 1 Introduction The reason is that when working in a large number of dimensions, one can solve for the black hole metric exactly, independent of the derivative expansion (which is naturally truncated), thus obtaining an exact solution to the Riemann problem which in- cludes possible viscous terms and is in general valid even when gradients of thermodynamic quantities are large (as is the case with discontinuous initial conditions). Our work is organized as follows. In section 2, we rederive the equations (1.1) by taking a large d limit of Einstein’s equations. We show how to rewrite them as the conservation – 3 – condition on a stress-tensor, ∂µT µν = 0. In section 3, we compare the large d stress tensor and equations of motion to those arising from the ﬂuid-gravity correspondence [26]. We ﬁnd that both eqs. (1.1) and the stress tensor T µν are equivalent to the hydrodynamic equations that come from the ﬂuid-gravity correspondence at large d, at least up to and including second order gradient corrections. In the same section we also construct an entropy current Jµ S using an area element of the black hole horizon and show that the divergence of the entropy current is positive ∂µJµ S ≥0 in this large d limit. In section 4, we solve the Riemann problem for eqs. (1.1) and derive the phase diagram given in ﬁgure 1. Finally, we conclude in section 5 with some directions for future research. Appendix A contains a short calculation of the entropy produced across a shock, while appendix B contains plots of auxiliary numerical results. JHEP08(2016)120 2 The holographic dual of the Riemann problem for large d We wish to construct a holographic dual of the Riemann problem. Consider the Einstein Hilb t ti We wish to construct a holographic dual of the Riemann problem. Consider the Einstein Hilbert action 1 Z (d 2)(d 1) S = −1 2κ2 Z √−g R + (d −2)(d −1) L2 ddx . (2.1) (2.1) A canonical stationary solution of the resulting equations of motion is the black brane solution d 1! A canonical stationary solution of the resulting equations of motion is the black brane solution ! ds2 = 2dt dr −r2 1 − 4πT (d −1)r d−1! dt2 + r2dx2 ⊥, (2.2) (2.2) where T is an integration constant which denotes the Hawking temperature. The solu- tion (2.2) is dual to a thermal state of a conformal ﬁeld theory with temperature T. For instance, the thermal expectation value of the stress tensor in such a state is given by ⟨T µν⟩=       (d −2)P(T) 0 . . . 0 0 P(T) . . . 0 ... ... ... ... 0 0 . . . P(T)       (2.3) (2.3) where where P(T) = p0 4πT d −1 d−1 (2.4) (2.4) is the pressure with p0 a theory dependent dimensionless parameter. (The indices µ and ν run over the d −1 dimensions of the (d −1)-dimensional CFT.) As discussed in [8] a dual description of the Riemann problem necessitates an initial black hole conﬁguration which is held at some ﬁxed temperature TL for all z < 0 and at a diﬀerent temperature TR for z > 0. This would correspond to a conﬁguration where the expectation value of the stress tensor is given by (2.3) with T = TL for z < 0 and by (2.3) with T = TR for z > 0. Since the initial black hole is out of equilibrium it will evolve in time. Its dual description will provide a solution for the time evolution of the stress tensor which we are after. Thus, our goal is to solve the equations of motion following from (2.1) and use them to construct the dual stress tensor. 2 The holographic dual of the Riemann problem for large d – 4 – An ansatz for the metric which is compatible with the symmetries and our initial conditions is given by ds2 = dt(2dr −gttdt −2gtzdz) + gzzdz2 + g⊥⊥dx2 ⊥, (2.5) (2.5) where the metric components are functions only of t, r, and z. (A more general ansatz which involves a transverse velocity can be found in [1].) A numerical solution of the equations of motion for gtt, gtz and gii (i = x⊥or z) with smoothened initial conditions has been obtained for d = 4 in [8] for relatively small initial temperature diﬀerences, (TL −TR)/(TL + TR) < 1. A solution for ﬁnite d > 4 and for large temperature diﬀerences, (TL −TR)/(TL + TR) ∼1 is challenging. JHEP08(2016)120 JHEP08(2016)120 In this work we use the methods developed in [1, 14] (see also [15–23]) to address the Riemann problem in the limit that d is very large. Such a limit can be understood as follows. In an appropriate gauge, the near boundary expansion of the metric gives gtt = r2 + O(r3−d) , gtz = O(r3−d) , gii = r2 + O(r3−d) . (2.6) (2.6) Thus, in the large d limit at any ﬁnite value of r, the spacetime looks like the AdS vacuum. Only by keeping R = rn ﬁnite with n ≡d −1 will the O(r−n) corrections to the metric remain observable. Our strategy is to solve the equations of motion in the ﬁnite R region subject to the boundary conditions (2.6). Following [1], we also use the scaling x⊥= χ/√n and z = ζ/√n so that in this coordinate system the line element takes the form ds2 = dt(2dr −gttdt −2gtζdζ) + gζζdζ2 + g⊥⊥dχ2 ⊥, (2.7) (2.7) where where where gtt r2 = X k=0 E(k) nk , gtζ = X k=1 J(k) nk , gii r2 = 1 n + X k=2 g(n) i nk . (2.8) gtt r2 = X k=0 E(k) nk , gtζ = X k=1 J(k) nk , gii r2 = 1 n + X k=2 g(n) i nk . (2.8) (2.8) (In a slight abuse of notation i is now either χ⊥or ζ.) We have used the letters E and J to emphasize these quantities’ (soon to be seen) close connection with an energy density and energy current in the dual hydrodynamic description. 2 The holographic dual of the Riemann problem for large d (In a slight abuse of notation i is now either χ⊥or ζ.) We have used the letters E and J to emphasize these quantities’ (soon to be seen) close connection with an energy density and energy current in the dual hydrodynamic description. One can now solve the equations of motion order by order in 1/n. The equations of motion are simply Einstein’s equations in the presence of a negative cosmological constant: RMN = −(d −1)gMN , (2.9) (2.9) – 5 – – 5 – setting L = 1 for convenience. Let a and b index the t, r, and ζ directions only, while i and j index the remaining perpendicular directions. Furthermore, let ˜Rab be the Ricci tensor with respect to the three dimensional metric in the t, r, and ζ directions. Then setting L = 1 for convenience. Let a and b index the t, r, and ζ directions only, while i and j index the remaining perpendicular directions. Furthermore, let ˜Rab be the Ricci tensor with respect to the three dimensional metric in the t, r, and ζ directions. Then Rab = ˜Rab + d −3 4 (∂a log g⊥⊥)(∂b log g⊥⊥) −d −3 2 ∇a∂bg⊥⊥ g⊥⊥ , (2.10) Rij = δij 5 −d 4 (∂ag⊥⊥)(∂ag⊥⊥) g⊥⊥ −1 2∇a∂ag⊥⊥ . (2.11) (2.10) (2.11) Imposing that the boundary metric is Minkowski and choosing a near boundary ex- pansion of the form (2.6) we ﬁnd Imposing that the boundary metric is Minkowski and choosing a near boundary ex- pansion of the form (2.6) we ﬁnd JHEP08(2016)120 gtt r2 = 1 −e R −1 n e2 R + log R R ∂ζj + j2 2R2 + O(n−2) , gtζ = 1 n j R + 1 n2 j2 R + log R R ∂ζ j2 e + 2f + j3 2R2e + O(n−3) , gζζ r2 = 1 n + 1 n2 j2 Re + O(n−3) , g⊥⊥ r2 = 1 n −1 n3 j2 Re + O(n−4) , (2.12) (2.12) where the O(n−2) correction to gtt and the O(n−3) contributions to gζζ are too long to write explicitly. The functions e and j are functions of t and ζ only and must satisfy the additional constraints (1.1). Equations (1.1) are identical to those obtained in [1, 14]. 3 Comparison with hydrodynamics Let us pause to understand (2.14). Within the context of the gauge-gravity duality it is possible to construct a solution to the Einstein equations which is perturbative in t, ζ and χ⊥derivatives of the metric components [26]. Such a perturbative solution to the equations of motion, which is available for any dimension d [27, 28], allows for a dual description of the theory in terms of ﬂuid dynamical degrees of freedom. 2 The holographic dual of the Riemann problem for large d We can rewrite them in terms of a conservation law where the O(n−2) correction to gtt and the O(n−3) contributions to gζζ are too long to write explicitly. The functions e and j are functions of t and ζ only and must satisfy the additional constraints (1.1). Equations (1.1) are identical to those obtained in [1, 14]. We can rewrite them in terms of a conservation law ∂µT µν = 0 (2.13) (2.13) where where T µν = e j −∂ζe j −∂ζe e + j2 e −2∂ζj + ∂2 ζe ! + ∂2 ζg −∂ζ∂tg −∂ζ∂tg ∂2 t g ! . (2.14) (2.14) where g is an arbitrary function. Likewise, the functions e2 and j2 must also satisfy a set of equations which can be obtained from the conservation of T µν 2 =   e2 j2 e + e −e2 −2j′′ + j + j2 j2 e + e −e2 −2j′′ + j + j2 T 11   + ∂2 ζg2 −∂ζ∂tg2 −∂ζ∂tg2 ∂2 t g2 ! . (2.15) + ∂2 ζg2 −∂ζ∂tg2 −∂ζ∂tg2 ∂2 t g2 ! . (2.15) (2.15) where T 11 = 2 1 − j e ′ j e ′ e(log(e) −3) + e + e2 1 −j2 e2 + 2j2 j e + e2 −4e −6j2 e + 4j′ ′′ −2 j2 −3j −j3 e2 ′ + j2 e j e ′ . (2.16) (2.16) We will use ′ and ∂ζ interchangeably in what follows. We will use ′ and ∂ζ interchangeably in what follows. We will use ′ and ∂ζ interchangeably in what follows. We will use ′ and ∂ζ interchangeably in what follows. We will use ′ and ∂ζ interchangeably in what follows. – 6 – 3.1 Stress tensor from ﬂuid-gravity correspondence To construct the dual hydrodynamic description of a slowly varying black hole, we boost the black hole solution (2.2) by a constant velocity uµ in the t, z, x⊥directions. The resulting line element is given by JHEP08(2016)120 ds2 (0) = 2uµdxµdr −r2 1 − 4πT (d −1)r d−1! uµuνdxµdxν + r2 (ηµν + uµuν) dxµdxν . (3.1) (3.1) (3.1) Allowing for uµ and T to become spacetime dependent implies that (3.1) will get corrected. By setting gradients of uµ and T to to be small, one can solve for the corrections to (3.1) order by order in derivatives so that the line element will take the schematic form ds2 = ds2 (0) + ds2 (1) + . . . (3.2) (3.2) where ds2 (i) denotes the ith order gradient corrections to the line element. (i) The stress tensor T µν which is dual to (3.1) takes the form T µν = X i T µν (i) (3.3) (3.3) also expanded in gradients. One ﬁnds [27, 28] also expanded in gradients. One ﬁnds [27, 28] T µν (0) = P(T) ((d −1)uµuν + ηµν) (3.4) (3.4) which is nothing but a boosted version of (2.3) and then, in the Landau frame, T µν (1) = −2ησµν , T µν (2) = (d −1)η 2πT (1 −τ0)u · Dσµν + σλµσλν −σαβσαβ d −2 Pµν −τ0 ωµλσλν + ωνλσµλ (3.5) T µ (1) = −2ησµν , T µν (2) = (d −1)η 2πT (1 −τ0)u · Dσµν + σλµσλν −σαβσαβ d −2 Pµν −τ0 ωµλσλν + ωνλσµλ (3 with with P µν = ηµν + uµuν , σµν = 1 2PµαPνβ (∂αuβ + ∂βuα) − 1 d −2P µν∂αuα , ωµν = 1 2P µαP νβ (∂αuβ −∂βuα) , u · Dσµν = PµαPνβuλ∂λσαβ + ∂αuα d −2σµν , (3.6) (3.6) – 7 – and η = (d −1)P 4πT , τ0 = Z ∞ 1 yd−3 −1 y(yd−1 −1)dy = 1 2 + O(d−2) . (3.7) (3.7) (Note that our deﬁnition of σµν is somewhat unconventional.) An initial analysis of third order gradient corrections has been carried out in [29] for d = 5. A full analysis of all third order transport terms for arbitrary dimension d is currently unavailable. 3.1 Stress tensor from ﬂuid-gravity correspondence Since (2.14) has been obtained from a large d limit of a gravitational dual theory, we expect that (2.14) coincides with (3.3) when the former is expanded in derivatives and the latter is expanded around large n = d −1. In short, we expect that taking a gradient expansion commutes with taking a large d limit. To make a direct comparison let us consider the hydrodynamic stress tensor (3.3) in the t, ζ, χ⊥coordinate system where the metric tensor takes the form JHEP08(2016)120 ds2 = −dt2 + dζ2 n + dχ2 ⊥ n . (3.8) (3.8) One important eﬀect of this rescaling is to keep the sound speed to be an order one quantity. Scaling the spatial component of the velocity ﬁeld by 1/√n, viz., One important eﬀect of this rescaling is to keep the sound speed to be an order one quantity. Scaling the spatial component of the velocity ﬁeld by 1/√n, viz., uµ = 1 q 1 −β2(t,ζ) n (1, β(t, ζ)) , (3.9) (3.9) and maintaining that ϵ = (d −2)P is ﬁnite in the large d limit, we ﬁnd, and maintaining that ϵ = (d −2)P is ﬁnite in the large d limit, we ﬁnd, σµν = n∂ζβ δµ ζ δνζ + O(n0) u · Dσµν = n β∂2 ζβ + ∂t∂ζβ δµ ζ δνζ + O(n0) −σαβσαβ d −2 Pµν = n (∂ζβ)2 δµ ζ δνζ + O(n0) (3.10) σµν = n∂ζβ δµ ζ δνζ + O(n0) u · Dσµν = n β∂2 ζβ + ∂t∂ζβ δµ ζ δνζ + O(n0) (3.10) u · Dσµν = n β∂2 ζβ + ∂t∂ζβ δµ ζ δνζ + O(n0) (3.10) (3.10) σλµσλν −σαβσαβ d −2 Pµν = n (∂ζβ)2 δµ ζ δνζ + O(n0) σλµσλν −σαβσαβ d −2 Pµν = n (∂ζβ)2 δµ ζ δνζ + O(n0) and thus, T µν = ϵ βϵ βϵ ϵ(1 + β2) + p ! + O n−1 (3.11) (3.11) where where p = −2ϵ∂ζβ + 2ϵ(∂ζβ)2 + ϵβ∂ζ2β + ϵ∂ζ∂tβ + O ∂3 (3.12) p = −2ϵ∂ζβ + 2ϵ(∂ζβ)2 + ϵβ∂ζ2β + ϵ∂ζ∂tβ + O ∂3 (3.12) (3.12) and O ∂3 denotes third order and higher derivative corrections. Note that this constitu- tive relation for the stress tensor includes and encodes the large d limit of the transport coeﬃcients (3.7). and O ∂3 denotes third order and higher derivative corrections. 3.1 Stress tensor from ﬂuid-gravity correspondence Note that this constitu- tive relation for the stress tensor includes and encodes the large d limit of the transport coeﬃcients (3.7). Now, we insert the redeﬁnitions e = ϵ −1 2∂2 ζϵ , j = βϵ + ∂ζϵ + 1 2∂t∂ζϵ , g = 1 2ϵ (3.13) (3.13) – 8 – into the large d constitutive relation for the stress tensor (2.14), use the large d stress tensor conservation equations (1.1), and throw out terms that have three or more derivatives. We claim that in this fashion, we recover the stress tensor (3.11) in the gradient expansion. Thus, the large d limit and the gradient expansion seem to commute. Note that while the conservation equations (1.1) are of second order in gradients of ζ and t, the stress tensor includes at least second order gradients. The implications of (3.13) are worth emphasizing. The equations of motion (1.1) are equivalent to the standard equations of motion of relativistic hydrodynamics when the lat- ter are expanded in a large d limit. When working with the e and j variables one obtains equations of motion which are second order in derivatives and therefore include dissipative eﬀects. When carrying out a frame transformation to the more traditional Landau frame, more derivatives will appear. When considering the stress tensor associated with the equa- tions of motion (1.1) one obtains more terms with higher gradients which do not contribute to the equations of motion. It would be interesting to see if one can construct an alternative to the Israel-Stewart theory using a “large d-frame” where gradients naturally truncate. JHEP08(2016)120 3.2 Entropy from gravity Within the context of our forthcoming analysis, it is instructive to compute the dual entropy production rate which is associated with the evolution of the horizon. Due to its teleological nature, it is usually diﬃcult to identify the location of the event horizon. However, in the large d limit the analysis is somewhat simpliﬁed. Let us look for a null surface of the form R = rh(t, ζ). The normal to such a surface is ΞMdxM = dR −∂trhdt −∂ζrhdζ . (3.14) (3.14) Demanding that Ξ2 R=rh = Demanding that Ξ2 rh = 0 implies, to leading order in the large d limit, that that Ξ2 R=rh = 0 implies, to leading order in the large d limit, that (3.15) rh = e . (3.15) The spacetime singularity which exists in our solution implies that an event horizon must be present. Since the only null surface available is (3.15), it must be the location of the event horizon. Subleading corrections to the location of the event horizon are given by The spacetime singularity which exists in our solution implies that an event horizon must be present. Since the only null surface available is (3.15), it must be the location of the event horizon. Subleading corrections to the location of the event horizon are given by rh = e + 1 n 4je′ −2(e′)2 −j2 2e + e2 −2j′ + 2e′′ + j′ log(e) ≡e + 1 nrh 1 . (3.16) (3.16) To compute the change in the black hole entropy over time we compute the area form of the event horizon. Following the prescription of [30], we ﬁnd that A = ϵµ1...µd (d −1)!Jµ1 S dxµ2 ∧. . . ∧dxµd (3.17) (3.17) where Jµ S = √ h 4GN Nµ Nt (3.18) (3.18) – 9 – – 9 – where h is the spatial (t = constant) part of the induced metric on the horizon Hµνdxµdxν = gMNdxMdxN R=rh (3.19) Hµνdxµdxν = gMNdxMdxN R=rh Hµνdxµdxν = gMNdxMdxN R=rh (3.19) and Nµ is deﬁned via ΞM∂M = NR∂R + Nµ∂µ . (3.20) ΞM∂M = NR∂R + Nµ∂µ . (3.20) (3.20) A short computation yields A short computation yields √ h = n−n−1 2 e + 1 n (rh 1 −e ln e) , Nµdxµ = −∂te dt −∂ζe dζ . (3.21) (3.21) JHEP08(2016)120 Nµdxµ = −∂te dt −∂ζe dζ . 1We thank R. Emparan for a discussion on this point. 3.2 Entropy from gravity Thus, ˜Jµ S = 16πGNn n−1 2 Jµ S = 4π n e, j −e′, . . . +4π n2 rh 1 −e ln e, j2 2e2 + log e (2e′ −j) + j2 e ′ log e + j2 −r′ h 1, . . . (3.22) (3.22) where we have normalized the entropy density so that it is compatible with our conventions for the energy density. The second law of black hole thermodynamics amounts to ∂µJµ S ≥0 . (3.23) (3.23) In our large d limit we ﬁnd that ∂µ ˜Jµ S = 8πe n2 ∂ζ j −∂ζe e 2 . (3.24) (3.24) The expectation from hydrodynamics, to second order in derivatives, is that the divergence of the entropy current is given by ∂µ ˜Jµ S = 2η T σ2 . (3.25) (3.25) (See for example (8) of ref. [31].) This expectation matches (3.24) on the nose. Note that to leading order in the large d limit the entropy current vanishes. This somewhat surprising feature of the large d limit follows from the fact that entropy production terms are suppressed by inverse powers of the dimension in the large d limit. Another way of understanding this suppression comes from thinking about the temperature T ∼e1/(d−1). In the large d limit, T is constant to leading order in d. From the thermodynamic relation de = Tds, it then follows that changes in energy are proportional to changes in entropy, and entropy conservation follows from energy conservation at leading order in a large d expansion.1 (See for example (8) of ref. [31].) This expectation matches (3.24) on the nose. Note that to leading order in the large d limit the entropy current vanishes. This somewhat surprising feature of the large d limit follows from the fact that entropy production terms are suppressed by inverse powers of the dimension in the large d limit. Another way of understanding this suppression comes from thinking about the temperature T ∼e1/(d−1). In the large d limit, T is constant to leading order in d. From the thermodynamic relation de = Tds, it then follows that changes in energy are proportional to changes in entropy, and entropy conservation follows from energy conservation at leading order in a large d expansion.1 – 10 – 4 Near equilibrium steady states We now analyze the dynamics controlled by the partial diﬀerential equations (1.1) which encode the dynamics of an out of equilibrium black hole (2.5) and its dual stress ten- sor (2.14). Various related holographic analyses can be found in [32–41]. As discussed in the introduction, the particular question we would like to address is a Riemann problem: what is the time evolution following from an initial condition (1.2)? We are particularly interested in the steady state solution which will emerge at late times. For convenience we will consider a reference frame for which jL = 0. Indeed, if e(x, t) and j(x, t) satisfy the conservation equations (1.1), then so do e(x −vt, t) and j(x −vt, t) + ve(x −vt, t). Thus, for constant values of e and j, we can choose a v such that j will be set to zero. The non-relativistic nature of the boost symmetry reﬂects the fact that the large d limit we have taken is eﬀectively a non-relativistic limit where the speed of light c ∼ √ d has been pushed oﬀto inﬁnity. JHEP08(2016)120 where β = tanh α is the ﬂuid velocity. 4.1 Rarefaction waves vs. shock waves Before addressing the Riemann problem in its entirety let us consider a simpliﬁed system which is less constrained. Consider (2.14) with gradient terms neglected. The resulting expression is the large d limit of the energy momentum tensor of an inviscid ﬂuid which is known to support (discontinuous) shock waves [2] for any ﬁnite value of d. While the solution to the full Riemann problem will consist of a pair of shock and/or rarefaction waves, we begin in this section with a single discontinuous shock wave moving with velocity s. Conservation of energy and momentum imply s[T tt] = [T tζ] , s[T tζ] = [T ζζ] , (4.1) (4.1) where [Q] = Ql −Qr and Qr/l specify the value of Q to the left or right of the shock respectively.2 The conservation conditions (4.1) are very general and are often referred to as the Rankine-Hugoniot (RH) relations. In our setup they reduce to sel −jl = ser −jr , sjl − el + j2 l el = sjr − er + j2 r er , (4.2) (4.2) where er/l and jr/l are the energy density and current immediately to the right or left of the shock. While these Rankine-Hugoniot relations hold for an arbitrary, piece-wise continuous ﬂuid proﬁle, in what follows, we are interested in the much simpler situation where e and j are constant functions away from the shocks. Amusingly, er satisﬁes a cubic equation,3 (4.3) (eljr −erjl)2 = eler(el −er)2 , (4.3) 2In this section we use subscripts r and l to denote values of quantities to the right or left of the shock. In other sections we use subscripts R and L to denote quantities in the right and left asymptotic regions. In the latter case there is generally an interpolating region which we denote with a 0 subscript. 3In general d, one ﬁnds the relation 2In this section we use subscripts r and l to denote values of quantities to the right or left of the shock. In other sections we use subscripts R and L to denote quantities in the right and left asymptotic regions. In the latter case there is generally an interpolating region which we denote with a 0 subscript. 3In general d, one ﬁnds the relation sinh2(αl −αr) = d −2 (d −1)2 (ϵl −ϵr)2 ϵlϵr , where β = tanh α is the ﬂuid velocity. 4.1 Rarefaction waves vs. shock waves – 11 – a plot of which as a function of jr resembles a ﬁsh: ﬁxing (el, jl), each value of s is mapped to a point on the (er, jr) plane. The collection of such points is given by a ﬁsh-like curve, an example of which is given in the left panel of ﬁgure 2. We make two observations about the ﬁsh. The vacuum (er, jr) = (0, 0) always lies on the cubic (4.3), corresponding to the fact that a shock can interpolate between any value of (el, jl) and the vacuum. Also (er, jr) = (el, jl) is the point of self-intersection of the cubic and has s = ±1 + jl/el. The physical content of this observation is that when (er, jr) is close to (el, jl) but still lies on the cubic, we can ﬁnd a close approximation to the ﬂuid proﬁle by linearizing the equations of motion. As we will describe in greater detail below, linearized ﬂuctuations correspond to damped sound modes, and indeed the two regions can be connected by sound waves propagating at the local sound speed s = ±1 + jl/el. JHEP08(2016)120 The shock solutions we found all solve the conservation equations (4.2). However, some of these solutions are unphysical in the following sense. Let us boost to a frame where the shock speed vanishes, s = 0. In half of the shock solutions, a quickly moving ﬂuid at low temperature is moving into a more slowly moving ﬂuid at higher temperature, converting kinetic energy into heat and producing entropy. We will refer to these shocks as “good” shocks. The other half of the solutions correspond to the time reversed process where a slowly moving ﬂuid at high temperature moves into a rapidly moving but cooler ﬂuid, turning heat into kinetic energy. This second solution, as we shall see shortly, should be discarded. Strictly speaking, entropy is conserved in the large d limit (see the discussion following equation (3.25)). A more formal way of understanding why one should discard the bad shocks is to restore the gradient corrections but take a limit where these are small. Let us assume that in the frame where the shock velocity is zero there is an approximately stationary conﬁguration such that time derivatives are much smaller than spatial deriva- tives. 4.1 Rarefaction waves vs. shock waves Boosting back to a shock with velocity s, we expect that e and j depend only on the combination ζ −st, i.e., j(t, ζ) = j(ζ −st) and likewise, e(t, ζ) = e(ζ −st). The equations of motion (1.1) become ordinary diﬀerential equations which can be integrated once to obtain e′ = −s(e −el) + (j −jl) , j′ = −s(j −jl) + e + j2 e −el −j2 l el . (4.4) (4.4) We have picked the two integration constants such that e′ and j′ vanish in the left asymp- totic region. The Rankine-Hugoniot conditions (4.2) imply that e′ and j′ also vanish in the right asymptotic region. As e′ and j′ themselves vanish in the left and right asymptotic regions, we can describe e′ and j′ well near these points by looking at a gradient expansion. Near the left asymptotic region e′ j′ ! ≈ −s 1 1 −j2 l e2 l 2jl el −s ! e −el j −jl ! ≡Ml e −el j −jl ! . (4.5) (4.5) – 12 – There is a similar looking equation for e′ and j′ near the right asymptotic region e′ j′ ! ≈Mr e −er j −jr ! . (4.6) (4.6) The solutions near (el, jl) and near (er, jr) have an exponential nature with the sign of the exponents depending on the eigenvalues of Ml and Mr appearing on the right hand side of (4.5) and (4.6) given by λr ± = ±1 + jr er −s , λl ± = ±1 + jl el −s . (4.7) (4.7) JHEP08(2016)120 We now observe that the signs of the eigenvalues of Ml and Mr determine whether the shock is a viable solution to the equations of motion. We now observe that the signs of the eigenvalues of Ml and Mr determine whether the shock is a viable solution to the equations of motion. • If both eigenvalues of Ml are negative, then e′ and j′ will not vanish as x →−∞. Thus we require that at least one eigenvalue of Ml is positive in order for a shock solution to exist. • If both eigenvalues of Ml are negative, then e′ and j′ will not vanish as x →−∞. Thus we require that at least one eigenvalue of Ml is positive in order for a shock solution to exist. 4.1 Rarefaction waves vs. shock waves • If we assume there is exactly one positive eigenvalue, then 1 + jl/el > s and −1 + jl/el < s. Note that the value 1 + jl/el corresponds to the slope of one of the characteristics (i.e. the local speed of one of the sound waves), and this condition implies that this characteristic will end on the shock. Since λl −is assumed to be negative, we have to tune one of the two integration constants of the system of diﬀerential equations to zero. This tuning means that generically the solution to the right of the shock will be a linear combination of both of the solutions near (er, jr). If both solutions are to be used, then it had better be that both eigenvalues of Mr are negative. (Otherwise, it will not be true that e′ and j′ vanish in the limit x →∞.) In particular, the larger of the two eigenvalues must be negative, which implies that 1 + jr/er < s. (In terms of characteristics, both will end on the shock.) Thus, we ﬁnd the constraint • If we assume there is exactly one positive eigenvalue, then 1 + jl/el > s and −1 + jl/el < s. Note that the value 1 + jl/el corresponds to the slope of one of the characteristics (i.e. the local speed of one of the sound waves), and this condition implies that this characteristic will end on the shock. Since λl −is assumed to be negative, we have to tune one of the two integration constants of the system of diﬀerential equations to zero. This tuning means that generically the solution to the right of the shock will be a linear combination of both of the solutions near (er, jr). If both solutions are to be used, then it had better be that both eigenvalues of Mr are negative. (Otherwise, it will not be true that e′ and j′ vanish in the limit x →∞.) In particular, the larger of the two eigenvalues must be negative, which implies that 1 + jr/er < s. (In terms of characteristics, both will end on the shock.) Thus, we ﬁnd the constraint 1 + jr/er < s < 1 + jl/el . 4.1 Rarefaction waves vs. shock waves (4.8a) (4.8a) • If both eigenvalues of Ml are positive, we still need at least one negative eigenvalue of Mr to be able to connect the solutions in the left and right asymptotic regions. More- over, for Mr to have two negative eigenvalues would be inconsistent with momentum conservation (4.2). An analysis similar to the previous one yields • If both eigenvalues of Ml are positive, we still need at least one negative eigenvalue of Mr to be able to connect the solutions in the left and right asymptotic regions. More- over, for Mr to have two negative eigenvalues would be inconsistent with momentum conservation (4.2). An analysis similar to the previous one yields • If both eigenvalues of Ml are positive, we still need at least one negative eigenvalue of Mr to be able to connect the solutions in the left and right asymptotic regions. More- over, for Mr to have two negative eigenvalues would be inconsistent with momentum conservation (4.2). An analysis similar to the previous one yields −1 + jr/er < s < −1 + jl/el . (4.8b) (4.8b) The constraints (4.8) choose the good shocks over the bad ones.4 The constraints (4.8) choose the good shocks over the bad ones.4 4In appendix A, we discuss a third RH relation one can write down for the entropy current. If the RH relations for energy and momentum are satisﬁed, the RH relation for the entropy current will typically be violated due to entropy production associated with viscous eﬀects. In the weak shock limit, we demon- strate that gradient corrections produce the entropy that leads to this violation of the third RH relation. Reversing the sign of the energy diﬀerence between the two asymptotic regions in eqs. (A.3) or (A.5), it is straighforward to see that a bad shock would lead to a decrease in entropy, at least in the simple case where s = 0 and jr = jl. – 13 – Figure 2. (Left panel) The solid blue curve corresponds to the solution to the Rankine-Hugoniot condition for (el, jl). Points on the curve correspond to diﬀerent values of s in (4.2). The regions jl/el < s < jl/el + 1 and s < jl/el −1 correspond to good shocks satisfying (4.8a) and (4.8b) respectively. 4.1 Rarefaction waves vs. shock waves (Center panel) The dashed line, which almost overlaps with the blue line at places, parameterizes the rarefaction solution (4.9) also associated with (el, jl). (Right panel) A plot of possible values of (er, jr) for a given a pair (el, jl) with good shocks preferred over the rarefaction solution and the rarefaction solution preferred over bad shocks. JHEP08(2016)120 Figure 2. (Left panel) The solid blue curve corresponds to the solution to the Rankine-Hugoniot condition for (el, jl). Points on the curve correspond to diﬀerent values of s in (4.2). The regions jl/el < s < jl/el + 1 and s < jl/el −1 correspond to good shocks satisfying (4.8a) and (4.8b) respectively. (Center panel) The dashed line, which almost overlaps with the blue line at places, parameterizes the rarefaction solution (4.9) also associated with (el, jl). (Right panel) A plot of possible values of (er, jr) for a given a pair (el, jl) with good shocks preferred over the rarefaction solution and the rarefaction solution preferred over bad shocks. Since bad shocks are not allowed, one may inquire as to the time evolution of a discon- tinuity with initial conditions which would have generated a bad shock. As it turns out, bad shocks can be replaced by the more physical rarefaction solutions [2]. The rarefaction solution assumes that between the asymptotic regions speciﬁed by (el, jl) and (er, jr), there is an interpolating solution where e and j are functions of ξ = ζ/t. As was the case for the shock wave, given el and jl, there is a one parameter family of allowed values of er and jr. These are given by er =el exp (±jl/el −1 ∓ξr) , jr =el(±1 + ξr) exp (±jl/el −1 ∓ξr) . (4.9) (4.9) The curve traced by (er, jr) also resembles a ﬁsh, and for moderate values of the shock parameters er and jr it closely follows the cubic curve corresponding to a shock solution. (See the central panel of ﬁgure 2.) The vacuum (0, 0) = (er, jr) solution can always be connected to (el, jl) through a rarefaction wave. The self-intersection point (er, jr) = (el, jl) has ξ = ∓1 + jl/el, again corresponding to a sound wave type interpolation between the two regions (er, jr) ≈(el, jl). 4.1 Rarefaction waves vs. shock waves The curve traced by (er, jr) also resembles a ﬁsh, and for moderate values of the shock parameters er and jr it closely follows the cubic curve corresponding to a shock solution. (See the central panel of ﬁgure 2.) The vacuum (0, 0) = (er, jr) solution can always be connected to (el, jl) through a rarefaction wave. The self-intersection point (er, jr) = (el, jl) has ξ = ∓1 + jl/el, again corresponding to a sound wave type interpolation between the two regions (er, jr) ≈(el, jl). Given that bad shocks are replaced by rarefaction waves, one should remove from the ﬁsh diagram (left panel of ﬁgure 2) the portion of the curve which corresponds to bad shocks and replace it with a curve corresponding to a rarefaction solution (central panel of ﬁgure 2). The resulting curve can be found on the right panel of ﬁgure 2: the belly of the – 14 – Figure 3. A graphical determination of the “good shocks” and “bad shocks”. The red ﬁsh cor- responds to (er, jr) while the blue ﬁsh is built from (el, 0). See the main text for a discussion. JHEP08(2016)120 Figure 3. A graphical determination of the “good shocks” and “bad shocks”. The red ﬁsh cor- responds to (er, jr) while the blue ﬁsh is built from (el, 0). See the main text for a discussion. ﬁsh and the lower part of its tail corresponds to a good shock and its back and upper tail to a rarefaction solution. One may compute the curve explicitly by imposing (4.8), but it can also be understood from a graphical viewpoint as we now explain. Recall that the self intersection point of the shock wave ﬁsh (solid curve on the left panel of ﬁgure 2) corresponds to a shock velocity, s, which takes the values of the local speed of sound, ±1+jl/el. On the tail, s is either larger than 1+jl/el (upper tail) or smaller than −1 + j/e (lower tail). Thus, on the tails, the eigenvalues are either both positive or both negative. The top portion of the tail has λ±l < 0 while the bottom portion of the tail has λ±l > 0. As a result, the top portion of the tail must be replaced by a rarefaction wave while the bottom portion can be a shock. 4.1 Rarefaction waves vs. shock waves To decide which portion of the body of the shock ﬁsh to replace by a rarefaction wave, one must study λ±r. Consider a second ﬁsh which exhibits the solution to the cubic (4.3) for a given value of (er, jr). We will call this second ﬁsh an r-ﬁsh and the ﬁrst an l-ﬁsh. Similar to the analysis of the tail of the l-ﬁsh, we ﬁnd that the bottom portion of the tail of the r-ﬁsh should be constructed from a rarefaction solution while the top portion from a shock. Consider an r-ﬁsh whose point of self intersection lies somewhere on the body of the l-ﬁsh. When the r-ﬁsh is drawn so that it intersects the back of the l-ﬁsh, the bottom portion of the r-ﬁsh’s tail will go through the point of self-intersection of the l-ﬁsh (see the left panel of ﬁgure 3). As the bottom portion of the tail of the r-ﬁsh is a rarefaction, the region (er, lr) can be connected to (el, jl) by a rarefaction. Reciprocally, since we’re describing a single shock or rarefaction interface between two regions, the back of the l-ﬁsh should be replaced by a rarefaction wave. We can run the argument again for an r-ﬁsh drawn to intersect the belly of the l-ﬁsh. We conclude that the belly of the l-ﬁsh must be a shock (see the right panel of ﬁgure 3). – 15 – Figure 4. Some diagramatic solutions to the Riemann problem. The blue ﬁsh corresponds to (eL, 0) while the red ﬁsh to (eR, jR). The solid line is a shock and the dashed line a rarefaction. The intermediate region is indicated by a black dot. Left panel: the shock solution of the right asymptotic region overlaps with the rarefaction solution of the left asymptotic region, so we get an SR type conﬁguration. Center panel: the rarefaction solution of the left and right regions overlap creating an RR type solution. Right panel: an SS type solution. Figure 4. Some diagramatic solutions to the Riemann problem. The blue ﬁsh corresponds to (eL, 0) while the red ﬁsh to (eR, jR). The solid line is a shock and the dashed line a rarefaction. The intermediate region is indicated by a black dot. Left panel: the shock solution of the right asymptotic region overlaps with the rarefaction solution of the left asymptotic region, so we get an SR type conﬁguration. 4.1 Rarefaction waves vs. shock waves Center panel: the rarefaction solution of the left and right regions overlap creating an RR type solution. Right panel: an SS type solution. JHEP08(2016)120 4.2 Solving the Riemann problem using ideal hydrodynamics As an example of the RS case, we take (eL, 0) and (eR, 0) as the asymptotic regions with eL > eR. The SR case is a left-right reﬂection of the RS case and therefore does not warrant further discussion. To estimate the values of e0 and j0 we can follow the strategy laid out in [12, 13]. For the left region we use the solution (4.9) with el = eL, jl = 0, er = e0 and jr = j0. For the right region we use (4.2) with el = e0, jl = j0, er = eR and jr = 0. We ﬁnd To estimate the values of e0 and j0 we can follow the strategy laid out in [12, 13]. For the left region we use the solution (4.9) with el = eL, jl = 0, er = e0 and jr = j0. For the right region we use (4.2) with el = e0, jl = j0, er = eR and jr = 0. We ﬁnd e0 = eRs2 , j0 = eRs(s2 −1) , 0 = 1 s −s −log eR eL s2 , (4.10) (4.10) JHEP08(2016)120 which, unsurprisingly, coincides with the large d limit of the hydrodynamic analysis of [12, 13]. As pointed out in [12] the rarefaction solution will cover the location of the original shock discontinuity whenever As pointed out in [12] the rarefaction solution will cover the location of the original shock discontinuity whenever eL eR ≥ 1 + √ 5 2 !2 exp(1) ∼7.11655 . (4.11) (4.11) At the point ζ = 0 in the rarefaction wave, the values of e and j are time independent (since any function of ζ/t will have a ﬁxed point at ζ = 0). Moreover for a conserved stress tensor T µν = T µν ζ t , the ﬁrst spatial derivative of T tζ and the ﬁrst and second spatial derivatives of T ζζ vanish at this ﬁxed point. Thus, one may think of the pressure at the ﬁxed point as a “short” steady state for long enough times. “Short” implies that the region is of small spatial extent. From this perspective one has split steady states for large enough initial temperature diﬀerences. 4.2 Solving the Riemann problem using ideal hydrodynamics Armed with our understanding of shock waves and rarefaction solutions, let us now tackle the Riemann problem we set out to solve. At t = 0, we consider a pair (eL, 0) which describes the ﬂuid for z < 0 and another pair (eR, jR) describing the ﬂuid for z > 0. For a single interpolating shock or rarefaction, we have seen that given (eL, 0) there is a one parameter family of solutions that determine (eR, jR). Thus, generically, there will not be a single shock or rarefaction solution that joins (eL, 0) to an arbitrary (eR, jR). However, we can connect the two regions using a pair of shock and/or rarefaction waves. That is, we could connect (eL, 0) to an intermediate regime with values of e and j given by (e0, j0) using a shock or rarefaction wave and another shock wave or rarefaction wave to connect the intermediate regime to the right asymptotic region (eR, jR). In all cases, given the initial conditions, the pair of rarefaction and/or shock waves should be such that they move away from each other. The strategy for determining which type of solution is allowed is to prefer good shocks over rarefaction solutions and rarefaction solutions over bad shocks. Thus, given a pair (eL, 0) and (eR, jR) we need to establish which of the four possibilities for the time evolution of the initial state is allowed: two shocks (SS), a rarefaction wave followed by a shock (RS), or the remaining two conﬁgurations which we will denote by SR and RR. To understand the possible solutions to the Riemann problem, let us ﬁrst consider two ﬁsh diagrams: one associated with (el, jl) = (eL, 0) (the l-ﬁsh) and another with (er, jr) = (eR, jR) (the r-ﬁsh). The points of overlap of the diagrams will give us the possible value of e0 and j0. We will always choose a point where the two disturbances are moving away from each other. See, for example, ﬁgure 4. Instead of plotting the r- and l-ﬁshes, we can obtain closed form expressions for the various types of solutions by solving (4.8) and (4.9) on a case by case basis. In the following we provide some simple examples of such expressions. – 16 – • RS conﬁgurations. • RS conﬁgurations. As an example of the RS case, we take (eL, 0) and (eR, 0) as the asymptotic regions with eL > eR. The SR case is a left-right reﬂection of the RS case and therefore does not warrant further discussion. 4.2 Solving the Riemann problem using ideal hydrodynamics Using eL = eR and jR > 0, we can ﬁnd simple solutions that involve two rarefaction waves.5 In this case, the NESS is characterized by e0 = eL exp −jR 2eL , j0 = jR 2 exp −jR 2eL , (4.16) (4.16) where the left moving rarefaction wave extends from ξ = −1 to ξ = ξ−while the right moving rarefaction wave extends from ξ = ξ+ to ξ = 1 with ξ+ −ξ−= 2 , ξ+ + ξ−= jR eR , (4.17) (4.17) JHEP08(2016)120 Similar to the RS case we ﬁnd that there is a ﬁxed point associated with the left moving wave whenever j jR 2eL ≥1 , (4.18) (4.18) with es = js = eL exp(−1) . (4.19) (4.19) We claim that given (eL, 0), the “phase diagram” of ﬁgure 1 immediately allows us to choose the correct conﬁguration of shocks and rarefaction waves for any (eR, jR). Indeed, following ﬁgure 4, the location of the self intersection point of the r-ﬁsh will determine the nature of the intersection of the r- and l-ﬁsh: if the intersection point of the r-ﬁsh lies above the l-ﬁsh we will always get an RR solution; if the intersection point of the r-ﬁsh is below the l-ﬁsh we get an SS solution; and RS and SR solutions will correspond to an intersection point of the r-ﬁsh in the body or tail of the l-ﬁsh respectively. Conformal invariance dictates that the phase diagram can depend on the only two dimensionless parameters of this problem, and we obtain the phase diagram in ﬁgure 1. Note that even though the r-ﬁsh and the l-ﬁsh intersect at (0, 0), we can always rule out an intermediate point that corresponds to a vacuum. The vacuum intersection point is always along the bodies of the two ﬁsh where we have λ−,l/r < 0 < λ+,l/r. As discussed, we can not in general connect the two asymptotic solutions if we do not have two eigenvalues of the same sign (positive for l and negative for r) in one of the regions. 4.2 Solving the Riemann problem using ideal hydrodynamics The values of e and j at the short steady state are given by At the point ζ = 0 in the rarefaction wave, the values of e and j are time independent (since any function of ζ/t will have a ﬁxed point at ζ = 0). Moreover for a conserved stress tensor T µν = T µν ζ t , the ﬁrst spatial derivative of T tζ and the ﬁrst and second spatial derivatives of T ζζ vanish at this ﬁxed point. Thus, one may think of the pressure at the ﬁxed point as a “short” steady state for long enough times. “Short” implies that the region is of small spatial extent. From this perspective one has split steady states for large enough initial temperature diﬀerences. The values of e and j at the short steady state are given by es = js = eL exp(−1) . (4.12) (4.12) • SS conﬁgurations. A simple example of the SS case has (eL, 0) on the left and (eL, jR) on the right with jR < 0. We compute the NESS by gluing two shock waves to an intermediate region with (e, j) = (e0, j0), similar to the RS case. Setting β = jR/eL, the intermediate NESS is given by e0 = eL 8 (8 + β2 −β p 16 + β2) , j0 e0 = β 2 , (4.13) (4.13) and the shock velocities for the left and right moving shocks, sL and sR respectively, are given by sL = 1 4(β − p 16 + β2) , (4.14) sR = 1 4(3β + p 16 + β2) . (4.15) (4.14) (4.15) – 17 – – 17 – • RR conﬁgurations. Using eL = eR and jR > 0, we can ﬁnd simple solutions that involve two rarefaction waves.5 In this case, the NESS is characterized by • RR conﬁgurations. 5As it turns out in the RR phase, there is a simple expression for the steady state for all values of eL, eR, jL and jR, 4.3 A numerical solution to the Riemann problem 4.3 In the previous sections we have obtained predictions for the evolution of e and j starting from an initial conﬁguration (1.2) and assuming that gradient corrections to the equations of motion are small. It is somewhat unfortunate that this assumption stands in stark contrast to the discontinuous jump in the initial state and one may inquire whether the analysis of the previous section is relevant for the problem at hand. In order to resolve this issue we solve the full equations of motion (1.1) numerically. We give numerical examples of the RR, SS, and RS phases described above. To our numerical accuracy, the diﬀerence in e0 and j0 between the ideal case which we have studied analytically and the case with gradients included which has been obtained numerically appears to disappear in the long time limit. In the previous sections we have obtained predictions for the evolution of e and j starting from an initial conﬁguration (1.2) and assuming that gradient corrections to the equations of motion are small. It is somewhat unfortunate that this assumption stands in stark contrast to the discontinuous jump in the initial state and one may inquire whether the analysis of the previous section is relevant for the problem at hand. In order to resolve this issue we solve the full equations of motion (1.1) numerically. We give numerical examples of the RR, SS, and RS phases described above. To our numerical accuracy, the diﬀerence in e0 and j0 between the ideal case which we have studied analytically and the case with gradients included which has been obtained numerically appears to disappear in the long time limit. JHEP08(2016)120 As it turns out, the equations (1.1) are easy to evolve numerically with canned PDE solvers, such as Mathematica’s NDSolve routine [42]. To obtain various solutions one can evolve the initial condition e = ⟨e⟩(1 + δe tanh(c sin(2πx/L))) , (4.20) j = ⟨j⟩(1 + δj tanh(c sin(2πx/L))) , (4.21) (4.20) (4.21) (4.21) in a periodic box of length L. (In appendix B, we use a more elaborate piecewise continuous initial condition.) For c suﬃciently large, the initial condition approaches a square wave. 4.2 Solving the Riemann problem using ideal hydrodynamics 5As it turns out in the RR phase, there is a simple expression for the steady state for all values of eL, eR, jL and jR, e0 = √eLeR exp jL 2eL −jR 2eR , j0 = e0 2 (ξ+ + ξ−) , where ξ+ −ξ−= 2 , ξ+ + ξ−= jL eL + jR eR −log eR eL . A ﬁxed point associated with a left moving rarefaction solution occurs whenever A ﬁxed point associated with a left moving rarefaction solution occurs whenever eR eL ≤exp jL eL + jR eR −2 with es = js = eL exp −1 + jL eL , and a ﬁxed point associated with the right moving rarefaction solution occurs whenever eR eL ≥exp jL eL + jR eR + 2 with es = −js = eR exp −1 + jR eR . – 18 – 4.3 A numerical solution to the Riemann problem 4.3 A numerical solution to the Riemann problem As long as the disturbance has not travelled a distance of order L, causality ensures that the behaviour of e and j are very close to that of an inﬁnite system where the values of e and j in the asymptotic region are ﬁxed at some constant value. If we denote these asymptotic values as eL and eR then δe = eL −eR eL + eR and ⟨e⟩= 1 2 (eL + eR) . (4.22) (4.22) We can similarly deﬁne ⟨j⟩and δj. We can similarly deﬁne ⟨j⟩and δj. In ﬁgures 5, 6, and 7, we have plotted typical results for numerical solutions to (1.1), corresponding to RS, SS, and RR conﬁgurations. The resulting values of e and j seem to approach the predicted values of e0 and j0 at long times — at least as far as our numerical precision can be trusted (see appendix B). In particular, in the RS case, we approach the steady state value (4.10); in the SS case, we approach (4.13); and in the RR case, we approach (4.16). As we discuss in greater detail in the next section, one place where gradient eﬀects show up and do not disappear as a function of time is in the shock width. One may speculate that the agreement between the predicted steady state in the absence of gradient corrections and the numerical results is associated to the fact that the gradient corrections, even though order one in our system of units, come with dimensionful coeﬃcients. In the language of the renormalization group, they conform to irrelevant couplings. Perhaps it is for this reason that at long enough time and in a large enough box, we may be able to ignore these corrections for the most part. – 19 – Figure 5. A numerical solution to the Riemann problem. The plots were obtained starting with an initial condition (B.5) with L = 8000, c = 300 and ⟨j⟩= 0. Only one half of the box, centered around the origin, is depicted. The dashed curve corresponds to values of e and j at t = 0 while the solid curve corresponds to values of e and j at t = 800. 4.3 A numerical solution to the Riemann problem The black, red and blue horizontal lines correspond to the predicted near equilibrium steady state associated with a rarefaction wave and shock pair (cf., equation (4.10)), a bad shock and good shock pair (cf., references [5, 7]), and a non thermodynamic shock pair (cf., reference [5]) respectively. The ﬁxed point associated with a rarefaction solution which exists for δe ≥0.7536 . . . is represented by a black dot. JHEP08(2016)120 Figure 5. A numerical solution to the Riemann problem. The plots were obtained starting with an initial condition (B.5) with L = 8000, c = 300 and ⟨j⟩= 0. Only one half of the box, centered around the origin, is depicted. The dashed curve corresponds to values of e and j at t = 0 while the solid curve corresponds to values of e and j at t = 800. The black, red and blue horizontal lines correspond to the predicted near equilibrium steady state associated with a rarefaction wave and shock pair (cf., equation (4.10)), a bad shock and good shock pair (cf., references [5, 7]), and a non thermodynamic shock pair (cf., reference [5]) respectively. The ﬁxed point associated with a rarefaction solution which exists for δe ≥0.7536 . . . is represented by a black dot. 4.4 Restoring gradient corrections In this section, we try to gain a better handle over the gradient corrections and their aﬀect on the predicted steady state values. The analysis here is incomplete and approximate. To overcome the deﬁciencies of paper and pencil estimates, we include some numerical solutions to the conservation equations (1.1) that provide support for the estimates. We will consider separately corrections to each of the features we found in the idealized limit: the steady state and asymptotic regions with constant e and j, a shock wave, a rarefaction wave, and the discontinuity at the edge of the rarefaction. Corrections to constant regions. Corrections to a constant e and j region are easiest to analyze. Assuming the ﬂuctuations are small, we look for linearized solutions of the form e = e0+δe exp(−iωt+ikζ) and j = j0+δj exp(−iωt+ikζ). We ﬁnd two propagating modes ω = ±1 + j0 e0 k −ik2 . (4.23) (4.23) – 20 – – 20 – Figure 6. Two numerical solutions to the Riemann problem in the RR case. The plots were obtained starting with a constant e initial condition, jL = 0, and ﬁxed β = jR/eL, with L = 8000 and c = 200. The dashed line corresponds to the solution at t = 0 and the solid blue line at t = 1000. The solid red curves are the rarefaction waves in the ideal limit, without gradient corrections. The horizontal black line is the predicted steady state value. JHEP08(2016)120 Figure 6. Two numerical solutions to the Riemann problem in the RR case. The plots were obtained starting with a constant e initial condition, jL = 0, and ﬁxed β = jR/eL, with L = 8000 and c = 200. The dashed line corresponds to the solution at t = 0 and the solid blue line at t = 1000. The solid red curves are the rarefaction waves in the ideal limit, without gradient corrections. The horizontal black line is the predicted steady state value. Figure 7. A numerical solution to the Riemann problem in the SS case. The plots were obtained starting with a constant e initial condition, jR = 0, and β = −jL/eL = −1, with L = 8000 and c = 200. The dashed line corresponds to the solution at t = 0 and the solid blue line at t = 1000. 4.4 Restoring gradient corrections The horizontal black line is the predicted steady state value. Figure 7. A numerical solution to the Riemann problem in the SS case. The plots were obtained starting with a constant e initial condition, jR = 0, and β = −jL/eL = −1, with L = 8000 and c = 200. The dashed line corresponds to the solution at t = 0 and the solid blue line at t = 1000. The horizontal black line is the predicted steady state value. – 21 – These two modes are damped sound modes whose speed is shifted by the ﬂuid velocity β = j/e. The gradient corrections appear here in the form of the damping term ik2 in the dispersion relation. Given this result, we anticipate that we will be able to correct a constant e and j region by taking an appropriate linear superposition of sound waves. The damping suggests that at long times the solution can only involve constant e and constant j. These two modes are damped sound modes whose speed is shifted by the ﬂuid velocity β = j/e. The gradient corrections appear here in the form of the damping term ik2 in the dispersion relation. Given this result, we anticipate that we will be able to correct a constant e and j region by taking an appropriate linear superposition of sound waves. The damping suggests that at long times the solution can only involve constant e and constant j. As a side comment, an odd thing about these mode relations is that they are exact. Recall that in ﬁrst order viscous hydro, we would typically solve an equation of the form ω2 + iΓk2ω −k2 = 0 for ω, in the case of vanishing background ﬂuid velocity. If this equation were treated as exact, the solutions for ω would be non linear in k and therefore have higher order contributions, i.e. O(k3), O(k4), etc., when expanded around small k. JHEP08(2016)120 Corrections to shocks. The gradient corrections should act to smooth a shock and give it some characteristic width. We estimate this width in a frame in which the shock is not moving, i.e. s = 0. In this frame, jr = jl and erel = j2 l . 6We found that when δe = 0.8 the relative error between (4.24) and the numerical solution grew to ∼13%. As δe gets closer to one numerical error is more diﬃcult to control. 4.4 Restoring gradient corrections We can ﬁnd a solution for the shock proﬁle in the case where the shock is weak er ∼el: e = ⟨e⟩ " 1 + δe tanh ζδe 2 −δe2 2 sech ζδe 2 2 log cosh ζδe 2 + O(δe3) # , (4.24) j = ⟨j⟩ " 1 + δe2 2 sech ζδe 2 2 + O(δe3) # , (4.25) (4.24) (4.25) where we have deﬁned ⟨e⟩≡er + el 2 , δe ≡er −el er + el , and ⟨j⟩≡jr + jl 2 . We can see in ﬁgure 8 that even for values of δe ∼1/2, that ⟨e⟩δe2/2 appears to be a good estimate for the slope of the shock.6 In appendix A, we show that this shock proﬁle produces, at the correct subleading order in a large d expansion, the correct (positive) amount of entropy predicted by the RH relations. We can see in ﬁgure 8 that even for values of δe ∼1/2, that ⟨e⟩δe2/2 appears to be a good estimate for the slope of the shock.6 In appendix A, we show that this shock proﬁle produces, at the correct subleading order in a large d expansion, the correct (positive) amount of entropy predicted by the RH relations. Corrections to a rarefaction. We will perform two estimates of gradient corrections to the rarefaction wave. The ﬁrst estimate is a correction to the interior of the wave far from the edges where it joins onto constant e and j regions. The second estimate is a correction to the discontinuity where the rarefaction joins a constant region. For the ﬁrst estimate, we assume an ansatz for the long time behavior of the rarefaction wave: e = e0(ξ) + log t t el(ξ) + 1 t e1(ξ) + O((log t)2/t2) , j = j0(ξ) + log t t jl(ξ) + 1 t j1(ξ) + O((log t)2/t2) , 6We found that when δe = 0.8 the relative error between (4.24) and the numerical solution grew to ∼13%. As δe gets closer to one numerical error is more diﬃcult to control. 6We found that when δe = 0.8 the relative error between (4.24) and the numerical solution grew to ∼13%. As δe gets closer to one numerical error is more diﬃcult to control. – 22 – Figure 8. A numerical simulation of stationary shocks. 4.4 Restoring gradient corrections We start from an initial condition e = ⟨e⟩(1 + δe tanh(c sin(2πx/L))), j = 1 with parameters L = 8000 and c = 1.2(Lδe/4π). We chose er and el to produce a stationary shock (el = √1−δe √1+δe, er = √1+δe √1−δe) using the RH relations. We then plot the value of the slope of the shock after the system has settled into a steady state. This is compared with the weak shock solution (4.24), given by the dashed red line. The inset plot shows the relaxation from the initial conditions to the steady state for δe = 0.23. JHEP08(2016)120 Figure 8. A numerical simulation of stationary shocks. We start from an initial condition e = ⟨e⟩(1 + δe tanh(c sin(2πx/L))), j = 1 with parameters L = 8000 and c = 1.2(Lδe/4π). We chose er and el to produce a stationary shock (el = √1−δe √1+δe, er = √1+δe √1−δe) using the RH relations. We then plot the value of the slope of the shock after the system has settled into a steady state. This is compared with the weak shock solution (4.24), given by the dashed red line. The inset plot shows the relaxation from the initial conditions to the steady state for δe = 0.23. where where e0 = c1 exp(∓ξ) , j0 = (±1 + ξ)c1 exp(∓ξ) , (4.26) el = 2c1 exp(∓ξ) −1 2c2 exp(∓ξ/2) , jl = ξel , (4.27) j1 = ± exp(∓ξ)(c1 −c2 exp(±ξ/2)) + ξe1 . (4.28) (4.27) (4.28) With an appropriate choice for the integration constant c1, the expressions for e0 and j0 become the same as we had before (4.9). There are subleading corrections that scale as 1/t and log(t)/t that depend on a second integration constant c2 and an arbitrary function e1(ξ), both presumably set by the initial conditions. Note that the combination ξe −j is independent of the arbitrary function e1(ξ) at order 1/t. In ﬁgure 9, the numerics conﬁrm that the corrections to ξe −j do indeed scale as 1/t. Last, we would like to heal the discontinuity at the edge of a rarefaction wave. The tanh function we found above heals the discontinuity in the shock case, making the question of what happens at the edge of a shock less pressing. 4.4 Restoring gradient corrections Consider a case where the rarefaction wave meets a steady state at ζ = 0, with the rarefaction region to the right and the steady state to the left. (We can always move the meeting point away from ζ = 0 by boosting the solution ζ →ζ +vt.) With the intuition that the second order gradients in the conservation equations are dominant and render the behavior similar to that of a heat equation with – 23 – Figure 9. A plot of δ(ξe −j) vs. time at three diﬀerent points in a single rarefaction wave. The quantity δ(ξe −j) is the diﬀerence between the zeroth order prediction (4.9) and numerics. The rarefaction wave spreads from ξl = −1 to ξr = 1. The three points correspond to ξ = −1/2 (red), ξ = 0 (purple) and ξ = 1/2 (green). The dashed line 1/(2t) is a guide to the eye. Inset: the rarefaction proﬁle at t = 3000. Dashed lines correspond to e while the solid lines correspond to j. The blue curve is numeric, while the red curve is the ideal result (4.9). JHEP08(2016)120 Figure 9. A plot of δ(ξe −j) vs. time at three diﬀerent points in a single rarefaction wave. The quantity δ(ξe −j) is the diﬀerence between the zeroth order prediction (4.9) and numerics. The rarefaction wave spreads from ξl = −1 to ξr = 1. The three points correspond to ξ = −1/2 (red), ξ = 0 (purple) and ξ = 1/2 (green). The dashed line 1/(2t) is a guide to the eye. Inset: the rarefaction proﬁle at t = 3000. Dashed lines correspond to e while the solid lines correspond to j. The blue curve is numeric, while the red curve is the ideal result (4.9). 1/ √ t broadening, we look for an approximate late time solution of the form e = e0 + 1 √ te1(χ) + O(t−1) , (4.29) j = j0 + j1 √ t + 1 t j2(χ) + O(t−3/2) , (4.30) (4.29) deﬁning χ ≡ζ/ √ t. We ﬁnd that j0 = ±e0, that j1 is constant, and that deﬁning χ ≡ζ/ √ t. We ﬁnd that j0 = ±e0, that j1 is constant, and that j′ 2(χ) = ∓e1(χ)e′ 1(χ) e0 + 4e′ 1 e0 ± 1 j1 . j′ 2(χ) = ∓e1(χ)e′ 1(χ) e0 + 4e′ 1 e0 ± 1 j1 . 4.4 Restoring gradient corrections This boundary condition ﬁxes the remaining integration constant associated with the ﬁrst order equation (4.32), and the solution for e1 is then The shifts send j1 →0 and c1 →c1 ∓3j2 1/8e0 in the equation (4.32). If we apply the boundary condition that both e1(χ) and e′ 1(χ) vanish in the steady state region χ → −∞, then we must set c1 = 0, and the resulting ﬁrst order diﬀerential equation becomes separable. To match onto the rarefaction region, we require that e′ 1 →±e0 as χ →∞. This boundary condition ﬁxes the remaining integration constant associated with the ﬁrst order equation (4.32), and the solution for e1 is then e1 = ± 2e0e−χ2/4 √π erfc(χ/2) . (4.33) (4.33) As we choose the rarefaction region to match onto the steady state at χ = 0, we conclude that the integration constant j1 in the original diﬀerential equation must be zero as well. We can check numerically that a 1/ √ t scaling is consistent with the behavior at the endpoints of a rarefaction solution. See ﬁgure 10. 4.4 Restoring gradient corrections Note that the relation j0 = ±e0 is consistent with a rarefaction meeting a steady state region at ζ = 0. These relations for the ji lead to a second order, nonlinear diﬀerential equation for e1: Note that the relation j0 = ±e0 is consistent with a rarefaction meeting a steady state region at ζ = 0. These relations for the ji lead to a second order, nonlinear diﬀerential equation for e1: e′′ 1 + χ 2 + ±e1 −j1 e0 e′ 1 + e1 2 ∓j1 4 = 0 . (4.31) (4.31) Remarkably, this equation can be written as a total derivative and integrated to yield ± e2 1 2e0 + e−χ2/4∂χ(eχ2/4e1) −j1e1 e0 ∓j1 4 χ = c1 , (4.32) (4.32) where c1 is another integration constant. The integration constants reﬂect a translation symmetry of both e1 and χ. We can shift χ →χ + j1/e0 and e1(χ) →e1(χ −j1/e0) ± j1/2. – 24 – Figure 10. A log log plot of δe,δj vs. time at the endpoints of a rarefaction wave, where δe = e−e0 and δj = j−j0 and e0 and j0 are from the zeroth order prediction (4.9). As in ﬁgure 9 the rarefaction wave spreads from ξl = −1 to ξr = 1. The four curves correspond to e(1) (red), e(−1) (purple) and j(1) (green) and j(−1) (orange). The dashed lines 0.43t−1/2 and 3t−1/2 are a guide to the eye. JHEP08(2016)120 Figure 10. A log log plot of δe,δj vs. time at the endpoints of a rarefaction wave, where δe = e−e0 and δj = j−j0 and e0 and j0 are from the zeroth order prediction (4.9). As in ﬁgure 9 the rarefaction wave spreads from ξl = −1 to ξr = 1. The four curves correspond to e(1) (red), e(−1) (purple) and j(1) (green) and j(−1) (orange). The dashed lines 0.43t−1/2 and 3t−1/2 are a guide to the eye. The shifts send j1 →0 and c1 →c1 ∓3j2 1/8e0 in the equation (4.32). If we apply the boundary condition that both e1(χ) and e′ 1(χ) vanish in the steady state region χ → −∞, then we must set c1 = 0, and the resulting ﬁrst order diﬀerential equation becomes separable. To match onto the rarefaction region, we require that e′ 1 →±e0 as χ →∞. 5 Discussion We presented a solution to the Riemann problem for the conservation equations (1.1). Through ﬂuid-gravity and the AdS/CFT correspondence, these equations describe, in a large d limit, both the dynamics of a black hole horizon and also the dynamics of a strongly interacting conformal ﬁeld theory. There are a number of possible future directions for research. The simplest is perhaps to include a transverse velocity. With a transverse velocity, in addition to the shock and rarefaction waves, there will in general be a contact discontinuity [13, 43–45]. It is known – 25 – (and perhaps intuitive given the similarity to a counter ﬂow experiment), that the contact discontinuity is in general unstable to the development of turbulence [46]. It would be interesting to see what precisely happens in our large d limit. Another more complicated extension is the inclusion of a conserved charge. The large d equations of motion in the presence of a conserved charge are available from ref. [14]. Once again, a contact disconti- nuity is expected (see for example [13]) although whether such a discontinuity is stable or unstable to turbulence is unclear. More ambitiously, one could consider what happens for the holographic dual of a superﬂuid or superconductor [19, 25, 47–51]. Another possible direction is the addition of higher curvature terms to the dual grav- itational description. One could presumably tune the d dependence of these terms such that higher order gradient corrections appear in the conservation equations (1.1) and also such that the ﬁrst and second order transport coeﬃcients are tuned away from the values examined in this paper. JHEP08(2016)120 Perhaps the most interesting direction for future study is the connection to black hole dynamics. What can we learn about black holes through the connection to hydrodynamics in a large d limit? Acknowledgments We would like to thank S. Bhattacharyya, S. Cremonini, J. Glimm, V. Hubeny, D. Huse, A. Lucas, A. Ori, M. Rangamani, and K. Schalm for discussion. M. S. and C. P. H. were supported in part by NSF Grant No. PHY13-16617. A. Y. was supported by the ISF under grant numbers 495/11, 630/14 and 1981/14, by the BSF under grant number 2014350, by the European commission FP7, under IRG 908049 and by the GIF under grant number 1156/2011. A Comment about entropy production across a shock In the ideal limit, in addition to conservation of energy and momentum, we can write down a conservation condition for the entropy current, ∂µ ˜Jµ S = 0 where ˜Jµ S = (ϵ + p)uµ/T. (A.1) (A.1) This conservation condition would naively seem to lead to an additional Rankine-Hugoniot relation across a single shock. As is well known in the hydrodynamics community (see for example [12]), since shocks create entropy this third Rankine-Hugoniot relation is violated. Let us parameterize a possible violation of the additional Rankine-Hugoniot relation by ∆. ∆= s[ ˜Jt S] −[ ˜Jζ S] (A.2) (A.2) where the square brackets are the same as those in (4.1). One ﬁnds where the square brackets are the same as those in (4.1). One ﬁnds ∆= 2π √ereld2 e2 r −e2 l −2erel log er el + O(d−3) . (A.3) (A.3) – 26 – – 26 – Equation (A.3) can be obtained by using a large d expression for the entropy current (3.22) along with the Rankine-Hugoniot relations for energy and momentum, (4.1) supplemented by (2.14) and (2.15). Note that in the asymptotic regions, the gradient terms will all vanish. (It is also possible to start with a ﬁnite d result, using for example refs. [12] or [13], and then take a large d limit directly.) The non-conservation of entropy (A.3) can be captured by the leading viscous correc- tions to the shock width (4.24) when the energy diﬀerence is small. Indeed, using (3.24) ∂µ ˜Jµ S = 8π d2 j2 0(e′)2 e3 + O(d−3) = 2πj2 0δe4 d2⟨e⟩ sech ζδe 2 4 + O(δe5, d−3) . (A.4) (A.4) JHEP08(2016)120 Integrating this divergence over the ζ direction leads to Z ∂µ ˜Jµ Sdζ = 16π⟨e⟩δe3 3d2 + O(δe4, d−3) , (A.5) (A.5) which agrees with a small δe expansion of (A.3). B A bestiary of plots In section 4.3 we studied the numerical solutions to the Riemann problem for various initial energy and velocity proﬁles associated with RR, RS and SS type solutions. In what follows we provide additional evidence that at late times the full numerical solution to the Riemann problem approaches the appropriate predicted steady state values e0 and j0 and ﬁxed point values es and js. B.1 RR conﬁgurations To generate an RR conﬁguration we used the initial data To generate an RR conﬁguration we used the initial data e = 1 , j =                      f(ζ) 0 ≤ζ < ℓ/4 0 ℓ/4 ≤ζ < L/2 −ℓ/4 f(ζ −L/2 −ℓ/2) L/2 −ℓ/4 ≤ζ < L/2 + ℓ/4 j∗ L/2 + ℓ/4 ≤ζ < L −ℓ f(ζ −L) L −ℓ≤ζ < L (B.1) (B.1) where f(ζ) = 1 2j∗ 1 −tanh c sin 2πζ ℓ . (B.2) (B.2) The analysis of section 4.2 predicts a steady state of the form The analysis of section 4.2 predicts a steady state of the form e0 = exp (−j∗/2) j0 = j∗ 2 exp (−j∗/2) . (B.3) (B.3) Once j∗≥2 one should ﬁnd a ﬁxed point with es = js = exp(−1). We ﬁnd that the numerical solution approaches the predicted states via power law behavior, see ﬁgure 11. Once j∗≥2 one should ﬁnd a ﬁxed point with es = js = exp(−1). We ﬁnd that the numerical solution approaches the predicted states via power law behavior, see ﬁgure 11. – 27 – Figure 11. Late time behavior of the steady state and ﬁxed point for RR type conﬁgurations. Top plots: the deviation of j(t, ζ = vt) from the predicted steady state value j0 for various values of v. The initial conditions are given by (B.1) with L = 20000, ℓ= 8000, and c = 300 and j∗= 1.8 for the top left plot and L = 8000, ℓ= 2000, c = 100 and j∗= 5 for the top right plot. Both the results roughly ﬁt a ∼tα asymptotic behavior with α ∼0.9. Bottom plots: the deviation of e and j from the predicted ﬁxed point value at ζ = 0 for various values of c. The initial conditions are given by (B.1) with L = 16000, ℓ= 4000 and j∗= 3. Both the time dependence of e/es −1 and j/js −1 can be ﬁt to a power law, ∼tα. For the energy density one ﬁnds that α gradually increases to α ∼0.8 as one approaches c = 300. For the energy current α decreases to α ∼1.1 at c = 300. JHEP08(2016)120 Figure 11. Late time behavior of the steady state and ﬁxed point for RR type conﬁgurations. B.2 SS conﬁgurations To generate an SS conﬁguration we used the initial data (B.1) with j∗< 0. The analysis of section 4.2 predicts a steady state of the form To generate an SS conﬁguration we used the initial data (B.1) with j∗< 0. The analysis of section 4.2 predicts a steady state of the form e0 = 1 8(8 + j2 ∗−j∗ p 16 + j2∗) , j0 e0 = j∗ 2 . (B.4) (B.4) See ﬁgure 12 for a comparison with the numerical data. See ﬁgure 12 for a comparison with the numerical data. B.3 RS conﬁgurations B.1 RR conﬁgurations Top plots: the deviation of j(t, ζ = vt) from the predicted steady state value j0 for various values of v. The initial conditions are given by (B.1) with L = 20000, ℓ= 8000, and c = 300 and j∗= 1.8 for the top left plot and L = 8000, ℓ= 2000, c = 100 and j∗= 5 for the top right plot. Both the results roughly ﬁt a ∼tα asymptotic behavior with α ∼0.9. Bottom plots: the deviation of e and j from the predicted ﬁxed point value at ζ = 0 for various values of c. The initial conditions are given by (B.1) with L = 16000, ℓ= 4000 and j∗= 3. Both the time dependence of e/es −1 and j/js −1 can be ﬁt to a power law, ∼tα. For the energy density one ﬁnds that α gradually increases to α ∼0.8 as one approaches c = 300. For the energy current α decreases to α ∼1.1 at c = 300. B.3 RS conﬁgurations We expect that numerical error is of order 10−7 −10−8. where f(ζ) = 1 2 (1 + e∗) + 1 2 (e∗−1) tanh c sin 2πζ ℓ + e∗. (B.6) (B.6) The analysis of section 4.2 predicts a steady state of the form The analysis of section 4.2 predicts a steady state of the form e0 = s2 , j0 = s(s2 −1) . (B.7) e0 = s2 , j0 = s(s2 −1) . (B.7) 0 = 1 s −s −log s2 e∗ . (B.8) e0 = s2 , j0 = s(s2 −1) . (B.7) with 0 = 1 s −s −log s2 e∗ . (B.8) (B.7) with 0 = 1 s −s −log s2 e∗ . (B.8) (B.8) According to the same analysis, once e∗≥ 1+ √ 5 2 2 exp(1) we will obtain a ﬁxed point at the origin with es = js = exp(−1). An analysis of the late time behavior of the numerical solution can be found in ﬁgure 13. According to the same analysis, once e∗≥ 1+ √ 5 2 2 exp(1) we will obtain a ﬁxed point at the origin with es = js = exp(−1). An analysis of the late time behavior of the numerical solution can be found in ﬁgure 13. B.3 RS conﬁgurations To generate an RS conﬁguration we used the initial data j = 0 , e =                  f(ζ) 0 ≤ζ < ℓ/4 e∗ ℓ/4 ≤ζ < L/2 −ℓ/4 f(ζ −L/2 −ℓ/2) L/2 −ℓ/4 ≤ζ < L/2 + ℓ/4 1 L/2 + ℓ/4 ≤ζ < L −ℓ f(ζ −L) L −ℓ≤ζ < L (B.5) (B.5) – 28 – Figure 12. Late time behavior of the steady state and ﬁxed point for SS type conﬁgurations. The plots show the deviation of e(t, ζ = vt) and j(t, ζ = vt) from the predicted steady state values e0 and j0 for various values of v. The initial conditions are given by (B.1) with L = 40000 (top) or L = 20000 (bottom), ℓ= 2000 and c = 100. The top plots correspond to j∗= −0.5 and the bottom ones to j∗= −2. We expect that numerical error is of order 10−7 −10−8. JHEP08(2016)120 Figure 12. Late time behavior of the steady state and ﬁxed point for SS type conﬁgurations. The plots show the deviation of e(t, ζ = vt) and j(t, ζ = vt) from the predicted steady state values e0 gure 12. Late time behavior of the steady state and ﬁxed point for SS type conﬁgurations. The Figure 12. Late time behavior of the steady state and ﬁxed point for SS type conﬁgurations. The plots show the deviation of e(t, ζ = vt) and j(t, ζ = vt) from the predicted steady state values e0 and j0 for various values of v. The initial conditions are given by (B.1) with L = 40000 (top) or L = 20000 (bottom), ℓ= 2000 and c = 100. The top plots correspond to j∗= −0.5 and the bottom ones to j∗= −2. We expect that numerical error is of order 10−7 −10−8. Figure 12. Late time behavior of the steady state and ﬁxed point for SS type conﬁgurations. The plots show the deviation of e(t, ζ = vt) and j(t, ζ = vt) from the predicted steady state values e0 and j0 for various values of v. The initial conditions are given by (B.1) with L = 40000 (top) or L = 20000 (bottom), ℓ= 2000 and c = 100. The top plots correspond to j∗= −0.5 and the bottom ones to j∗= −2. B.4 Error analysis In sections B.1 and B.3 we have ﬁt the late time approach of the data to the predicted steady state and (or) ﬁxed point values to a power law behavior. The ﬁt was done using Math- ematica’s NonLinearModelFit routine [42]. In detail, the late time data was discretized into order 1 time steps which were then ﬁt to a a/tα curve with a and α as parameters. The standard errors for the ﬁt were usually of order 10−3 to 10−4. Fits involving very – 29 – Figure 13. Late time behavior of the steady state and ﬁxed point for RS type conﬁgurations. Top plots: the deviation of e(t, ζ = vt) from the predicted steady state value e0 for various values of v. The initial conditions are given by (B.5) with L = 16000, ℓ= 2000, and c = 100 and e∗= 4 for the top left plot and e∗= 9 for the top right plot. Bottom plots: the deviation of e and j from the predicted ﬁxed point value at ζ = 0 for various values of c. The initial conditions are given by (B.1) with L = 16000, ℓ= 4000 and e∗= 9. Both the time dependence of e/es −1 and j/js −1 can be ﬁt to a power law, ∼tα. For the energy density one ﬁnds α ∼0.77. For the energy current α ∼1.1. JHEP08(2016)120 Figure 13. Late time behavior of the steady state and ﬁxed point for RS type conﬁgurations. Top plots: the deviation of e(t, ζ = vt) from the predicted steady state value e0 for various values of v. The initial conditions are given by (B.5) with L = 16000, ℓ= 2000, and c = 100 and e∗= 4 for the top left plot and e∗= 9 for the top right plot. Bottom plots: the deviation of e and j from the predicted ﬁxed point value at ζ = 0 for various values of c. The initial conditions are given by (B.1) with L = 16000, ℓ= 4000 and e∗= 9. Both the time dependence of e/es −1 and j/js −1 can be ﬁt to a power law, ∼tα. For the energy density one ﬁnds α ∼0.77. For the energy current α ∼1.1. small values of the slope parameter c in (B.2) and (B.6) (cf., the bottom plots of ﬁgures 11 and 13) often had large standard errors. Open Access. 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https://openalex.org/W3003437270	https://www.nature.com/articles/s41598-020-58659-3.pdf	English	null	Resistance to change: AMR gene dynamics on a commercial pig farm with high antimicrobial usage	Scientific reports	2,020	cc-by	9,176	Resistance to change: AMR gene dynamics on a commercial pig farm with high antimicrobial usage Jolinda Pollock1,2, Adrian Muwonge 2, Michael R. Hutchings1, Geoffrey Mainda2, Barend M. Bronsvoort 2, David L. Gally2 & Alexander Corbishley 2* OPEN Group antimicrobial administration is used to control disease in livestock, but we have little insight into how this impacts antimicrobial resistance (AMR) gene dynamics. Here, a longitudinal study was carried out during a single production cycle on a commercial pig unit with high historic and current antimicrobial usage. Quantitative PCR, 16S rRNA gene metabarcoding and shotgun metagenomic sequencing were used to track faecal AMR gene abundance and diversity and microbiome alpha diversity. Shotgun metagenomic sequencing identified 144 AMR genes in total, with higher AMR gene diversity present in young pigs compared to dry sows. Irrespective of in-feed antibiotic treatment or changes in microbiome diversity, mean AMR gene copy number was consistently high, with some AMR genes present at copy numbers comparable to the bacterial 16S rRNA gene. In conclusion, AMR gene prevalence and abundance were not influenced by antibiotic use, either during the production cycle or following whole-herd medication. The high levels of certain genes indicate they are widely disseminated throughout the microbial population, potentially aiding stability. Despite the high and relatively stable levels of resistance genes against the main antimicrobials used, these compounds continue to control production limiting diseases on this unit. Antimicrobial agents are used regularly in many agricultural systems worldwide to improve the health, welfare and productivity of livestock1,2. This has led to concerns about the anthropogenic selection of antimicrobial-resistant bacteria in livestock systems2–4, particularly due to the potential transfer of antimicrobial resistance (AMR) genes from livestock to humans5–7 and into the environment8,9. In the United Kingdom, 52% of all antimicro- bials sold for livestock were used in the pig and poultry sectors, with tetracyclines being reported as the highest sold antibiotic class10. Given the substantial use of antimicrobials in pig production, the association between antimicrobial use and AMR has been an area of intensive study. Specifically, antimicrobial administration has been associated with increased AMR in sentinel bacteria such as Escherichia coli11–13, Staphylococcus aureus14,15, and Campylobacter16, Salmonella17 and Enterococcus18 species. Molecular studies have revealed a higher richness and diversity of AMR genes in pigs administered oxytetracycline in-feed19, with farm origin being associated with AMR gene abundances in faeces at slaughter using both quantitative PCR20 and metagenomic21 datasets. However, there are currently few longitudinal studies of AMR in livestock systems, specifically for pig produc- tion22, with information on antimicrobial use in these systems often derived from national figures, rather than from farm medicines records12,21. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Animal and Veterinary Sciences, Scotland’s Rural College (SRUC), Edinburgh, United Kingdom. 2The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom. email: alexander.corbishley@roslin.ed.ac.uk Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 Materials and Methods Study Farm y In order to study the impact of antimicrobial usage on AMR gene abundance and diversity, a 600 sow Landrace x Large White commercial farrowing to finishing unit (United Kingdom) with high historic and current antimicro- bial usage was recruited. In the three months prior to the study period, a total of 389.1 mg/Population Correction Unit (PCU) of antibiotics were used on this farm. This compares to a UK average in pigs of 183 mg/PCU in 2016 and 131 mg/PCU in 2017, making this a high antibiotic usage farm10. Detailed antimicrobial usage records and additional herd information are available in Supplementary Materials 1. This study received ethical approval from the Royal (Dick) School of Veterinary Studies Veterinary Ethics Research Committee. All methods were conducted in accordance with institutional and national guidelines and regulations. Sampling. Faecal samples were taken from this unit during two time periods (Phase 1 and Phase 2) as described below. All samples were collected in universal tubes and stored at −20 °C on-site, prior to batch trans- portation to the laboratory on dry ice and storage at −80 °C until processing. Phase 1: Faecal sampling during a single production cycle. Pooled faecal samples from the pen floors contain- ing medicated young pigs (referred to as “young pig accommodation”) were collected weekly from just prior to farrowing on 26th October 2016 to 5th April 2017 (W1 to W25), when the studied batch were sent for slaughter. The sampling design and periods of in-feed medication are summarised in Fig. 1. Weekly faecal samples were also taken randomly from the dry sow barn floor (referred to as “dry sow accommodation”) (n = 6) to establish background levels of AMR genes in a group of adult pigs that were not routinely medicated with antimicrobials. Further medication and sampling details are available in Supplementary Materials 1. Phase 2: Faecal sampling during and after a partial depopulation. In an effort to improve health status and reduce antimicrobial use, a partial depopulation was carried out on this farm in May 2017. This involved removing all the young pigs (i.e. nursing piglets, weaners, growers and finishers) from the unit, fumigating the young pig accom- modation with formaldehyde and administering in-feed chlortetracycline and tiamulin to all the breeding sows for a period of six weeks. Resistance to change: AMR gene dynamics on a commercial pig farm with high antimicrobial usage Jolinda Pollock1,2, Adrian Muwonge 2, Michael R. Hutchings1, Geoffrey Mainda2, Barend M. Bronsvoort 2, David L. Gally2 & Alexander Corbishley 2 OPEN Here, we examine microbial population dynamics and both AMR gene abundance and diversity on a single pig farm during a six-month production cycle. Faecal samples were collected weekly from a batch of young pigs that were followed from birth to slaughter. Group treatments during this time included acidified water, dietary zinc supplementation and a prolonged period of in-feed chlortetracycline, followed by tylosin administration. Faecal samples from dry sow accommodation containing unmedicated pigs were taken in parallel. Finally, fae- cal samples were taken from dry sows during and after a partial depopulation event which involved antibiotic administration to every pig on the farm to assess if this practice impacted on AMR gene abundance. Microbiome alpha diversity, AMR gene abundance and AMR gene diversity were measured by 16S rRNA gene metabarcoding, quantitative PCR (qPCR) and shotgun metagenomic sequencing, respectively. 1Animal and Veterinary Sciences, Scotland’s Rural College (SRUC), Edinburgh, United Kingdom. 2The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom. *email: alexander.corbishley@roslin.ed.ac.uk www.nature.com/scientificreports/ Figure 1. Summary of pig production cycle, antimicrobial administration and sampling points. Faecal sampling (red arrows) was carried out from farrowing pens containing the pregnant sows only (W1), and then post-farrowing from both the sow and the nursing pigs during co-housing (W2-W4). Weaners were mixed and moved into new accommodation prior to sampling on W5 and were moved to larger accommodation for the growing/finishing period prior to sampling on W14. Samples subject to metagenomic sequencing are indicated (Meta 1 to Meta 3). No sampling was carried out on W6 due to access issues. Figure 1. Summary of pig production cycle, antimicrobial administration and sampling points. Faecal sampling (red arrows) was carried out from farrowing pens containing the pregnant sows only (W1), and then post-farrowing from both the sow and the nursing pigs during co-housing (W2-W4). Weaners were mixed and moved into new accommodation prior to sampling on W5 and were moved to larger accommodation for the growing/finishing period prior to sampling on W14. Samples subject to metagenomic sequencing are indicated (Meta 1 to Meta 3). No sampling was carried out on W6 due to access issues. Results AMR gene abundances during a full production cycle. Temporal fluctuations in AMR gene copy num- ber and bacterial load (16S rRNA gene) can be observed in both the young pig (Fig. 2a) and dry sow (Fig. 2b) accommodation. Time point was a statistically significant explanatory variable for the 16S rRNA gene and all five AMR genes quantified in this study (Table 1). In the dry sow accommodation, high levels of AMR genes were detected in the absence of group antimicrobial administration.i g p Prior to weaning and during co-housing (W1-W4), the levels of all five AMR genes were similar when com- paring the nursing sow and young pig samples, in the presence of similar faecal bacterial load (Fig. 2a). During acidified water and zinc administration, an increase in dfrA1 counts occurred in the young pig accommodation between W5 (mean 4.17 log10 copies/g DM, standard error of the mean (SEM) 0.80) and W7 (mean 5.58 log10 copies/g DM, SEM 0.65), and a decrease in ermA between W5 (mean 6.83 log10 copies/g DM, SEM 1.44) and W7 (2.70 log10 copies/g DM, SEM 1.17). There were no marked changes in the levels of tetB, ermB and tetQ during acidified water and zinc administration. i Chlortetracycline administration (W8-W10) was associated with a decrease in tetB gene copy number in the young pig accommodation between W9 (mean 6.95 log10 copies/g DM, SEM 0.79) and W10 (mean 3.29 log10 copies/g DM, SEM 0.79) with no significant changes in tetQ, ermA, ermB or dfrA1 levels being observed dur- ing chlortetracycline administration. Tylosin administration (W11-W25) did not have an effect on AMR gene counts in the young pig accommodation following withdrawal from chlortetracycline. Increases in the majority of AMR genes being observed were seen from W21 to W24 in both the young pig and dry sow accommodation, which coincided with an increase in bacterial load, suggesting that this increase was independent of antimicrobial administration (see Supplementary Materials 3 for detailed statistical model outputs). AMR gene abundances and microbial diversity. 16S rRNA gene metabarcoding was carried out to provide context on faecal microbiota development in the young pigs and whether changes in alpha diversity were linked to AMR gene abundances. Notable stepwise increases in measures of alpha diversity (both SIs and ISIs) occurred during the first 4 weeks of life (Fig. 3), highlighting an expected rapid increase in microbiota complexity. www.nature.com/scientificreports/ www.nature.com/scientificreports/ copy number per gram of dry faeces. Further details on methodology and primer/probe sequences are shown in Supplementary Materials 1 and 2. copy number per gram of dry faeces. Further details on methodology and primer/probe sequences are shown in Supplementary Materials 1 and 2. 16S rRNA gene metabarcoding. All DNA extracts were prepared for 16S rRNA gene metabarcoding targeting the V3 hypervariable region, as described in previous work23 and in Supplementary Materials 1. The sequence files generated (Edinburgh Genomics, UK) with the primers removed, are publicly available through the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA557844. Using a mock bacterial community (20 Strain Even Mix Genomic Material ATCC®MSA-1002, ATCC, United States), the mean sequenc- ing error rate was calculated as 0.01%.h g The generated sequences were processed using cutadapt24 and mothur25 (URL: https://www.mothur.org/wiki/ MiSeq_SOP. Accessed January 2018) as described previously23. Here, unique sequences were binned into opera- tional taxonomic units (OTUs) using a database-independent approach. A mean of 125,312 sequences per sample were retained after quality control and were subsampled to 10,000 sequences per sample for analysis. Both the Inverse Simpson index (ISI) and Shannon index (SI) were calculated for each sample to assess alpha diversity. Shotgun metagenomic sequencing. Three time points were selected that corresponded to differing antimicrobial treatment phases in the young pigs (Fig. 1). Triplicate samples from both the dry sow and young pig accommo- dation (n = 18) were submitted for shotgun metagenomic sequencing (Edinburgh Genomics, UK) with further details on library preparation and analysis26–34 available in Supplementary Materials 1. The sequence files are publicly available on the European Nucleotide Archive (ENA) under study accession number PRJEB34736. Statistical analysis of alpha diversity and gene abundance data. Temporal changes in both gene copy number and alpha diversity indices were assessed statistically using repeated measures analysis of variance (Genstat 16, VSN International, UK) with the data obtained from the dry sow and young pig accommodation faecal samples. To assess the impact of the antimicrobial treatments on gene copy number and alpha diversity indices in the young pig accommodation, least significant differences were used for multiple comparisons of means. if For the dry sow analyses, the values calculated from samples collected between W1-W25 were included in the statistical models. www.nature.com/scientificreports/ For the young pig analyses, the values calculated from samples collected between W5-W25 were included in the statistical models, since W1 samples were obtained from pregnant sows only and samples obtained between W2-W4 were obtained when the piglets were still grouped by litter and were not yet assigned to their rearing pens. For the young pig analyses, the pen was included as a factor to assess any differences between the triplicate pen samples. Materials and Methods Study Farm In order to establish the impact of this management practice on AMR gene abundance and diversity, faecal samples were obtained from the dry sow accommodation during the final week of antibiotic administration (W36), two months after treatment (W45) and five months after treatment (W57) (n = 6). Sample preparation and analysis. DNA extractions and dry matter calculations. DNA extractions were carried out using the DNeasy PowerSoil Kit (Qiagen, UK) following the manufacturer’s instructions. In parallel, approximately 1 g of homogenised faeces were weighed out and placed into a drying oven overnight at 60 °C. After re-weighing, the percentage of dry matter (% DM) was determined. Quantitative PCR. Five AMR genes associated with the main antibiotics used on the unit were targeted for longitudinal analysis based on initial end point screening of 36 AMR genes. These were tetB and tetQ (tetracy- cline), ermA and ermB (tylosin) and dfrA1 (trimethoprim). Additionally, quantification of the 16S rRNA gene was included as a proxy of overall bacterial load. Plasmids containing gene fragments of tetB, tetQ, ermA, ermB, dfrA1 and the 16S rRNA gene were generated in-house and used as standards for absolute quantification of gene Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 Results During the partial depopulation, the dry sows received 1500 ppm chlortetracycline and 500 ppm tiamulin in-feed, whilst the nursing sows received 1875 ppm chlortetracycline and 625 ppm tiamulin from W30 to W36 (black coloured points). Figure 2. Quantification of AMR genes. AMR gene copy numbers in faecal samples obtained from the (A) young pig and (B) dry sow accommodation including the partial depopulation. During the study period, the following routine group medication regimens were used in the young pigs: toltrazuril (30 mg/head oral) at 4 days old, zinc (2500 ppm in feed) between 4 and 6 weeks old, acidified water (Baynes Evacide S 0.2%) between 3 and 7 weeks old, chlortetracycline (300 ppm in feed) from 6 to 8 1/2 weeks old and tylosin (100 ppm in feed) from 8 1/2 weeks old until slaughter. During the partial depopulation, the dry sows received 1500 ppm chlortetracycline and 500 ppm tiamulin in-feed, whilst the nursing sows received 1875 ppm chlortetracycline and 625 ppm tiamulin from W30 to W36 (black coloured points). Dry sow accommodation Young pig accommodation F-statistic P-value F-statistic P-value tetB 12.16 <0.001 2.05 0.030 tetQ 11.83 <0.001 5.60 <0.001 ermA 7.19 <0.001 2.01 0.034 ermB 30.02 <0.001 8.94 <0.001 dfrA1 20.69 <0.001 2.59 0.006 16S rRNA 13.30 <0.001 4.84 <0.001 ISI 5.63 <0.001 1.79 0.065 SI 5.79 <0.001 1.78 0.065 Table 1. Summary of statistical model outputs (F-statistics and P-values) for assessing temporal shifts in gene abundances and microbiome diversity. ISI = Inverse Simpson. Index. SI = Shannon Index. Dry sow accommodation Young pig accommodation F-statistic P-value F-statistic P-value tetB 12.16 <0.001 2.05 0.030 tetQ 11.83 <0.001 5.60 <0.001 ermA 7.19 <0.001 2.01 0.034 ermB 30.02 <0.001 8.94 <0.001 dfrA1 20.69 <0.001 2.59 0.006 16S rRNA 13.30 <0.001 4.84 <0.001 ISI 5.63 <0.001 1.79 0.065 SI 5.79 <0.001 1.78 0.065 Table 1. Summary of statistical model outputs (F-statistics and P-values) for assessing temporal shifts in gene abundances and microbiome diversity. ISI = Inverse Simpson. Index. SI = Shannon Index. Table 1. Summary of statistical model outputs (F-statistics and P-values) for assessing temporal shifts in gene abundances and microbiome diversity. ISI = Inverse Simpson. Index. SI = Shannon Index. Table 1. Summary of statistical model outputs (F-statistics and P-values) for assessing temporal shifts in gene abundances and microbiome diversity. ISI = Inverse Simpson. Index. SI = Shannon Index. Results Even in faecal samples with low alpha diversity, comparable numbers of AMR genes were observed when compar- ing to nursing sow samples (Fig. 2a) and dry sow accommodation samples (Fig. 2b). AMR gene diversity over the production cycle. A total of 144 AMR genes were detected on the farm (Fig. 4a), 21 of which were ubiquitous across sampling location and time point. The majority of the 60 genes that were present in both locations were more abundant (however, in the same order of magnitude) in the young pig accommodation. There was noticeably higher AMR gene richness within the young pig accommodation com- pared to the dry sow accommodation, with 77 AMR genes found solely in the former, compared to just 7 genes that were unique to the latter. Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 www.nature.com/scientificreports .nature.com/scientificreports/ Figure 2. Quantification of AMR genes. AMR gene copy numbers in faecal samples obtained from the (A) young pig and (B) dry sow accommodation including the partial depopulation. During the study period, the following routine group medication regimens were used in the young pigs: toltrazuril (30 mg/head oral) at 4 days old, zinc (2500 ppm in feed) between 4 and 6 weeks old, acidified water (Baynes Evacide S 0.2%) between 3 and 7 weeks old, chlortetracycline (300 ppm in feed) from 6 to 8 1/2 weeks old and tylosin (100 ppm in feed) from 8 1/2 weeks old until slaughter. During the partial depopulation, the dry sows received 1500 ppm chlortetracycline and 500 ppm tiamulin in-feed, whilst the nursing sows received 1875 ppm chlortetracycline and 625 ppm tiamulin from W30 to W36 (black coloured points). www.nature.com/scientificreports/ Figure 2. Quantification of AMR genes. AMR gene copy numbers in faecal samples obtained from the (A) Figure 2. Quantification of AMR genes. AMR gene copy numbers in faecal samples obtained from the (A) young pig and (B) dry sow accommodation including the partial depopulation. During the study period, the following routine group medication regimens were used in the young pigs: toltrazuril (30 mg/head oral) at 4 days old, zinc (2500 ppm in feed) between 4 and 6 weeks old, acidified water (Baynes Evacide S 0.2%) between 3 and 7 weeks old, chlortetracycline (300 ppm in feed) from 6 to 8 1/2 weeks old and tylosin (100 ppm in feed) from 8 1/2 weeks old until slaughter. Results The Inverse Simpson and Shannon indices of the faecal microbiomes obtained from the young pig accommodation and dry sow accommodation. On W1, faecal samples were obtained from pregnant sows only (n = 6) and on W2–4, faecal drops were obtained from both nursing sows (n = 6) and nursing pigs (n = 6) during co-housing. Figure 3. Alpha diversity indices of the faecal microbiota. The Inverse Simpson and Shannon indices of the faecal microbiomes obtained from the young pig accommodation and dry sow accommodation. On W1, faecal samples were obtained from pregnant sows only (n = 6) and on W2–4, faecal drops were obtained from both nursing sows (n = 6) and nursing pigs (n = 6) during co-housing. aminoglycoside and sulphonamide resistance. These proportional changes were accompanied by an increase in AMR genes, from 94 genes in the young pig accommodation on W7 to 120 genes on W9 and 32 genes in the dry sow accommodation on W7 to 60 genes on W9. The proportion of reads associated with tetracycline resistance declined between W7 and W9, despite the young pigs starting chlortetracycline treatment during this time. aminoglycoside and sulphonamide resistance. These proportional changes were accompanied by an increase in AMR genes, from 94 genes in the young pig accommodation on W7 to 120 genes on W9 and 32 genes in the dry sow accommodation on W7 to 60 genes on W9. The proportion of reads associated with tetracycline resistance declined between W7 and W9, despite the young pigs starting chlortetracycline treatment during this time. AMR gene diversity and antimicrobial treatment. In the young pig accommodation, there were both high levels and numbers (i.e. >30) of genes conferring cross-class resistance (CCR) during zinc and acidified water (W7) and chlortetracycline administration (W9). This number dropped by the end of the production cycle, despite the young pigs having spent several months on in-feed antibiotics (Fig. 4c). Genes associated with CCR were not detected in the dry sow accommodation on W7 and W9, with only a single gene detected on W25. Nearly half i.e. 35 of the 77 genes found solely in the young pig accommodation were associated with CCR and appeared to be related to acidified water and zinc administration, rather than in-feed antibiotic (chlortetracycline and tylosin) administration. AMR gene quantification during and after a partial depopulation. Results When examining the proportion of reads associated with each antibiotic class, AMR genes associated with tetracycline, macrolide (MLS) and aminoglycoside resistance predominated across all samples (Fig. 4b). With a few exceptions, these proportional results were consistent across the triplicate biological replicates. These propor- tional results were also broadly similar for the dry sows and young pigs on and between W7 and W25, despite the fact that the young pigs received tylosin from weeks 11 to 25. Th b h h f d d h h b l l d The biggest change in the proportion of reads associated with each antimicrobial class occurred on W9 in both sampling locations. In the young pig accommodation, there was a proportional increase in reads associated with cross-class resistance (CCR). In the dry sow accommodation, the increase was seen in reads associated with Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 www.nature.com/scientificreports/ Young pig accommodation Sow barn Inverse Simpson index Shannon index W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21 W22 W23 W24 W25 W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21 W22 W23 W24 W25 0 50 100 150 2 3 4 5 6 Week of sampling Diversity index Sows None Acidified water Acidified water + chlortetracycline Chlortetracycline Tylosin Figure 3. Alpha diversity indices of the faecal microbiota. The Inverse Simpson and Shannon indices of the faecal microbiomes obtained from the young pig accommodation and dry sow accommodation. On W1, faecal samples were obtained from pregnant sows only (n = 6) and on W2–4, faecal drops were obtained from both nursing sows (n = 6) and nursing pigs (n = 6) during co-housing. Young pig accommodation Sow barn Inverse Simpson index Shannon index W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21 W22 W23 W24 W25 W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21 W22 W23 W24 W25 0 50 100 150 2 3 4 5 6 Week of sampling Diversity index Sows None Acidified water Acidified water + chlortetracycline Chlortetracycline Tylosin Figure 3. Alpha diversity indices of the faecal microbiota. Results To improve the health status of the farm, all the young pigs were removed (partial depopulation) and the sows treated with in-feed chlortetracy- cline and tiamulin. During this time period, all the pigs on the farm were treated with antibiotics simultaneously. During the last week of treatment (W36), and two (W45) and five (W57) months after cessation of treatment, bacterial load and AMR gene copy number in these samples did not change in response to the partial depopula- tion and antibiotic treatment (Fig. 2b). Discussion When designing this study, our first hypothesis was that there would be a reservoir of AMR genes within the faecal microbiome, which would increase in both abundance and diversity in response to in-feed antimicro- bial administration. Furthermore, we anticipated that AMR gene abundance would be inversely related to faecal microbiome diversity. Whilst large cross-sectional studies have been instrumental in furthering our understand- ing of AMR gene abundance and diversity across different livestock systems12,21,35, they have not provided the granularity of data and medicines usage history required to test these hypotheses. For this reason, we chose to undertake a longitudinal dissection of a single commercial pig unit, which included three group in-feed antibiotic treatment regimens and a partial depopulation event. Measured AMR genes consistently high across the unit. Although previous work has demonstrated high baseline levels of specific AMR genes36 and phenotypic resistance in Escherichia coli isolates37,38 in unmedi- cated pigs, our initial expectation was that AMR gene abundances would markedly increase in response to anti- microbial administration as a result of selective pressure being exerted on specific genes39. In agreement with Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 www.nature.com/scientificreports w.nature.com/scientificreports/ Figure 4. AMR gene abundance and diversity described by metagenomic data. (A) Log-transformed normalised gene abundance counts from both the dry sow and young pig accommodation (each bar is the sum of nine samples), (B) relative abundances of genes conferring resistance to particular antibiotic classes and (C) the sum of the absolute read counts from the three replicate samples conferring resistance to particular antibiotic classes. www.nature.com/scientificreports w.nature.com/scientificreports/ Figure 4. AMR gene abundance and diversity described by metagenomic data. (A) Log-transformed normalised gene abundance counts from both the dry sow and young pig accommodation (each bar is the sum of nine samples), (B) relative abundances of genes conferring resistance to particular antibiotic classes and (C) the sum of the absolute read counts from the three replicate samples conferring resistance to particular antibiotic classes. www.nature.com/scientificreports/ Figure 4. AMR gene abundance and diversity described by metagenomic data. (A) Log-transformed normalised gene abundance counts from both the dry sow and young pig accommodation (each bar is the sum of nine samples), (B) relative abundances of genes conferring resistance to particular antibiotic classes and (C) the sum of the absolute read counts from the three replicate samples conferring resistance to particular antibiotic classes. Figure 4. AMR gene abundance and diversity described by metagenomic data. Discussion In fact, the larg- est increases in AMR gene abundance were seen across all genes and in both classes of stock at the same time (W21-W24) which coincided with an increase in 16S rRNA gene copies, hence suggesting an as yet undefined environmental influence on bacterial load and consequently AMR gene abundance. A previous cross-sectional study highlighted that only 10–42% of the variation in AMR gene levels could be explained by factors included in statistical models (including lifetime antimicrobial exposure), suggesting that AMR gene levels are strongly influenced by a variety of other elements43. Whilst these could be related to feed changes, the fact that the dry sows and young pigs were housed differently and fed different diets44 would suggest that this effect is due to some other factors affecting the entire farm, such as housing and management35,45,46, environmental conditions46 or the introduction of an infectious agent. Microbiome alpha diversity was not affected by antimicrobial exposure. Similar to the studied AMR genes, fluctuations in microbiome alpha diversity were apparent in both dry sow and young pig accom- modation samples. These changes were clearly more pronounced in the young pigs, as the alpha diversity of the faecal microbiome increased from nursing to finishing, which has been shown previously47,48. However, changes in the microbiome associated with antimicrobial treatment were not observed. Previous work has demonstrated that sub-therapeutic administration of chlortetracycline and tylosin had no impact on alpha diversity indices47,49. Whilst changes in relative abundances of specific taxa have been observed in response to tylosin administration, it was previously reported that these shifts were temporary, suggesting that the gut microbiota post-weaning seems to be resilient to perturbation by antimicrobial agents47. Despite using markedly higher levels of chlortetracycline and tylosin (300 ppm and 100 ppm, respectively), our results also showed that antimicrobial treatment did not impact on microbiome diversity in the young pigs. AMR gene abundances are high in nursing piglets with low microbiome diversity. With respect to our second hypothesis, even when the young pig faecal microbiome was at its least diverse during the suck- ling period, the AMR gene levels were comparable to that of the nursing sows. Although microbiome diversity increased dramatically in the piglets during the first few weeks of life, as reflected in previous studies47,50–53, this was not associated with changes in AMR gene prevalence. Discussion (A) Log-transformed normalised gene abundance counts from both the dry sow and young pig accommodation (each bar is the sum of nine samples), (B) relative abundances of genes conferring resistance to particular antibiotic classes and (C) the sum of the absolute read counts from the three replicate samples conferring resistance to particular antibiotic classes. previous work, we showed high levels of all studied AMR genes in the non-medicated dry sows, presumably as a consequence of the strong selection pressure exerted by this farm’s history of high antimicrobial usage. The levels of the five AMR genes studied in the young pigs reflected those of the sow population, whilst prolonged exposure to two different in-feed antibiotic regimens in the young pigs and a combined in-feed antibiotic regi- men in the dry sows had no marked effect on AMR gene counts, potentially suggesting that they have reached saturation within the faecal bacterial populations39. Curiously, the antibiotics used (chlortetracycline and tylosin) were still clinically effective on this farm, suggesting that despite a high abundance of resistance genes within the faecal microbiome, that they were not present, or at least not active, within the organisms of clinical inter- est, i.e. Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Actinobacillus pleuopneumoniae and Lawsonia intracellularis. Interestingly, some of the targeted AMR genes (i.e. tetQ and ermB) were present in higher copy numbers than the 16S rRNA gene at the majority of the studied time points. This is consistent with the work of other groups, Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 www.nature.com/scientificreports/ which has demonstrated overall AMR gene abundances of 0.77–5.1440 and 0.54–3.1 gene copies41 per 16S rRNA gene in agricultural faecal and wastewater samples. It is worth noting that these AMR gene copy numbers are orders of magnitude higher than in samples obtained from other environments, such as sediments, soil and river water41,42. Collectively, these findings suggest that some bacteria are carrying multiple copies of particular AMR genes. Changes in AMR gene abundances were not associated with antimicrobial exposure. Week-to-week fluctuations in AMR gene abundance in both young pigs and dry sows were apparent, however the most marked changes were generally not associated with antimicrobial administration. Discussion We expected, as others have proposed, that changes in the microbiota would influence AMR gene levels20,44,54, but in fact the high levels of studied AMR genes in the young pigs reflected that of the farm’s sow population. The most obvious explanation for this is a combination of vertical and horizontal transmission of bacteria at or shortly after birth55,56. The presence of comparable levels of AMR genes in both sows and piglets and the associated large differences in microbiome diversity suggest that the AMR genes studied appear to either be widespread across multiple taxa or highly concentrated within dominant taxa present throughout all stages of microbiota development. High AMR gene diversity and cross-class resistance genes in young pigs. Metagenomic sequenc- ing revealed a diverse set of AMR genes in the presence and absence of antimicrobial treatment, which reflects findings in other recent work19. Specifically, genes associated with tetracycline and macrolide resistance pre- dominated. This is not surprising given the history of high levels of tetracycline and macrolide use on this farm. Reassuringly, there was no evidence of co-selection between antimicrobial classes following chlortetracycline or tylosin administration. This is a significant finding and relevant to the principles of antimicrobial stewardship, where veterinary surgeons are actively discouraged from using fluoroquinolones and 3rd/4th generation cephalo- sporins, so as to minimise the risk that resistance to these critically important antibiotics in human medicine is selected for in livestock. What was evident, were the large numbers of genes associated with cross-class resistance (CCR) in the young pig accommodation samples taken on W7 and W9 and how these reduced by W25 and were almost absent from the unmedicated sows. The presence of these genes was not associated with the administration of chlortetracy- cline and tylosin in this study. Although the current study design does not allow us to disentangle temporal effects versus the effect of different treatments, it is interesting to note that the CCR genes were already highly abundant at W7, following a period of in-feed zinc administration and the use of acidified water. Copper and zinc salts are commonly administered in-feed at supranutritional levels due to their antimicrobial properties, with increasing doses of zinc oxide being previously shown to increase the abundance of both tetracycline and sulphonamide resistance genes in weaned pigs57,58.h g p g The metagenomic sequencing results for the dry sow accommodation on W9 were unexpected. Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 Discussion All three biological replicates demonstrated an increase in the proportion of reads associated with sulphonamide and aminoglycoside resistance. The richness of AMR genes also doubled at this time point compared to W7, despite no antimicrobial administration to this group of pigs. An increase in AMR gene diversity was also seen in the young pig accommodation at the same time point, however the increase was seen in beta-lactam resistance genes, CCR genes and genes associated with other antimicrobial classes. This is unlikely to be an artefact, given that this observation was seen across two different locations at the same time and five of the six biological replicates. This Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 www.nature.com/scientificreports/ shift appears to be a consequence of an undefined environmental factor affecting the whole farm, where the two groups of pigs respond differently with regards to faecal AMR gene diversity. Persistently high abundances of AMR genes after partial depopulation. The partial depopulation at the end of the study involved considerable antibiotic use, with every sow on the farm receiving in-feed tiamulin and chlortetracycline. The treated sows were followed for five months after this treatment, during a period where antibiotic use on the farm declined nearly twenty-fold. Despite such dramatic changes in antibiotic usage, there were no marked changes in AMR gene abundances. This therefore begs the question as to how long (if at all) it would take to see reductions in AMR gene levels following the reduction or cessation of antimicrobial use on farms with a previous history of high-level use. We propose that the long-term use of these treatments has led to wide and stable dissemination of relevant resistance genes throughout the gastrointestinal microbial population of the pigs. Conclusion AMR gene abundance and diversity on this unit were high in both dry sows and young pigs, likely as a conse- quence of historic antimicrobial usage. In this context, in-feed antibiotic administration (chlortetracycline and tylosin) did not affect faecal AMR gene abundance or diversity, suggesting that genes under positive selection in the presence of these antibiotics have already become stably integrated into the faecal microbiome. This would also potentially explain the failure of AMR gene levels to decline following dramatic reductions in antimicrobial use on the farm. Acidified water and zinc supplementation were linked to an increase in AMR gene diversity, which importantly included CCR genes, which decayed quickly after their withdrawal. This study did not identify which bacteria carried these AMR genes or indeed which genes were being expressed. Future work therefore needs to determine in what organisms these genes are not only present, but also active. Data availability Th d y The sequence data sets generated and analysed during the current study are publicly available on the NCBI Sequence Read Archive (16S rRNA gene metabarcoding data - BioProject accession number PRJNA557844) and the European Nucleotide Archive (shotgun metagenomic data - Study accession number PRJEB34736). References UK-VARSS. UK Veterinary Antibiotic Resistance and Sales y p 11. Lugsomya, K. et al. Antimicrobial resistance in commensal Escherichia coli isolated from pigs and pork derived from far routinely using or not using in-feed antimicrobials. Microb. 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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-58659-3. Correspondence and requests for materials should be addressed to A.C. 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 Internationa License, which permits use, sharing, adaptation, distribution and reproduction in any medium o 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 th article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to th 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 th copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Competing interests The authors declare no competing interests. Author contributions A.C., D.L.G., M.H., J.P. and G.M. conceived of and designed the experiment. J.P., G.M. and A.C. collected the samples. G.M. and J.P. carried out the laboratory work described. A.C., M.R.H., B.M.B., A.M. and J.P. carried out the data analysis. A.C. and J.P. wrote the manuscript, and all authors contributed to the review and editing of the manuscript. Scientific Reports \| (2020) 10:1708 \| https://doi.org/10.1038/s41598-020-58659-3 www.nature.com/scientificreports/ Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-58659-3. Correspondence and requests for materials should be addressed to A.C. 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. 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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. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-58659-3. Correspondence and requests for materials should be addressed to A.C. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W4206687377	https://ejournal.unsri.ac.id/index.php/jp/article/download/15672/pdf	Indonesian	null	PERAN PROVINSI KEPULAUAN BANGKA BELITUNG DALAM PENGEMBANGAN SISTEM EKONOMI KERAKYATAN SAAT PANDEMI COVID-19 MENURUT UNDANG-UNDANG 1945	Jurnal profit	2,021	cc-by-sa	3,778	P-ISSN: 2355-7176 E-ISSN: 2620-8504 P-ISSN: 2355-7176 E-ISSN: 2620-8504 JURNAL PROFIT: Kajian Pendidikan Ekonomi dan Ilmu Ekonomi https://ejournal.unsri.ac.id/index.php/jp Volume 8 (2), 2021, 95-101 P-ISSN: 2355-7176 E-ISSN: 2620-8504 JURNAL PROFIT: Kajian Pendidikan Ekonomi dan Ilmu Ekonomi https://ejournal.unsri.ac.id/index.php/jp Volume 8 (2), 2021, 95-101 Abstract This research study aims to determine the Strategy for the development of a People's Economy (Economic Civic) and is very relevant to do considering that the People's Economy is businesses in the economic field, where the main achievement target is not financial gain. This study uses qualitative research methods, namely collecting data from scientific articles, research reports or other matters relevant to the topic. The results of the study conclude that UMKM in the role of the populist economy in the Province of the Bangka Belitung Islands through entrepreneurial programs, business unit development, improving the quality of human resources, and utilizing technology and information so taht they can have a positive impact on increasing products. Sejarah Artikel: diterima : 26 Oktober 2021 direvisi : 9 November 2021 disetujui : 11 November 2021 Sejarah Artikel: diterima : 26 Oktober 2021 direvisi : 9 November 2021 disetujui : 11 November 2021 Kata-kata Kunci: Sistem Ekonomi Kerakyatan, Kebijakan Hukum, Kesejahteraan Abstrak PERAN PROVINSI KEPULAUAN BANGKA BELITUNG DALAM PENGEMBANGAN SISTEM EKONOMI KERAKYATAN SAAT PANDEMI COVID-19 MENURUT UNDANG-UNDANG 1945 Muhamad Hijran1, Dini Oktariani2 1Program Studi Ekonomi, Universitas Bangka Belitung 2Program Studi Manajemen, Universitas Bangka belitung Kata-kata Kunci: Sistem Ekonomi Kerakyatan, Kebijakan Hukum, Kesejahteraan PENDAHULUAN Pandemi Covid-19 di Indonesia terjadi pertama kali pada Tanggal 2 Maret 2020, pandemi sudah menyebar ke 34 provinsi dengan Jawa Timur, DKI Jakarta dan Sulawesi Selatan sebagai provinsi paling terpapar. Upaya pencegahan penyebaranpun dilakukan dan dikampanyekan oleh berbagai bidang pemerintah dan tenanga kesehatan. Setidaknya ada dua cara yang menjadi kunci pengendalian penularan dan penyebaran Covid-19 yaitu dengan cara menjaga jarak dan rajin mencuci tangan dengan sabun dan memperbanyak wastafel portable yang diadakan secara mandiri oleh masyarakat. Pemerintahpun akhirnya melakukan Pembatasan Sosial Berskala Besar (PSBB) serta mengkampanyekan Stay at home. Sektor UMKM pun terdampak parah. Berdasarkan data dari kementerian koperasi yang memaparkan bahwa sebanyak 1.785 koperasi dan 163.713 pelaku Usaha Mikro Kecil dan Menengah (UMKM) terdampak pandemi virus corona (Antara, Mei 2020). Sektor UMKM yang paling terdampak yakni makanan dan minuman. Kementerian Koperasi dan UMKM mengatakan bahwa koperasi yang bergerak pada bidang jasa dan produksi juga paling terdampak pada pandemi COVID-19. Para pengusaha UMKM merasakan turunnya penjualan, kekurangan modal, dan terhambatnya distribusi. Sedikitnya 39,9 persen UMKM memutuskan mengurangi stok barang selama pembatasan sosial berskala besar (PSBB) akibat Covid-19. Sementara itu 16,1 persen UMKM memiih mengurangi karyawan akibat toko fisik ditutup. Sektor UMKM mengalami dampak yang cukup dalam akibat pandemi Covid-19. Dampak yang signifikanpun terjadi terhadap perekonomian di Indonesia. Dari semua usaha mikro kecil hingga koperasi sangat terdampak dengan adanya wabah virus corona. Penjualan menurun, permodalan, pesanan menurun, kesulitan bahan baku, dan kredit macet. Ekonomi tiba-tiba ambruk dalam sekejap. Bersarkan Undang-undang Nomor 20 Tahun 2008 tentang Usaha Mikro Kecil menengah, Usaha Mikro adalah usaha produktif milik perseorangan dan atau badan usaha perseorangan yang memenuhi kriteria usaha mikro sebagaimana diatur dalam Undang-Undang. Sedangkan Usaha Kecil adalah usaha ekonomi produktif yang berdiri sendiri, yang dilakukan oleh perseorangan atau badan usaha yang berdiri sendiri, yang dilakukan oleh perseorangan atau badan usaha yang bukan merupakan anak perusahaan atau bukan cabang perusahaan yang sesuai dengan kriteria usaha kecil sebagaimana diatur dalam Undang-Undang. Sedangkan Usaha Menengah adalah usaha ekonomi produkrif yang berdiri sendiri, yang dilakukan oleh perseorangan atau badan usaha yang bukan merupakan anak perusahaan atau cabang perusahaan dengan jumlah kekayaan atau hasil penjualan tahunan sebagaimana diatur dalam Undang_undang.Selain itu pula Undang-Undang Dasar 1945 pasal 33 merupakan payung hukum dalam pelaksanaan sistem ekonomi kerakyatan. Secara historis konsep ekonomi kerakyatan merupakan gagasan di bidang ekonomi yang dikembangkan oleh Soekarno dan Hatta pada tahun 1930-an. Soekarno menggunakan istilah ekoomi rakyat yang berhadapan dengan sistem ekonomi monopoli. Abstrak Kajian penelitian ini bertujuan untuk mengetahui Strategi pengembangan Ekonomi kerakyatan (Economic Civic) dan sangat relevan dilakukan mengingat Ekonomi Kerakyatan merupakan usaha-usaha di bidang ekonomi, dimana target pencapaian utamanya bukanlah keuntungan finansial semata. Penelitian ini mengunakan metode penelitian kualitatif, yakni mengumpulkan data-data dari artikel ilmiah, laporan penelitian atau hallai yang relevan dengan topik. Hasil penelitian menyimpulkan bahwa UMKM di Provinsi Kepulauan Bangka Belitung untuk meningkatkan pendapatan melalui program entreprenuer, pengembangan unit usaha, peningkatan kualitas sumber daya manusia, dan pemanfaatan teknologi dan informasi sehingga bisa memberikan dampak positif pada peningkatan penjualan produk. Cara mengutip : Hijran, M., Oktariani, D. (2021). Peran Provinsi Kepulauan Bangka Belitung dalam Pengembangan Sistem Ekonomi Kerakyatan saat Pandemi Covid-19 Menurut Undang-Undang 1945. Jurnal PROFIT: Kajian Pendidikan Ekonomi dan Ilmu Ekonomi, 8(2), 95-101. https://doi.org/10.36706/jp.v8i2.15672 95 Co Author Muhamad-hijran@ubb.ac.id Muhamad Hijran Co Author M h d hi DOI: doi.org/10.36706/jp.v8i2.15672 Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101 PENDAHULUAN Penelitian Sri Edi Swansono (2014: 89) mengemukakan bawha pendapat Mohamad hatta tentang ekonomi kerakyatan menggunakan istilah perekonomian ra’jat dan ekonomi ra’jat merujuk pada pemahaman tentang gras-rootseconomy atau ekonomi berbasis rakyat (people-based economy) dan ekonomi terpusat pada kepentingan rakyat (people-centered economy). Pemikiran Mohammad Hatta tersebut dijabarkan dalam beberapa tulisan yang dimuat pada harian Daulat Ra’jat: “Pengaroeh Kolonial kapital di Indonesia” yang dimuat pada 20 November 1932; “Pendirian Kita” dimuat tanggal 10 September 1932; “Krisis Dunia dan Nasib Ra’jat Indonesia” dimuat tanggal 20 September 1932 dan yang paling monumental adalah tulisannya 10 juni 1934 yang berjudul “Ekonomi Ra’jat dalam Bahaya”. Latar bekalang tulisan-tulisan Mohammad hatta pada era 1932 sampai dengan tahun 1934 diatas di dorong keprihatinan beliau dengan politik yang diterapkan Belanda, dimana kebijakan perekonomian tidak berpusat pada kepentingan rakyat dimaksud. Pembangunan ekonomi di Indonesia landasan pemikirannya terkandung pada pasal 33 Undang-Undang Dasar 1945. Makna secara Holistik menyatakan bahwa peran Negara dalam menjamin segala apa yang terkandung pada bumi Indonesia harus dikuasai negara dan diperuntukan untuk kemakmuran dan kesejahteraan rakyat. Menurut Sabil (2014: 51) Besar kecilnya perekonomian rakyat tergantung pada tingkat pertumbuhan ekonomi atau nominal produk domestik bruto (PDB). Berbagai cabang Produksi yang penting bagi negara dan yang menguasai hajat hidup orang banyak, dikuasi oleh negara sebagai modal usaha mencapai kemakmuran rakyat. Untuk mencapai kemakmuran tersebut, hadir pandangan mengenai ekonomi kerakyatan. Hal tersebut sejalan dengan pendapat Hasmawati (2018: 54) menyatakan bahwa ekonomi kerakyatan adalah kegiatan yang 96 DOI: doi.org/10.36706/jp.v8i2.15672 memberikan kesempatan partisipasi yang luas kepada masyarakat untuk pelaksanan dan pengembangan yang baik. Sehingga memenuhi syarat khususnya untuk mewujudkan Sila Ke 5 (lima), “Keadilan sosial Bagi Seluruh Rakyat”. Konsep ekonomi kerakyatan ini sebagai salah satu bentuk usaha untuk menumbuhkan kesejahteraan melalui pemberdayaan masyarakat. Untuk mencapai kesejahteraan tersebut diperlukan sinergi antara pemerintahan pusat maupun daerah. Menurut Wibawa dan yusnita (2018:2) peneliti sependapat bawha pembangunan ekonomi berbasis potensi daerah pada dasarnya menitikberatkan pada penguatan kemampuan lembaga ekonomi kerakyatan seperti Usaha Mikro, Kecil, dan Menengah (UMKM) dengan menciptakan sentra pengembangan produk unggulan daerah berbasis budaya dan potensi daerah. Oleh sebab itu, diperlukannya memberdayakan potensi UMKM yang secara berkelanjutan, ideal, dan tentunya insklusif. Bersdasarkan data Kementerian Koperasi dan UMKM per April 2021, jumlah UMKM saat ini mencapai 64, 2 juta dengan kontribusi terhadap PDB sebesar 61,07% atau senilai 8.573,89 triliun rupiah. Kontribusi UMKM terhadap perekonomian Indonesia meliputi kemampuan menyerap 97% dari total tenaga kerja yang ada serta dapat menghimpun sampai 60,4% dari total investasi. PENDAHULUAN Namun tingginya jumlah UMKM di Indonesia juga tidak terlepas dari tantangan yang ada. Di Indonesia pandemi Covid-19 berdampak terhadap beberapa sektor, tidak terkecuali sektor ekonomi. Sehingga menyebabkan perekonomian indonesia menjadi lemah dikarenakan beberapa usaha gulung tikar. Dalam pembangunan ekonomi daerah, Khusunya Provinsi Kepulauan Bangka Belitung sesuai data Badan Pusat Statistik triwulan I-2021 tumbuh 0, 97% persen, mengalami perlambatan bila dibandingkan triwulan I-2020 (y-on-y) yang tumbuh sebesar 1,36. Melemahnya perekonomian ini, menyebabkan UMKM mengalamai resesi sehingga diharapkan segera bangkit dari keterpurukan karena peran vitalnya UMKM dalam roda perekonomian daerah, bahkan maupun nasional. Peneliti sependapat dengan Halim (2020: 158) bahwa Peran UMKM memiliki makna yang sangat penting untuk daerah, khusunya sebagai salah satu pendorong pertumbuhan ekonomi daerah. Oleh karena itu, diperlukannya perhatiaan khusus untuk para pelaku UMKM. Seperti apa yang di uatarakan oleh Helmalia dan Afrinawati (2018: 237) bahwa UMKM bisa menjadi prioritas daari sistem ekonomi kerakyatan untuk menanggulangi permasalahan seperti kemiskinan dan pengangguran. Nilai ekonomi dari satu produk UMKM diperlukannya inovasi dan kreativitas bukan hanya mengandalkan harga saja. Menurut Sumarni (2017: 139) Untuk meningkatkan Kemampuan UMKM di Indonsia agar produk yang dihasilkan mampu bersaing di pasar global yang harus ditempuh pemerintah yaitu dengan cara meningkatkan neraca perdagangnnya. Di era New Normal di masa Pandemi Covid-19 dan teknologi yang semakin maju, Produk UMKM harus bisa beradaptasi dengan perkembangan zaman sehingga nantinya bisa bersaing baik didalam negeri dan luar negeri. Tetapi kenyatan dilapangan, banyak sekali kendala yang harus dibenahi. Penelitian Thaha (2020: 149) melaporkan bahwa 163. 713 pelaku UMKM pada sektor makanan dan minuman terkena dampak pandemi Covid-19. Disinilah perlunya keharmonisan antara pemerintah pusat, pemerintah daerah serta para pelaku UMKM untuk menanggulangi permasalahan yang ada sehingga bisa teratasi dengan baik. Penelitian Hardilawati (2020: 97) menunjukan strategi bertahan UMKM di tengah Pandemi Covid-19 yang meliputi rekomendasi untuk melakukan perdagangan secara e-commerce, melakukan pemasaran secara digital, perbaikan kualitas produk, penambahan pelayanan konsumen, dan optimalisasi hubungan pemasaran pelanggan. Hasil penelitian diatas telah menjelaskan bahawa dampak dari pandemi Covid-19 untuk para pelaku UMKM ini diperlukanya Pendampingan dalam strategi menghadapi pandemi Covid-19. Penelitian Andayani et al (2021 : 13) menjelaskan dalam menjalankan Stategi pelaku UMKM dalam mengahadapi Covid-19 dapat dilakukan melalui pemasaran produk secara online sehingga menghasilkan penghematan pada sektor produksi, distribusi dan pemasaran konvensional. Disinilah diperlukannya kreativitas dari pelaku UMKM dalam memanfaatkan teknologi. PENDAHULUAN Sejalan dengan Pendapatnya Sutinah et al (2020 : 206) Para pelaku UMKM di dorong untuk belaajr memahami tentang karakteristik pelaku konsumen ke arah pasar digital dengan memasarkan produk UMKM melalui teknologi digital. Inilah urgensi penjualan produk UMKM sangat diperlukan dengan memanfaatkan marketing digital yang berdampak positif sehingga UMKM dapat tetap berjalan dan menghasilkan nilai ekonomi. 97 DOI: doi.org/10.36706/jp.v8i2.15672 Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101 Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101 Berdasarkan latar belakang diatas, penelitian ini bertujuan untuk melihat bagaimana peran Provinsi Kepulauan Bangka Belitung dalam pengembangan sistem ekonomi kerakyatan saat pandemi Covid-19. Peneliti berasumsi bahwa penelitian tersebut belum pernah dilakukan dengan kebermanfaatn hasil penelitian bagi Provinsi Kepulauan Bangka Belitung. METODE Penelitian ini menggunakan metode Literature review. Teknik ini dilakukan dengan tujuan untuk mengungkapkan berbagai teori-teori yang relevan dengan permasalahan yang sedang dihadapi sebagai bahan rujukan dalam pembahasan hasil peneliti. Kumpulan data Studi Literatur yang terdiri dari artikel jurnal, textbook, handbook, arsip maupun regulasi adalah cara untuk menyelesaikan persoalan dengan menelusuri sumber-sumber tulisan yang pernah dibuat sebelumnya. Pengumpulan Literature review digunakan beberapa tahapan diantaranya pencarian artikel berdasarkan topik garis besar, pengelompokkan artikel berdasarkan relevansi dengan topik. Setelah artikel terkumpul selanjutnya peneliti mengelompokkan sejumlah artikel yang telah didapatkan berdasarkan relevansi topik.kemudian artikel yang dikelompokkan peneliti analisis penjelasan struktur mengenai ketertarikan artikel dan topik peneliti. Lalu penulis membandingkan apabila ada jurnal saling berhubungan dan penambahan textbook lain bersifat memperkuat dan menambah ketajaman pembahasan hasil penelitian. Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101 UMKM yang terdampak COVID-19 Begitu cepatnya penyebaran Covid-19, dampak perlambatan perekonomi secara global mulai dirasakan oleh Indonesia. Banyak pelaku UMKM tidak segan-segan untuk meliburkan sampai ada yang mengeluarkan karyawannya bahkan tidak sedikit yang menutup sementara usahanya. Salah satu penyebabnya adalah penurunan omzet penjualan. Work Form Homeatau dikenal dengan singakatan WFH juga berpengaruh terhadap penurunan pendapatan UMKM. Pelaku UMKM mengalami penurunan pendapatan yang drastis akibat penerapan Phisical Distancing dan penerapan Pemberlakuan Pembatasan Kegiatan Masyarakat (PPKM). Pembatasan aktifitas masyarakat sangat berpengaruh pada aktifitas bisnis yang kemudian berimbas pada perekonomian di Indonesia. Para pelanggan menutup diri bahkan menjaga jarak untuk saling bertemu dengan masyarakat dan berdampak pada aktifitas bisnis yang mengakibatkan ekonomi menjadi turun. Kegiatan interaksi fisik antara sesama warga masyrakat menjadi berkurang dan membuat masyarakat mengurangi aktifitas ekonomi secara drastis. Hal ini sangat berpengaruh terhadap berbagai sektor perekonomian, salah satunya yaitu UMKM. Penelitian Rosita (2020) Keadaan ini sangat mengkhawatirkan mengingat pertumbuhan ekonomi suatu negara sangat berpengaruh dan sangat tergantung pada ketahanan UMKM saat mengahadapi krisis. Dengan adanya pandemi seperti ini, tidak membuat perekonomian daerah menurun, bahkan seluruh daerah di Indonesia merasakan hal yang sama tidak terkecuali di Kepulauan Bangka Belitung. Kondisi UMKM di Provinsi Kepulauan Bangka Belitung juga mengalami kerugian bahkan ada yang mengalami penurunan omzet penjualan karena adanya pandemi Covid-19. Penelitian Soetjipto (2020 : 97) melaporkan bahwa penyebab penurunan omzet pelaku UMKM, yaitu 63% disebabkan oleh daya beli konsumen menurun, 46% konsumen takut membeli disertai adanya aturan PSBB, dan 42% adanya aturan jam operasional toko. Dampak dari penurunan omzet, 33% pelaku UMKM memilih menghentikan produksi, 19% menghabiskan stok barang yang dikembalikan, 8% melakukan produksi ketika ada pesanan, dan 27% membuka dagangan dengan protokol kesehatan yang ketat. Usaha Pelaku UMKM dalam menyiasati situasi pandemi, yaitu 38% melakukan pemasaran melalui media sosial, 35% menggunakan promosi berbayar yang telah tersedia secara online, 13% menitipkan produk secara konvensional dan 6% tetap membuka tempat jualan. Disinilah diperlukannya perhatian dari berbagai stekholder dalam membatu para pelaku UMKM, untuk berkolaborasi sehingga bisa tetap bisa bertahan dalam masa pandemi. Provinsi Kepulauan Bangka Belitung sendiri memiliki peran untuk menanggulangi permasalahan tersebut dengan beberapa program. Salah satunya dilihat dari data Laporan Akuntabilitas Kinerja Instansi Pemerintah 98 Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101 DOI: doi.org/10.36706/jp.v8i2.15672 (LAKIP) Dinas Koperasi, Usaha Kecil dan Mengah Tahun 2020 yaitu program peningkatan pemberdayaan usaha kecil. UMKM yang terdampak COVID-19 Keluaran kegiatan ini adalah 100 produk UKM di Kota Pangkalpinang, Kabupaten Bangka, Bangka tengah, Bangka selatan, dan Bangka Barat yang di fasilitasi dalam pameran virtual di Kabupaten Bangka tepatnya di Pantai Tiung Kecamatan Belinyu dan 60 Produk UKM di kabupaten Belitung dan Belitung Timur yang di fasilitasi dalam pameran virtual di kabupaten Belitung. Selain itu pula terfasilitasinya 500 Produk UKM melalui Market Place di Bukalapak dan 200 Produk UKM di Market Place Bli-Bli. Sejalan dengan Penelitian Hardilawati (2020) menjelaskan bahwa strategi untuk bertahan di tengah Pandemi Covid-19 untuk para pelaku UMKM yaitu rekomendasi dengan melakukan perdagangan secara e-commerce, melakukan pemasaran secara digital, perbaikan kualitas produk, penambahan pelayanan konsumen, dan optimalisasi hubungan pemasaran pelanggan. Sementara itu Fadlillah (2020: 344) menjelaskan bahwa pelaku UMKM bangkalan yang memiliki usaha minuman Boba mengalami penurunan omzet penjualan, sehingga menuntut pelaku usaha tersebut melek teknologi untuk dapat melakukan strategi pemasaran. Program Selanjutnya yaitu Pengembangan, penguatan dan perlindungan usaha kecil. Keluaran kegiatan ini adalah terpasilitasinya 200 pelaku usaha melalui setifikasi halal, dan terlaksananya pendataan UMKM di 47 Kecamatan Se-Provinsi Kepulauan Bangka Belitung yang dituangkan dalam 1 (satu) dokumen pendataan. Selain itu pula penyelenggaraan pelatihan perkoperasian dan UKM (DAK) di ikuti oleh 21 orang yang mengikuti pelatihan tata dan prosedur ekspor bagi pengurus dan aggota koperasi (pelatihan daring), 44 pelaku usaha yang mengikuti pelatihan kewirausahaan bidang UKM Ekspor, 428 pelaku usaha yang mengikuti pelatihan pengelolalaan bisnis saaatn pandemi covid-19, 29 pelaku usaha yang mengikuti pelatihaan kewirausahaan bidang keterampilan teknis pengolahaan hasil perikanan dan 23 orang yang mengikuti pelatihan sertifikasi dan uji kompetensi juru buku. Penelitian Elfizon et al (2020: 237) mengungkapkan bahwa penerapan pemasaran produk melalui e-commerce berdampak positif terhadap pelaku industri rumah tangga. Aplikasi yang dapat digunakan pelaku UMKM dalam menjual pemasaran produknya bisa meliputi Bukalapak, Lazada, Shoppe Tokopedia, dan lain-lain. Sementara itu untuk penggunaan digital marketing bisa memanfaatkan media sosial, seperti Tiktok, Facebook, Instagram, Twitter dan Whatsapp sehingga bisa memberikan dampak positif pada peningkatan penjualan produk. Penggunaaan media sosial merupakan sarana promosi bagi konsumen, sehingga diharapkan dengan adanya media sosial akan meningkatkan penjualan produk bagi perusahaan. Media sosial sendiri merupakan sudah menjadi trend dalam komunikasi pemasaran untuk sekarang ini. Penelitian Narto (2020: 53) Menunjukan bahwa strategi pemasaran produk di tengah pandemi Covid-19 dilakukan melalui berbasis Online dengan mempertahankan harga produk dan meningkatkan kualitas untuk memperoleh loyalis Konsumen. Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101 Pembahasan Provinsi Kepulauan Bangka Belitung termasuk salah satu provinsi yang memiliki potensi yang sangat besar. Salah satunya pertumbuhan UMKM menunjukan peningkatan setiap tahunnya dimana ini merupakan kemajuan bagi perekonomian. Disatu sisi, ada permasalahan para pelaku UMKM yang sangat komplek, seperti yang di ungkapkan oleh Herawati et al (2020:17) potensi dan permasalahan UMKN yang meliputi: UMKM belum memiliki jaringan pemasaran, modal dari kemampuan pribadi, berjalan mandiri tanpa dukungan dinas terkait, sarana produksi masih manual, banyaknya pesaing usaha dengan produk yang sama, situasi pandemi wabah penyakit, dan globalisasi pasar. Penelitian Fauziyah (2020:157) menjelaskan bahwa munculnya era digitalisasi akan memberikan dampak perkembangan dan pertumbuhan bagi UMKM Pemerintah daerah sendiri, melakukan upaya dan usaha untuk mengatasi permasalahan tersebut. Provinsi Kepulauan Bangka Belitung adalah memberikan bantuan pemodalan, pengemasan dan pemasaran. Melalui Dinas Koperasi, Usaha Kecil dan Mengah Provinsi Kepulauan Bangka Belitung memiliki beberapa program untuk peningkatan pemberdayaan usaha kecil, selain itu ada pelatihan pelayanan teknis latihan perkoperasian, usaha mikro, kecil, dan menengah. Para pelaku UMKM sendiri mengikuti beberapa program pemerintah daerah Provinsi Kepulauan Bangka Belitung dengan berbagai macam kegiatan. Salah satunya mengadakan pelatihan untuk pelaku UMKM dengan bertujuan untuk mendorong kompetensi wirausaha sehingga menimbulkan kreatifitas dan kemampuan dalam 99 DOI: doi.org/10.36706/jp.v8i2.15672 mencapai kesuksusesan bisnisnya. Salah satu programnya peningkatan pusat layanan usaha terpadu UMKM dengan 150 pelaku usaha yang didampingi melalui klinik usaha terpadu dan pengembangan sentra usaha kepada 3 kelompok yaitu salah satu contohnya Bimtek peningkatan produktifitas dan daya saing kerajinan tangan daun Mengkuang di Desa Delas. Faktor lainnya yang dapat mempengaruhi dalam membangkitakn kinerja pelaku UMKM adalah kebijakan pemerintah daerahnya. Beberapa indikator kebijakan pemerintah daerah dalam mengembangkan UMKM adalah salah satunya kebijakan fasilitas permodalan. Mengutip pernyataan dari Gubernur Kepulauan Bangka Belitung, Erzaldi Rosman telah melakukan berbagai strategi untuk membuat UMKM tetap bertahan dan bangkit terutama di masa pandemi ini. Oleh karenanya, beliau berupaya mendorong sistem ekonomi kerakyatan melalui UMKM. Dengan adanya Pemodalan dan Pelatihan untuk para pelaku UMKM dari hasil kordinasi dan kolaborasi antara pemerintah pusat dan daerah diharapakan mempunyai produk yang bersaing dalam negeri dan berorientasi ekspor sehingga menjadi penggerak perekonomian dan pemerataan pembangunan di Provinsi Kepulauan Bangka Belitung. Realisasi KUR di Bangka Belitung Khusu untuk UMKM sampai sekarang ini di manfaatkan oleh hampir 20.000 pelaku UMKM. Capaian ini merupakan komitmen dan semangat lembaga perbankan dan perusahaan dalam hal ini ini BUMN, yang membantu fasilitasi askes permodalan. Pembahasan Selain itu pula yang harus diperhatikan adalah bagaimana upaya untuk mendampingi atau membina para pelaku UMKM agar dapat melaksanakan usahanya dengan baik. Dengan pendampingan terus-menerus akan memberikan semangat kepada pelaku UMKM. Oleh karena itu, pemerintahan Provinsi Kepulauan Bangka Belitung melakukan beberapa langkah untuk meningkatkan kembali UMKM ini diantaranya pemasaran digitalisasi, memfasilitasi UMKM untuk melakukan penjualan pada market place baik secara nasional maupun internasional dan promosi melalui media elektronik dan media online serta melakukan pameran virtual guna memperkenalkan produk unggulan Provinsi Kepulauan Bangka Belitung. Selain itu, mendorong UMKM untuk meningkatkan daya saing melalui fasilitasi halal, fasilitasi kemasan produk, bantuan alat pendukung produksi, mendekatkan pada akses pembiayaan serta menajalin kemitraan dengan lembaga lainnya untuk bersinergi dalam pengembangan UMKM. Selain itu pula melakukan pelatihan produktivitas untuk membuat kerajinan yang diorientasikan untuk kebutuhan lokal, nasional dan ekspor. SIMPULAN Berdasarkan hasil analisis yang dilakukan oleh peneliti bahwa Peran Provinsi Kepulauan Bangka Belitung dalam pengembangan sistem ekonomi kerakyatan saat pandemi covid-19 yaitu mengadakan beberapa program dengan membutuhkan strategi pemasaran dalam perekonomiannya. Sehingga tujuan dari ekonomi kerakyatan bisa berjalan dengan lancar dan dapat mencapai tujuan dari Alenia ke IV Pembukaan UUD 1945. Dalam hal ini Provinsi Kepulauan Bangka Belitung melakukan kolaborasi dan pendampingan secara berkala antar berbagai pelaku kepentingan. Beberapa programnya, yaitu program peningkatan pemberdayaan usaha kecil, Pengembangan, penguatan dan perlindungan usaha kecil serta penyelenggaraan pelatihan perkoperasian dan UKM (DAK). Sehingga dengan adanya beberapa program dengan jejaring pemasaran digital diharapkan mampu meningkatkan pendapatan para pelaku ekonomi di Kepulauan Provinsi Bangka Belitung sehingga meningkat dan ekonomi kerakyatan bisa berjalan dengan maksimal. UCAPAN TERIMA KASIH Penulis ucapkan terima kasih kepada Tim yang sudah memberikan dukungan sehingga artikel ini bisa selesai tepat waktu, DAFTAR PUSTAKA Andayani, Indah:, Roesminingsih, Maria Veronika; Yulianingsih, Wiwin. (2021) Strategi Pemberdayaan Masyarakat Pelaku UMKM di Masa Pandemi Covid-19. Jurnal Pendidikan Nonfromal. Vol. 16 (1) DOI: http://dx.doi.org/10.17977/um041v16i1p12-20 DAFTAR PUSTAKA DAFTAR PUSTAKA Andayani, Indah:, Roesminingsih, Maria Veronika; Yulianingsih, Wiwin. (2021) Strategi Pemberdayaan Masyarakat Pelaku UMKM di Masa Pandemi Covid-19. Jurnal Pendidikan Nonfromal. Vol. 16 (1) DOI: http://dx.doi.org/10.17977/um041v16i1p12-20 100 DOI: doi.org/10.36706/jp.v8i2.15672 Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101 Elfizon, Ganefri, dan Asrul Huda. (2020). Pemberdayaan Masyarakat dalam Pemasaran Produk Industri Rumah Tangga melalui Penerapan E-Commerce di Kenagarian Vii koto Talago Kecamatan Guguak Kabupaten 50 kota. Jurnal Teknik Elektro dan Vokasional. Vol. 6 (2) https://doi.org/10.24036/jtev.v6i2.109134 Fadlillah Syaiful, F., & Elihami, E. (2020). PENERAPAN ANALISIS SWOT TERHADAP STRATEGI PEMASARAN USAHA MINUMAN KAMSIA BOBA MILIK ABDULLAH DI TENGAH PANDEMI COVID- 19 DI KABUPATEN BANGKALAN. Jurnal Edukasi Nonformal, 1(1), 343-359.https://ummaspul.e- journal.id/JENFOL/article/view/583 Fauziyah. (2020). Tantangan UMKM dalam mengahadapi Revolusi Industri 4.0 ditinjau dari Aspek Marketing dan Accounting. Jurnal Manajemen dan Kewirausahaan. Vol. 5 (2) 155-172 https://doi.org/10.32503/jmk.v5i2.1008 Halim, Abdul. (2020). Pengaruh Pertumbuhan usaha Mikro, Kecil Dan Menengah Terhadap Pertumbuhan Ekonomi Kabupaten Mamuju. Jurnal Ilmiah Ekonomi Pembangunan. Vol. 1 (2) 157-172 https://stiemmamuju.e-journal.id/GJIEP/article/view/39/30 Hardilawati, Wan Lauran. (2020). Strategi Bertahan UMKM di Tengah Pandemi Covid-19. Jurnal Akuntasi & Ekonomika. Vol. 10 (1) 89-98 https://doi.org/10.37859/jae.v10i1.1934 Hasmawati, Fifi (2018). Ekonomi Kerakyatan Berbasis Potensi Lokal. Jurnal Pemberdayaan Masyarakat. Vol. V (5) 54-65 http://dx.doi.org/10.37064/jpm.v6i1.4986 Helmalia & Afrinawati. (2018). Pengaruh E-Comerce Terhadap Peningkatan Pendapatan Usaha Mikro Kecil dan Menengah di Kota padang. Jurnal Ekonomi dan Bisnis Islam. Vol. 3 (2) 237-246 https://doi.org/10.15548/jebi.v3i2.182 Herawati, L, Dayal Gustopo & Prima Vitasari, (2020). Identifikasi Permasalahan Penjualan dengan Metode SWOT pada UKM Gula Merah. Jurnal Teknologi dan Manajemen Industri. Vol. 6 (1) 17-20 https://doi.org/10.36040/jtmi.v6i1.2625 Narto & Gatot Basuki HM. (2020). Penguatan Strategi Pemasaran Pudak di tengah Pandemi Covid-19 untuk meningkatkan Keunggulan Bersaing Usaha Mikro Kecil Menengah Kota Gresik. Jurnal Intech. Vol. 6 (1) 48-54 http://dx.doi.org/ 10.30656/intech.v6i1.2195 Rosita, Rahmi. (2020). Pengaruh Pandemi Covid-19 Terhadap Umkm Di Indonesia.Jurnal Lentera Bisnis. Vol. (9) No 2 109-110 doi : 10.34127/jrlab.v9i2.380 Sabil. (2014). Sistem Ekonomi Kerakyatan sebagai Landasan Pembangunan Ekonomi Indonesia Melalui UKM, Koperasi dan Pemerintah Daerah. Jurnal Moneter. Vol. I (1) 51-57 https://doi.org/10.31294/moneter.v1i1.938 Soetjipto, Noer. (2020). Ketahanan UMKM Jawa Timur Melintasi Pandemi Covid-19. Yogyakar Soetjipto, Noer. (2020). Ketahanan UMKM Jawa Timur Melintasi Pandemi Covid-19. Yogyakarta: K-Media Sumarni. (2017). Peranan UMKM (Usaha Mikro, Kecil dan Menengah) Terhadap Perekonomian Indonesia. Jurnal Manajemen FE-UB. Vol. 005 (1) 138-145 Sumarni. (2017). Peranan UMKM (Usaha Mikro, Kecil dan Menengah) Terhadap Perekonomian Indonesia. Jurnal Manajemen FE-UB. Vol. 005 (1) 138-145 https://ejournal.borobudur.ac.id/index.php/manajemen/article/view/438/435 Sutinah, Bagong suyanto, dan Ratna Azis P. (2020). DAFTAR PUSTAKA Pemberdayaan Pelaku UMKM Merespon Pergeseran Karakteristik Konsumen di Era Digital. Jurnal Layanan Masyarakat. Vol. 4 (1) 202- 207 http://dx.doi.org/10.20473/jlm.v4i1.2020.202-207 Swasono Sri Edi, (2014) Kebangsaan, Kerakyatan, dan Kebudayaan dalam “Prosiding Kongres Pancasila VI:Penguatan, Sinkronisasi, Harmonisasi, Integrasi Pelembagaan dan Pembudayaan Pancasila dalam Rangka Memperkokoh Kedaulatan Bangsa”, Ambon, 31 Mei - 01 Juni 2014, hlm. 89. Thaha, Abdurahman Firdaus. (2020). Dampak Covid-19 Terhadap UMKM di Indonesia. Jurnal Brand. Vol. 2 (1) 147-153 https://ejournals.umma.ac.id/index.php/brand Wibawa, Dian & Yusnita, M. (2019). Peran UMKM sebagai Penyangga Perekonomian Berbasis Kerakyatan. Holistic Jurnal of Management Research. Vol. 4 (2) 1-9 https://journal.ubb.ac.id/index.php/holistic/article/view/1430 101 DOI: doi.org/10.36706/jp.v8i2.15672 Hijran, M., Oktariani, D. Jurnal PROFIT, 8 (2) 2021, 95-101
https://openalex.org/W4247617192	http://www.epj-conferences.org/10.1051/epjconf/20135204009/pdf	English	null	First results of the SPHERE experiment	EPJ web of conferences	2,013	cc-by	213	EPJ Web of Conferences DOI: 10.1051/ , epjconf 201 / 04009 (2013) 35204009 52 EPJ Web of Conferences DOI: 10.1051/ , epjconf 201 / 04009 (2013) 35204009 52 EPJ Web of Conferences DOI: 10.1051/ , epjconf 201 / 04009 (2013) 35204009 52 / C ⃝Owned by the authors, published by EDP Sciences, 2013 pj / C ⃝Owned by the authors, published by EDP Sciences, 2013 pj / aser101@inbox.ru First results of the SPHERE experiment Sergey Shaulov1,a 1Lebedev Physical Institute of the Russian Academy of Sciences, Russia Sergey Shaulov1,a 1Lebedev Physical Institute of the Russian Academy of Sciences, Russia Abstract. First results of the balloon-borne experiment SPHERE are presented. The pri- mary spectrum in the energy range 1016 – 5 · 1017 eV is compared with data of other ex- periments. The primary energies were reconstructed using characteristics of the Vavilov- Cherenkov radiation from extensive air showers, reﬂected from a snow surface. Slides The slides of the talk can be found on the website of the symposium ISVHECRI 2012: https://indico.desy.de/conferenceOtherViews.py?view=standard&confId=4594 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2 0 , which . permits unrestricted use, distributi and reproduction in any medium, provided the original work is properly cited. on, Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20125204009
https://openalex.org/W4393444199	https://journal.lembagakita.org/index.php/jemsi/article/download/2215/1623	Indonesian	null	Analisis Efektivitas Perencanaan Anggaran Perguruan Tinggi Agama Islam dalam Mencapai Tujuan Strategis	JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi)	2,024	cc-by-sa	4,301	Pendahuluan Perguruan tinggi agama Islam dihadapkan pada tantangan unik dalam merumuskan dan melaksanakan perencanaan anggaran yang sesuai dengan prinsip-prinsip Islam dan memenuhi tujuan-tujuan strategisnya (Joudar et al., 2023). Hasil analisis menunjukkan bahwa pemahaman mendalam tentang prinsip-prinsip Islam dalam konteks keuangan serta integrasi yang kuat antara nilai-nilai agama dan tujuan-tujuan akademik menjadi landasan penting dalam merancang perencanaan anggaran yang efektif bagi perguruan tinggi agama Islam (To et al., 2022). ) Dalam kerangka manajemen keuangan, perencanaan anggaran yang efektif membutuhkan keterlibatan yang aktif dari berbagai pihak di dalam institusi, termasuk pimpinan, staf administratif, dan akademisi (Ginting, et al., 2023). Selain itu, penggunaan teknologi informasi dan sistem informasi manajemen keuangan turut berperan penting dalam meningkatkan transparansi, akuntabilitas, dan efisiensi dalam proses perencanaan anggaran di perguruan tinggi agama Islam (Qudah et al., 2023). Perencanaan anggaran yang efektif di perguruan tinggi agama Islam membutuhkan pendekatan holistik yang mempertimbangkan aspek keuangan, manajerial, strategis, serta nilai-nilai agama. Integrasi yang kuat antara perencanaan anggaran dan tujuan strategis institusi, didukung oleh partisipasi aktif dari seluruh pemangku kepentingan dan pemanfaatan teknologi informasi yang tepat, merupakan langkah kunci dalam mencapai efektivitas perencanaan anggaran yang diinginkan (Gandrita, 2023). Abstrak: Perencanaan anggaran di perguruan tinggi agama Islam memiliki peran penting dalam mencapai tujuan strategis. Artikel ini bertujuan untuk menganalisis efektivitas perencanaan anggaran dalam konteks tersebut melalui metode literature review. Melalui telaah terhadap literatur terkait, artikel ini mengidentifikasi faktor-faktor yang memengaruhi efektivitas perencanaan anggaran di perguruan tinggi agama Islam, termasuk aspek keuangan, manajerial, dan strategis. Hasil analisis menunjukkan bahwa integrasi antara perencanaan anggaran dengan tujuan strategis menjadi kunci keberhasilan dalam mencapai efektivitasnya. Temuan ini memberikan kontribusi penting dalam memperkuat praktik perencanaan anggaran di perguruan tinggi agama Islam guna meningkatkan pencapaian tujuan strategis yang diinginkan. Kesimpulannya, upaya untuk meningkatkan efektivitas perencanaan anggaran memerlukan pemahaman yang mendalam tentang dinamika internal dan eksternal yang memengaruhi perguruan tinggi agama Islam serta integrasi yang kokoh antara perencanaan anggaran dan tujuan strategis institusi. Keywords: Perencanaan Angaran, Perguruan Tinggi Agama Islam, Keuangan. Keywords: Perencanaan Angaran, Perguruan Tinggi Agama Islam, Keuangan. JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. Analisis Efektivitas Perencanaan Anggaran Perguruan Tinggi Agama Islam dalam Mencapai Tujuan Strategis Article’s History: Article’s History: y Received 12 December 2023; Received in revised form 26 December 2023; Accepted 1 January 2024; Published 1 April 2024. All rights reserved to the Lembaga Otonom Lembaga Informasi dan Riset Indonesia (KITA INFO dan RISET). Suggested Citation: Suggested Citation: Zikrawahyu. (2024). Analisis Efektivitas Perencanaan Anggaran Perguruan Tinggi Agama Islam dalam Mencapai Tujuan Strategis. JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi). JEMSI (Jurnal Ekonomi, Manajemen, Dan Akuntansi), 10 (2). 1032-1039. https://doi.org/10.35870/jemsi.v10i2.2215 Analisis Efektivitas Perencanaan Anggaran Perguruan Tinggi Agama Islam dalam Mencapai Tujuan Strategis Zikrawahyu UIN Mahmud Yunus Batusangkar dededwahyu@gmail.com Article’s History: Received 12 December 2023; Received in revised form 26 December 2023; Accepted 1 January 2024; Published 1 April 2024. All rights reserved to the Lembaga Otonom Lembaga Informasi dan Riset Indonesia (KITA INFO dan RISET). Tinjauan Pustaka Alhammadi (2022) menyoroti pentingnya pengelolaan keuangan yang baik dalam konteks perencanaan anggaran di perguruan tinggi agama Islam. Studi ini menekankan bahwa alokasi dana yang efisien dan tepat 1032 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. Volume 10 (2) April Tahun 2024, Hal 1032-10 sasaran merupakan aspek penting yang dapat memengaruhi efektivitas perencanaan anggaran di institusi tersebut. Aspek ini mempertimbangkan bagaimana dana dialokasikan dan digunakan untuk mendukung tujuan strategis institusi. sasaran merupakan aspek penting yang dapat memengaruhi efektivitas perencanaan anggaran di institusi tersebut. Aspek ini mempertimbangkan bagaimana dana dialokasikan dan digunakan untuk mendukung tujuan strategis institusi. g Studi-studi terdahulu, seperti yang dilakukan oleh Menne et al. (2022), menyoroti peran penting aspek manajerial dalam efektivitas perencanaan anggaran. Manajemen sumber daya manusia yang efisien, proses pengambilan keputusan yang tepat, dan struktur organisasi yang jelas merupakan faktor-faktor kunci yang dapat memengaruhi kinerja perencanaan anggaran di institusi pendidikan tinggi agama Islam. Selain itu, integrasi antara perencanaan anggaran dan tujuan strategis institusi menjadi fokus penting dalam penelitian literatur, seperti yang diungkapkan oleh Mujiatun et al. (2023). Penelitian ini menunjukkan bahwa institusi yang mampu mengintegrasikan perencanaan anggaran dengan visi, misi, dan tujuan strategisnya cenderung lebih sukses dalam mencapai pencapaian yang diinginkan. Selanjutnya, pemahaman atas prinsip-prinsip Islam dalam konteks keuangan juga telah menjadi fokus studi dalam merancang perencanaan anggaran yang sesuai dengan nilai-nilai agama. Rahahleh et al. (2019) menyoroti pentingnya memahami prinsip-prinsip keuangan Islam, seperti keadilan, transparansi, dan penghindaran riba, sebagai landasan untuk merancang perencanaan anggaran yang konsisten dengan identitas institusi. Dengan demikian, tinjauan pustaka ini memberikan pandangan yang komprehensif tentang faktor-faktor yang memengaruhi perencanaan anggaran di perguruan tinggi agama Islam. Metodelogi Penelitian ini mengadopsi metode literature review untuk menganalisis efektivitas perencanaan anggaran di perguruan tinggi agama Islam. Tahap pertama melibatkan pengumpulan sumber-sumber literatur yang relevan, seperti jurnal akademik, buku, dan laporan riset yang dipublikasikan sejak tahun 2019. Selanjutnya, seleksi literatur dilakukan untuk memilih sumber-sumber yang paling relevan dan berkualitas tinggi sesuai dengan kriteria yang telah ditetapkan, seperti relevansi topik, kredibilitas sumber, dan konteks perguruan tinggi agama Islam. Tahap analisis literatur dilanjutkan dengan menyintesis temuan-temuan utama yang berkaitan dengan faktor-faktor yang memengaruhi efektivitas perencanaan anggaran di institusi-institusi tersebut serta mengidentifikasi pola dan tren yang muncul dalam penelitian terdahulu. Langkah terakhir melibatkan interpretasi terhadap temuan-temuan literatur serta pembentukan kesimpulan yang relevan dengan tujuan penelitian. Metode literature review ini diharapkan dapat memberikan pemahaman yang komprehensif tentang efektivitas perencanaan anggaran di perguruan tinggi agama Islam berdasarkan penelitian-penelitian dan temuan-temuan terkini yang telah dipublikasikan sejak tahun 2019. Dengan demikian, penelitian ini akan memberikan kontribusi penting dalam meningkatkan pemahaman kita tentang bagaimana perencanaan anggaran dapat secara efektif mendukung pencapaian tujuan strategis di institusi-institusi pendidikan tinggi agama Islam. Hasil dan Pembahasan Hasil Integrasi antara Perencanaan Anggaran dan Tujuan Strategis Integrasi antara perencanaan anggaran dan tujuan strategis merupakan aspek krusial yang telah menjadi fokus dalam penelitian literatur terkait manajemen keuangan di perguruan tinggi agama Islam. Temuan utama dari literature review menunjukkan bahwa integrasi yang kokoh antara perencanaan anggaran dengan tujuan strategis institusi memiliki dampak signifikan terhadap efektivitas perencanaan anggaran itu sendiri (Poon et al., 2020). Penelitian menyoroti bahwa perguruan tinggi agama Islam yang berhasil mengintegrasikan perencanaan anggaran dengan tujuan-tujuan strategisnya memiliki kecenderungan yang lebih tinggi untuk mencapai pencapaian yang diinginkan. Dengan kata lain, ketika perencanaan anggaran diarahkan secara langsung menuju pencapaian tujuan strategis institusi, proses perencanaan anggaran tersebut menjadi lebih efektif dan menghasilkan dampak yang lebih positif bagi institusi tersebut (Glavina et al., 2021). Integrasi antara perencanaan anggaran dan tujuan strategis memungkinkan institusi untuk mengalokasikan sumber daya secara lebih efisien dan efektif sesuai dengan prioritas-prioritas strategis yang telah ditetapkan. Hal ini membantu institusi dalam mengoptimalkan penggunaan dana yang terbatas serta memastikan bahwa setiap investasi keuangan mendukung pencapaian tujuan-tujuan strategis jangka panjang institusi (Tok & Yesuf, 2022). Dalam konteks manajemen keuangan di perguruan tinggi agama Islam, integrasi antara perencanaan anggaran dan tujuan strategis juga memainkan peran penting dalam memperkuat arah dan identitas institusi. Dengan memastikan bahwa setiap keputusan keuangan didasarkan pada pertimbangan yang terkait dengan visi, misi, dan nilai-nilai institusi, integrasi ini membantu menciptakan keselarasan antara praktik keuangan dan identitas lembaga (Wijayanti & Setiawan, 2022). Dengan demikian, integrasi antara perencanaan anggaran dan tujuan strategis tidak hanya meningkatkan efektivitas perencanaan anggaran, tetapi juga mengarah pada penciptaan nilai tambah yang signifikan bagi institusi pendidikan tinggi agama Islam. Oleh karena itu, pemahaman dan implementasi integrasi ini merupakan langkah penting dalam memperkuat kinerja keuangan dan pencapaian tujuan strategis institusi. Hasil Faktor-Faktor yang Mempengaruhi Efektivitas Perencanaan Anggaran Faktor-faktor yang memengaruhi efektivitas perencanaan anggaran di perguruan tinggi agama Islam merupakan topik yang telah diperdebatkan dalam literatur. Penelitian sebelumnya menyoroti beberapa aspek kunci yang berperan dalam menentukan kesuksesan perencanaan anggaran di institusi tersebut. Salah satu aspek utama yang disoroti adalah aspek keuangan, yang melibatkan alokasi dana yang efisien dan tepat sasaran. Studi oleh Alhammadi (2022) menekankan pentingnya pengelolaan keuangan yang baik dalam konteks perencanaan anggaran di perguruan tinggi agama Islam. Selain aspek keuangan, aspek manajerial juga memainkan peran penting dalam efektivitas perencanaan anggaran. Pengelolaan sumber daya manusia yang efisien, pengambilan keputusan yang tepat, dan struktur organisasi yang jelas merupakan faktor-faktor yang dapat memengaruhi kinerja perencanaan anggaran. Studi- studi terdahulu telah menunjukkan bahwa proses manajerial yang baik dapat meningkatkan efektivitas perencanaan anggaran di institusi pendidikan tinggi agama Islam (Menne et al., 2022). Selanjutnya, aspek strategis juga menjadi fokus utama dalam memahami efektivitas perencanaan anggaran. Integrasi antara perencanaan anggaran dengan visi, misi, dan tujuan strategis institusi menjadi kunci 1033 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. penting dalam mencapai keberhasilan. Penelitian literatur menunjukkan bahwa perguruan tinggi agama Islam yang berhasil mengintegrasikan perencanaan anggaran dengan tujuan-tujuan strategisnya cenderung lebih sukses dalam mencapai pencapaian yang diinginkan (Mujiatun et al., 2023). Dalam konteks faktor-faktor yang memengaruhi efektivitas perencanaan anggaran, pemahaman mendalam tentang prinsip-prinsip Islam juga memainkan peran krusial. Rahahleh et al., (2019) menyoroti pentingnya memahami prinsip-prinsip keuangan Islam, seperti keadilan, transparansi, dan penghindaran riba, dalam merancang perencanaan anggaran yang sesuai dengan nilai-nilai agama. Integrasi prinsip-prinsip Islam ini dapat membantu memperkuat integritas dan keberlanjutan perencanaan anggaran di perguruan tinggi agama Islam. g Dengan memperhatikan faktor-faktor ini, institusi pendidikan tinggi agama Islam dapat mengembangkan strategi perencanaan anggaran yang lebih efektif dan sesuai dengan nilai-nilai mereka. Integrasi yang kuat antara aspek keuangan, manajerial, strategis, dan nilai-nilai agama dapat membantu meningkatkan kualitas perencanaan anggaran di institusi tersebut. Pemahaman atas Prinsip-Prinsip Islam dalam Konteks Keuangan Pemahaman yang mendalam tentang prinsip-prinsip Islam dalam konteks keuangan memainkan peran krusial dalam perencanaan anggaran di perguruan tinggi agama Islam. Studi yang dilakukan oleh Saleh et al., (2023) menyoroti pentingnya memperhatikan prinsip-prinsip Islam, seperti keadilan, transparansi, dan penghindaran riba, dalam merancang perencanaan anggaran yang sesuai dengan nilai-nilai agama. Aspek keadilan dalam konteks keuangan Islam menekankan pentingnya adil dalam alokasi dan pengelolaan dana. Prinsip ini menuntut bahwa setiap keputusan keuangan harus memperhatikan kebutuhan dan kepentingan semua pihak yang terlibat, termasuk mahasiswa, staf, dan masyarakat yang dilayani oleh perguruan tinggi agama Islam. Transparansi merupakan aspek lain yang sangat diperhatikan dalam prinsip-prinsip keuangan Islam. Prinsip ini menekankan pentingnya keterbukaan dan akuntabilitas dalam pengelolaan dana institusi. Transparansi memastikan bahwa setiap pengeluaran dan penerimaan dana dapat dipertanggungjawabkan secara jelas kepada seluruh pemangku kepentingan. 1034 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. Penghindaran riba, atau bunga dalam konsep keuangan Islam, menjadi prinsip yang mendasar dalam merancang perencanaan anggaran di perguruan tinggi agama Islam. Prinsip ini menuntut bahwa institusi harus menghindari praktik keuangan yang bertentangan dengan prinsip-prinsip keuangan Islam, termasuk penggunaan bunga dalam pinjaman atau investasi. Dengan memahami dan mengimplementasikan prinsip-prinsip Islam dalam perencanaan anggaran, perguruan tinggi agama Islam dapat memastikan bahwa kegiatan keuangan mereka sejalan dengan nilai-nilai agama dan prinsip-prinsip yang diberlakukan dalam Islam. Hal ini tidak hanya meningkatkan integritas institusi, tetapi juga memperkuat ikatan antara praktik keuangan dan identitas religius institusi tersebut. Oleh karena itu, pemahaman yang mendalam tentang prinsip-prinsip Islam dalam konteks keuangan merupakan hal yang penting dan relevan dalam merancang perencanaan anggaran di perguruan tinggi agama Islam. Peran Teknologi Informasi dalam Perencanaan Anggaran Perkembangan teknologi informasi telah membawa dampak signifikan dalam proses perencanaan anggaran di perguruan tinggi agama Islam. Penelitian yang dilakukan oleh Nugraha et al. (2022) menyoroti peran penting sistem informasi keuangan dan teknologi terkait dalam meningkatkan efektivitas perencanaan anggaran di institusi-institusi tersebut. Pemanfaatan teknologi informasi tidak hanya membawa keuntungan operasional, tetapi juga memberikan manfaat yang besar dalam hal transparansi, akuntabilitas, dan efisiensi. Salah satu manfaat utama teknologi informasi dalam perencanaan anggaran adalah peningkatan transparansi. Dengan adanya sistem informasi keuangan yang terintegrasi, informasi tentang penggunaan dana dapat diakses dengan mudah oleh pihak-pihak yang berkepentingan, seperti pimpinan, dewan pengawas, dan masyarakat umum. Hal ini membantu meningkatkan tingkat transparansi dan memastikan bahwa penggunaan dana dilakukan dengan penuh akuntabilitas. Selain itu, teknologi informasi juga memperkuat akuntabilitas dalam proses perencanaan anggaran. Melalui sistem informasi keuangan yang terotomatisasi, institusi dapat melacak setiap transaksi keuangan dengan rinci dan menghasilkan laporan keuangan secara berkala. Dengan demikian, tanggung jawab atas pengelolaan anggaran menjadi lebih terstruktur dan dapat dipertanggungjawabkan dengan lebih baik. Pemanfaatan teknologi informasi juga meningkatkan efisiensi dalam pengelolaan anggaran. Automatisasi proses-proses administratif, seperti pengajuan dan persetujuan anggaran, dapat mengurangi waktu dan biaya yang diperlukan dalam proses tersebut. Selain itu, integrasi antara sistem informasi keuangan dengan sistem- sistem lain, seperti manajemen inventaris dan sumber daya manusia, memungkinkan pengambilan keputusan yang lebih cepat dan akurat. Secara keseluruhan, peran teknologi informasi dalam perencanaan anggaran di perguruan tinggi agama Islam tidak dapat diabaikan. Dengan memanfaatkan teknologi informasi dengan bijaksana, institusi dapat meningkatkan transparansi, akuntabilitas, dan efisiensi dalam pengelolaan anggaran mereka. Hal ini tidak hanya menghasilkan manfaat operasional, tetapi juga memperkuat integritas dan reputasi institusi di mata masyarakat. Keterlibatan Pihak-Pihak Terkait dalam Proses Perencanaan Anggaran Keterlibatan pihak-pihak terkait memainkan peran krusial dalam memastikan keberhasilan proses perencanaan anggaran di perguruan tinggi agama Islam. Studi yang dilakukan oleh Abbas et al., (2019) menekankan pentingnya keterlibatan aktif dari berbagai pihak di dalam institusi, termasuk pimpinan, staf administratif, dan akademisi, dalam merancang dan melaksanakan perencanaan anggaran yang efektif. Pimpinan institusi memiliki peran utama dalam menetapkan arah dan prioritas dalam perencanaan anggaran. Keterlibatan mereka dalam proses perencanaan anggaran memastikan bahwa kebijakan dan tujuan strategis institusi tercermin dalam alokasi dana yang tepat. Selain itu, dukungan dan komitmen dari pimpinan juga penting untuk memastikan bahwa perencanaan anggaran mendapat sumber daya dan dukungan yang cukup. p Keterlibatan staf administratif juga sangat penting dalam proses perencanaan anggaran. Mereka memiliki pengetahuan yang luas tentang operasi harian institusi dan kebutuhan keuangan yang terkait. Melalui kolaborasi yang baik antara staf administratif dan pimpinan, perencanaan anggaran dapat disusun dengan mempertimbangkan semua aspek yang relevan dan memastikan bahwa sumber daya dialokasikan secara efisien. 1035 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. Selain itu, keterlibatan akademisi juga penting dalam memastikan bahwa perencanaan anggaran mencerminkan kebutuhan dan prioritas akademik institusi. Mereka memiliki wawasan yang penting tentang program-program akademik dan kegiatan pendidikan lainnya yang memerlukan dukungan keuangan. Melalui partisipasi aktif akademisi dalam proses perencanaan anggaran, kebutuhan akademik dapat diperhitungkan dengan baik dan dialokasikan sesuai. g Kolaborasi dan komunikasi yang baik antara berbagai departemen dan unit di perguruan tinggi agama Islam juga merupakan faktor kunci dalam memastikan keberhasilan perencanaan anggaran. Koordinasi yang efektif antara berbagai pihak memungkinkan informasi dan masukan yang relevan dapat dipertukarkan dengan lancar, sehingga keputusan perencanaan anggaran dapat dibuat secara lebih baik dan berdasarkan pada pemahaman yang komprehensif. Dengan demikian, keterlibatan aktif dari berbagai pihak di dalam institusi merupakan faktor penting dalam memastikan perencanaan anggaran yang berhasil. Kolaborasi dan komunikasi yang baik antara pimpinan, staf administratif, dan akademisi membantu memastikan bahwa perencanaan anggaran mencerminkan visi, misi, dan kebutuhan sehari-hari dari perguruan tinggi agama Islam tersebut. Pengaruh Konteks Eksternal terhadap Perencanaan Anggaran Pengaruh konteks eksternal, termasuk regulasi pemerintah dan kondisi ekonomi, memainkan peran penting dalam proses perencanaan anggaran di perguruan tinggi agama Islam, seperti yang disoroti dalam penelitian literatur oleh Menne et al., (2023). Regulasi pemerintah yang berkaitan dengan pendanaan dan kebijakan pendidikan dapat mempengaruhi cara institusi merencanakan, mengalokasikan, dan menggunakan anggaran mereka. Misalnya, perubahan kebijakan pemerintah terkait dana pendidikan atau bantuan keuangan dapat memaksa perguruan tinggi untuk menyesuaikan rencana anggaran mereka agar sesuai dengan persyaratan baru yang diberlakukan. Di samping itu, kondisi ekonomi yang berubah-ubah juga dapat berdampak signifikan pada perencanaan anggaran institusi. Ketika kondisi ekonomi sedang tidak stabil, institusi pendidikan tinggi agama Islam mungkin menghadapi tantangan tambahan dalam mengelola dan mengalokasikan sumber daya keuangannya. Perencanaan anggaran yang responsif terhadap kondisi ekonomi eksternal ini dapat membantu institusi untuk mengelola risiko keuangan dan menjaga stabilitas keuangan mereka dalam jangka panjang. Pemahaman yang baik tentang konteks eksternal, termasuk regulasi pemerintah dan kondisi ekonomi, memungkinkan institusi untuk mengambil keputusan perencanaan anggaran yang lebih informan dan adaptif. Dengan memantau perubahan dalam lingkungan eksternal mereka, institusi dapat mengidentifikasi peluang dan ancaman potensial yang mungkin mempengaruhi keberhasilan perencanaan anggaran mereka. Sebagai hasilnya, mereka dapat mengadaptasi strategi perencanaan anggaran mereka agar tetap relevan dan efektif dalam menghadapi dinamika eksternal yang terus berubah. Dalam konteks ini, penelitian literatur menunjukkan bahwa institusi pendidikan tinggi agama Islam yang memiliki pemahaman yang mendalam tentang dinamika konteks eksternal mereka cenderung lebih siap dan mampu untuk merespons perubahan-perubahan tersebut. Oleh karena itu, pengelolaan perencanaan anggaran yang efektif tidak hanya memperhitungkan faktor-faktor internal institusi, tetapi juga memperhatikan pengaruh dan tuntutan dari lingkungan eksternal mereka. Dengan memperhatikan temuan-temuan tersebut, institusi pendidikan tinggi agama Islam dapat memperbaiki praktik perencanaan anggaran mereka untuk mencapai tujuan strategis yang lebih efektif. Pembahasan Faktor-faktor yang memengaruhi efektivitas perencanaan anggaran di perguruan tinggi agama Islam merupakan topik yang telah diperdebatkan dalam literatur. Studi sebelumnya menyoroti beberapa aspek kunci yang berperan dalam menentukan kesuksesan perencanaan anggaran di institusi tersebut. Salah satu aspek utama yang disoroti adalah aspek keuangan, yang melibatkan alokasi dana yang efisien dan tepat sasaran. Studi oleh Alhammadi (2022) menekankan pentingnya pengelolaan keuangan yang baik dalam konteks perencanaan anggaran di perguruan tinggi agama Islam. Selain aspek keuangan, aspek manajerial juga memainkan peran penting dalam efektivitas perencanaan anggaran. Pengelolaan sumber daya manusia yang efisien, pengambilan keputusan yang tepat, dan struktur organisasi yang jelas merupakan faktor-faktor yang dapat memengaruhi kinerja perencanaan anggaran. Studi-studi terdahulu telah menunjukkan bahwa proses manajerial yang baik dapat meningkatkan efektivitas perencanaan anggaran di institusi pendidikan tinggi agama Islam (Menne et al., 2022). 1036 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. Selanjutnya, aspek strategis juga menjadi fokus utama dalam memahami efektivitas perencanaan anggaran. Integrasi antara perencanaan anggaran dengan visi, misi, dan tujuan strategis institusi menjadi kunci penting dalam mencapai keberhasilan. Penelitian literatur menunjukkan bahwa perguruan tinggi agama Islam yang berhasil mengintegrasikan perencanaan anggaran dengan tujuan-tujuan strategisnya cenderung lebih sukses dalam mencapai pencapaian yang diinginkan (Mujiatun et al., 2023). Dalam konteks faktor-faktor yang memengaruhi efektivitas perencanaan anggaran, pemahaman mendalam tentang prinsip-prinsip Islam juga memainkan peran krusial. Rahahleh et al., (2019) menyoroti pentingnya memahami prinsip-prinsip keuangan Islam, seperti keadilan, transparansi, dan penghindaran riba, dalam merancang perencanaan anggaran yang sesuai dengan nilai-nilai agama. Dengan memperhatikan faktor-faktor ini, institusi pendidikan tinggi agama Islam dapat mengembangkan strategi perencanaan anggaran yang lebih efektif dan sesuai dengan nilai-nilai mereka. Integrasi yang kuat antara aspek keuangan, manajerial, strategis, dan nilai-nilai agama dapat membantu meningkatkan kualitas perencanaan anggaran di institusi tersebut. Pengaruh konteks eksternal, termasuk regulasi pemerintah dan kondisi ekonomi, juga memainkan peran penting dalam proses perencanaan anggaran di perguruan tinggi agama Islam, seperti yang disoroti dalam penelitian literatur oleh Menne et al., (2023). Regulasi pemerintah yang berkaitan dengan pendanaan dan kebijakan pendidikan dapat mempengaruhi cara institusi merencanakan, mengalokasikan, dan menggunakan anggaran mereka. Misalnya, perubahan kebijakan pemerintah terkait dana pendidikan atau bantuan keuangan dapat memaksa perguruan tinggi untuk menyesuaikan rencana anggaran mereka agar sesuai dengan persyaratan baru yang diberlakukan. Di samping itu, kondisi ekonomi yang berubah-ubah juga dapat berdampak signifikan pada perencanaan anggaran institusi. Pembahasan Ketika kondisi ekonomi sedang tidak stabil, institusi pendidikan tinggi agama Islam mungkin menghadapi tantangan tambahan dalam mengelola dan mengalokasikan sumber daya keuangannya. Perencanaan anggaran yang responsif terhadap kondisi ekonomi eksternal ini dapat membantu institusi untuk mengelola risiko keuangan dan menjaga stabilitas keuangan mereka dalam jangka panjang. Pemahaman yang baik tentang konteks eksternal, termasuk regulasi pemerintah dan kondisi ekonomi, memungkinkan institusi untuk mengambil keputusan perencanaan anggaran yang lebih informan dan adaptif. Dengan memantau perubahan dalam lingkungan eksternal mereka, institusi dapat mengidentifikasi peluang dan ancaman potensial yang mungkin mempengaruhi keberhasilan perencanaan anggaran mereka. Sebagai hasilnya, mereka dapat mengadaptasi strategi perencanaan anggaran mereka agar tetap relevan dan efektif dalam menghadapi dinamika eksternal yang terus berubah. Dalam konteks ini, penelitian literatur menunjukkan bahwa institusi pendidikan tinggi agama Islam yang memiliki pemahaman yang mendalam tentang dinamika konteks eksternal mereka cenderung lebih siap dan mampu untuk merespons perubahan-perubahan tersebut. Oleh karena itu, pengelolaan perencanaan anggaran yang efektif tidak hanya memperhitungkan faktor-faktor internal institusi, tetapi juga memperhatikan pengaruh dan tuntutan dari lingkungan eksternal mereka. Dengan memperhatikan temuan-temuan tersebut, institusi pendidikan tinggi agama Islam dapat memperbaiki praktik perencanaan anggaran mereka untuk mencapai tujuan strategis yang lebih efektif. Kesimpulan Institusi yang dapat menyesuaikan diri dengan perubahan dalam lingkungan eksternal memiliki kesempatan yang lebih baik untuk menjaga stabilitas keuangan dan mencapai tujuan strategis mereka. Dalam rangka memperkuat praktik perencanaan anggaran di perguruan tinggi agama Islam, penting bagi institusi untuk memperhatikan semua faktor yang telah dibahas secara komprehensif. Integrasi, pemahaman prinsip-prinsip Islam, pemanfaatan teknologi informasi, keterlibatan pihak-pihak terkait, dan respons terhadap konteks eksternal merupakan langkah-langkah kunci dalam merancang dan melaksanakan perencanaan anggaran yang efektif dan sesuai dengan nilai-nilai serta tujuan institusi. Dengan demikian, praktik perencanaan anggaran yang kokoh dan adaptif akan membantu perguruan tinggi agama Islam untuk mencapai keseimbangan yang optimal antara keberlanjutan keuangan dan pencapaian tujuan strategis jangka panjang mereka. Kesimpulan Dari paparan mengenai faktor-faktor yang memengaruhi efektivitas perencanaan anggaran di perguruan tinggi agama Islam, serta integrasi antara perencanaan anggaran dengan tujuan strategis, pemahaman atas prinsip-prinsip Islam dalam konteks keuangan, peran teknologi informasi, keterlibatan pihak- pihak terkait, dan pengaruh konteks eksternal, beberapa simpulan penting dapat ditarik. 1. Integrasi antara perencanaan anggaran dan tujuan strategis institusi merupakan langkah penting untuk mencapai efektivitas perencanaan anggaran. Institusi yang mampu mengalokasikan sumber daya secara efisien sesuai dengan visi, misi, dan tujuan strategisnya memiliki peluang lebih besar untuk mencapai keberhasilan. 1. Integrasi antara perencanaan anggaran dan tujuan strategis institusi merupakan langkah penting untuk mencapai efektivitas perencanaan anggaran. Institusi yang mampu mengalokasikan sumber daya secara efisien sesuai dengan visi, misi, dan tujuan strategisnya memiliki peluang lebih besar untuk mencapai keberhasilan. 2. Pemahaman yang mendalam tentang prinsip-prinsip Islam dalam konteks keuangan memberikan landasan yang kuat bagi perencanaan anggaran yang sesuai dengan nilai-nilai agama. Prinsip-prinsip seperti keadilan, transparansi, dan penghindaran riba menjadi pedoman yang penting dalam merancang perencanaan anggaran yang konsisten dengan identitas institusi. g p gg y g g 3. Pemanfaatan teknologi informasi memiliki peran signifikan dalam meningkatkan transparansi, akuntabilitas, dan efisiensi dalam pengelolaan anggaran. Institusi yang mampu memanfaatkan 3. Pemanfaatan teknologi informasi memiliki peran signifikan dalam meningkatkan transparansi, akuntabilitas, dan efisiensi dalam pengelolaan anggaran. Institusi yang mampu memanfaatkan 1037 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. , Volume 10 (2) April Tahun 2024, Hal 1032-10 teknologi informasi dengan baik dapat meningkatkan kualitas dan responsivitas perencanaan anggaran mereka. teknologi informasi dengan baik dapat meningkatkan kualitas dan responsivitas perencan anggaran mereka. 4. Keterlibatan aktif dari berbagai pihak terkait, termasuk pimpinan, staf administratif, akademisi, dan departemen lainnya, merupakan faktor penting dalam memastikan bahwa perencanaan anggaran mencerminkan kebutuhan dan prioritas institusi secara menyeluruh. 4. Keterlibatan aktif dari berbagai pihak terkait, termasuk pimpinan, staf administratif, akademisi, dan departemen lainnya, merupakan faktor penting dalam memastikan bahwa perencanaan anggaran mencerminkan kebutuhan dan prioritas institusi secara menyeluruh. 5. Pengaruh konteks eksternal, seperti regulasi pemerintah dan kondisi ekonomi, dapat mempengaruhi strategi perencanaan anggaran institusi. Institusi yang dapat menyesuaikan diri dengan perubahan dalam lingkungan eksternal memiliki kesempatan yang lebih baik untuk menjaga stabilitas keuangan dan mencapai tujuan strategis mereka. 5. Pengaruh konteks eksternal, seperti regulasi pemerintah dan kondisi ekonomi, dapat mempengaruhi strategi perencanaan anggaran institusi. Referensi Abbas, J., I. Hussain, S. Hussain, S. Akram, I. Shaheen, and B. Niu. "The Impact of Knowledge Sharing and Innovation on Sustainable Performance in Islamic Banks: A Mediation Analysis through a SEM Approach." Sustainability 11, no. 15 (2019): 4049. https://doi.org/10.3390/su11154049. Al Rahahleh, N., M. Ishaq Bhatti, and F. Najuna Misman. "Developments in Risk Management in Islamic Finance: A Review." Journal of Risk and Financial Management 12, no. 1 (2019): 37. https://doi.org/10.3390/jrfm12010037. Alhammadi, S. "Analyzing the Role of Islamic Finance in Kuwait Regarding Sustainable Economic Development in COVID-19 Era." Sustainability 14, no. 2 (2022): 701. https://doi.org/10.3390/su14020701. Gandrita, D. M. "Improving Strategic Planning: The Crucial Role of Enhancing Relationships between Management Levels." Administrative Sciences 13, no. 10 (2023): 211. https://doi.org/10.3390/admsci13100211. Ginting, A. H., I. Widianingsih, R. Mulyawan, and H. Nurasa. "Village Government’s Risk Management and Village Fund Administration in Indonesia." Sustainability 15, no. 24 (2023): 16706. https://doi.org/10.3390/su152416706. Glavina, S., I. Aidrus, and A. Trusova. "Assessment of the Competitiveness of Islamic Fintech Implementation: A Composite Indicator for Cross-Country Analysis." Journal of Risk and Financial Management 14, no. 12 (2021): 602. https://doi.org/10.3390/jrfm14120602. Joudar, F., Z. Msatfa, O. Metwalli, M. Mouabid, and B. Dinar. "Islamic Financial Stability Factors: An Econometric Evidence." Economies 11, no. 3 (2023): 79. https://doi.org/10.3390/economies11030079. Menne, F., S. Mardjuni, M. Yusuf, M. Ruslan, A. Arifuddin, and I. Iskandar. "Sharia Economy, Islamic Financial Performance and Factors That Influence It—Evidence from Indonesia." Economies 11, no. 4 (2023): 111. https://doi.org/10.3390/economies11040111. Mohamad Saleh, M. S., A. Mehellou, and B. Omar. "The Influence of Islamic Values on Sustainable Lifestyle: The Moderating Role of Opinion Leaders." Sustainability 15, no. 11 (2023): 8640. https://doi.org/10.3390/su15118640. 1038 JEMSI (Jurnal Ekonomi, Manajemen, dan Akuntansi) E-ISSN: 2579-5635, P-ISSN: 2460-5891 Volume 10 (2) April Tahun 2024, Hal 1032-1039. Mujiatun, S., B. Trianto, E. F. Cahyono, and Rahmayati. "The Impact of Marketing Communication and Islamic Financial Literacy on Islamic Financial Inclusion and MSMEs Performance: Evidence from Halal Tourism in Indonesia." Sustainability 15, no. 13 (2023): 9868. https://doi.org/10.3390/su15139868. Nugraha, K., M. Arief, S. B. Abdinagoro, and P. Heriyati. "Factors Influencing Bank Customers’ Orientations toward Islamic Banks: Indonesian Banking Perspective." Sustainability 14, no. 19 (2022): 12506. https://doi.org/10.3390/su141912506. Poon, J., Y. W. Chow, M. Ewers, and R. Ramli. "The Role of Skills in Islamic Financial Innovation: Evidence from Bahrain and Malaysia." Journal of Open Innovation: Technology, Market, and Complexity 6, no. 3 (2020): 47. https://doi.org/10.3390/joitmc6030047. Qudah, H., S. Malahim, R. Airout, M. Alomari, A. A. Hamour, and M. Referensi Alqudah. "Islamic Finance in the Era of Financial Technology: A Bibliometric Review of Future Trends." International Journal of Financial Studies 11, no. 2 (2023): 76. https://doi.org/10.3390/ijfs11020076. Tok, E., A. J. Yesuf, and A. Mohamed. "Sustainable Development Goals and Islamic Social Finance: From Policy Divide to Policy Coherence and Convergence." Sustainability 14, no. 11 (2022): 6875. https://doi.org/10.3390/su14116875. Tok, E., and A. J. Yesuf. "Embedding Value-Based Principles in the Culture of Islamic Banks to Enhance Their Sustainability, Resilience, and Social Impact." Sustainability 14, no. 2 (2022): 916. https://doi.org/10.3390/su14020916. Wijayanti, R., and D. Setiawan. "Social Reporting by Islamic Banks: The Role of Sharia Supervisory Board and the Effect on Firm Performance." Sustainability 14, no. 17 (2022): 10965. https://doi.org/10.3390/su141710965. 1039
https://openalex.org/W4381510028	https://scipost.org/10.21468/SciPostPhys.14.6.165/pdf	English	null	Nonexistence of motility induced phase separation transition in one dimension	SciPost physics	2,023	cc-by	7,298	Abstract We introduce and study a model of hardcore particles obeying run-and-tumble dynamics on a one-dimensional lattice, where particles run in either +ve or −ve x-direction with an effective speed v and tumble (change their direction of motion) with a constant rate ω when assisted by another particle from right. We show that the coarse-grained dynamics of the system can be mapped to a beads-in-urn model called misanthrope process where particles are identified as urns and vacancies as beads that hop to a neighbouring urn situated in the direction opposite to the current. The hop rate is same as the magnitude of the particle current; we calculate it analytically for a two-particle system and show that it does not satisfy the criteria required for a phase separation transition. Nonexistence of phase separation in this model, where tumbling dynamics is rather restricted, necessarily imply that motility induced phase separation transition can not occur in other models in one dimension with unconditional tumbling. Copyright I. Mukherjee et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. Received 12-09-2022 Accepted 18-04-2023 Published 21-06-2023 Check for updates doi:10.21468/SciPostPhys.14.6.165 Contents 1 Introduction 2 2 The restricted tumbling model 3 3 Mapping of RTM to a beads-in-urn model 3 4 Summary 8 A Appendix 9 References 11 Received 12-09-2022 Accepted 18-04-2023 Published 21-06-2023 Check for updates doi:10.21468/SciPostPhys.14.6.165 Received 12-09-2022 Accepted 18-04-2023 Published 21-06-2023 Check for updates doi:10.21468/SciPostPhys.14.6.165 Copyright I. Mukherjee et al. This work is licensed under the Creative Commons Attribution 4.0 International License. Published by the SciPost Foundation. SciPost Phys. 14, 165 (2023) Indranil Mukherjee, Adarsh Raghu and Pradeep Kumar Mohanty⋆ Indranil Mukherjee, Adarsh Raghu and Pradeep Kumar Mohanty⋆ Department of Physical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246 India Department of Physical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246 India ⋆pkmohanty@iiserkol.ac.in 1 Introduction An important class of nonequilibrium systems is that of active matter systems (AMS) [1] where the individual constituents are self-propelled; instances of such systems include bird flocks [2], bacterial colonies [3], photophoretic colloidal suspensions [4] and actin filaments [5] etc. They exhibit a number or interesting features like large number-fluctuations [1], clustering and pattern formation [4]. A major area of interest in the study of AMS has been the so-called motility-induced phase separation (MIPS) [6–13] which refers to spatially separated high and low density regimes. Such aggregation or clustering of particles has been observed experimen- tally in many active matter systems [4]. Relevance of the aggregation process has also been proposed as a mechanism of formation bacterial biofilms [3], which are sources of infection. Occurrence of MIPS relies on an argument that effective velocity of active particles de- crease in crowded or high density regions formed either by explicit dependence of local den- sity or merely by exclusion. Naturally such a slowing down of movement further increases the density of particles and gives rise to a feedback loop allowing the stable high density (liquid- like) regions to form and coexist with a low density (gas-like) phase elsewhere. MIPS has been widely investigated in simulations and apparent phase separation has been observed. Theoretical investigations of this phenomenon have thus far concentrated on continuum mod- els [8–10] where motility parameters, such as particle flux or velocity are characterized as functions of the coarse-grained local density [6, 7]. Lattice models of active particles have been studied in one and two dimensions numerically [14–16] with run and tumble particles (RTPs). RTPs move at a fixed speed along the direction of their orientation (a run) until they tumble and change their orientation. In one dimension (1D), the two orientations (say, ±) are usually referred to as the internal degrees of the particle (spin), which flips with a certain rate. Analytical studies of these lattice models are limited. Thompson et. al. [13] have in- troduced a model of self propelled particles with RTP dynamics; in 1D. These models exhibit inhomogeneous density profiles when particle velocities depend on their position. Recently Slowman et. al. [17, 18] have obtained an exact solution for two RTPs and found jamming induced attraction between the particles of the opposite spins, which indicates that, for many particle systems, a phase separated state might originate from these attractive interactions. Later, Dandekar et. al. Contents 1 Introduction 2 2 The restricted tumbling model 3 3 Mapping of RTM to a beads-in-urn model 3 4 Summary 8 A Appendix 9 References 11 1 Introduction 2 2 The restricted tumbling model 3 3 Mapping of RTM to a beads-in-urn model 3 4 Summary 8 A Appendix 9 References 11 11 1 SciPost Phys. 14, 165 (2023) 2 The restricted tumbling model We introduce a generic model of RTPs on an one dimensional periodic lattice with sites labeled by i = 1,2,... L. The sites are either empty (represented by τi = 0) or occupied by at most one RTP τi = ± having orientation (spin) ±. Particles follow a run dynamics, +0 p+ ⇋ q+ 0+; −0 p− ⇋ q−0−, (1) (1) (1) where RTPs move forward or backward with rates p± and q± respectively. Along with this, they can tumble and change their spin with rate ω as follows, where RTPs move forward or backward with rates p± and q± respectively. Along with this, they can tumble and change their spin with rate ω as follows, + ± ω →−±; −± ω →+ ± . (2) (2) Tumbling is restricted here in the sense that only those particles which are assisted from right by other particles can tumble their direction. This restriction helps us getting an approximate steady state of the system without tampering the main aim: the proposition that a stable MIPS state can not be sustained in 1D. Since frequent tumbling of particles helps the system to clear jamming, a proof of nonexistence of MIPS in our model necessarily guarantees its nonexistence in any other model that has more liberal tumbling dynamics. Hereafter we refer to the model following dynamics (1) and (2) as restricted tumbling model (RTM).i Although RTM is defined for generic rates (p±,q±) we study the case p± = q∓where the run dynamics exhibit a symmetry transformation, namely simultaneous interchange of par- ity (left ⇋right) and spin (+ ⇋−), that keeps the dynamics invariant. This symmetry was present for both run- and tumble-dynamics in 1D lattice models studied earlier [17,19]. When p± = q∓, it is also ensured that in the limit when lattice spacing vanishes [25], a single par- ticle dynamics of RTM reduces to that of a RTP moving in continuum space with same speed v = p−−q−= q+−p+ along +ve and −ve x-directions. Note that, under parity transformation (left ⇋right) the tumbling dynamics of our model is modified as tumbling now occurs for only those particles which are assisted by other particles from left. But, for p± = q∓, a left-assisted tumbling dynamics leads to the same steady state as the right-assisted tumbling. 2 The restricted tumbling model This can be verified easily from the exact mapping of these models to the corresponding beads-in-urn models (see later discussions). A special case of RTM with p+ = α = q−, p−= 0 = q+ and unrestricted tumbling dynamics + ω ⇋ ω −was studied earlier by Slowman et. al. [17] and an exact steady state solution was obtained for a system of two RTPs. It turned out that these two particles experience an effective attractive interaction in the steady state when their spins are opposite; it is envisaged that this attraction might be the source of MIPS states observed in corresponding hydrodynamic models. In comparison, in Eq. (2) we have dropped one of the transition +0 ω ⇋ ω −0; as a consequence, particles do not tumble if they are not assisted by a right neighbour. 1 Introduction [19] have obtained a mean-field solution of RTPs in 1D which turned out to be a good approximation when tumbling rate is large. An element of surprise in the formation of a phase separated state without any explicit attractive interaction has generated much excitement to the study of MIPS and raised questions about the stability of such states in 1D in absence of any explicit interaction or spatial potential. Recent works have added to the doubt by showing that MIPS phase transition in 2D belongs to the Ising universality class [20–22] which does not have a counterpart in one dimension. In this article we argue and show explicitly using 1D lattice models of RTPs that indeed MIPS transition can not occur in 1D; the inhomogeneous states observed in numerical simulations and in hydrodynamic models are only long lived transient states. First we introduce a generic model of hardcore RTPs in 1D with a restricted tumbling dynamics and show that its coarse-grained dynamics can be mapped to a beads-in-urn model, namely a misanthrope process [23, 24] where beads hop to their neighbouring urn, situated in the opposite direction of the particle current, with a rate same as the magnitude of current. The functional form of hop rate is determined from the exact steady state results of the model with only two RTPs. To determine if MIPS transition is possible, we use the following criterion. If a system of hardcore particles phase separates as its density ρ crosses a threshold ρ∗then the maximum density at which it remains homogeneous is ρ∗. Since systems with homogeneous densities are well described in the grand canonical ensemble (GCE) by a unique chemical potential µ (or fugacity z = eµ), we argue that phase separation transition is possible in a 2 SciPost Phys. 14, 165 (2023) system when its density in GCE attains a maximum value ρ∗= Max[ρ(z)] which is less than unity (the density of a fully occupied lattice). Nonexistence of MIPS transition in restricted tumbling model would imply that MIPS can not occur in any other RTP model in 1D where tumbling occurs more frequently. 3 Mapping of RTM to a beads-in-urn model Any microscopic configurations {τi} of RTM can be viewed as urns containing beads –each particle is an urn that contains beads which are uninterrupted sequence of 0s (vacancies) to 3 SciPost Phys. 14, 165 (2023) … + 0 0 – + 0 0 0 – 0 + 0 – 0 0 – ... … + – + – + – – ... p+ q- … ... u(3,1) u(1,2) (a) + w w u(2,0) J(2,0) … + – + – + – – ... (b) J(3,1) J(1,2) Figure 1: (a) Mapping lattice model of RTPs to an urn model. (b) Effective coarse- grained dynamics: hop rate of a bead u(mk, mk+1) from urn k (with mk particles) to k + 1 (with mk+1 particles) is assumed to be same as the local bead current J(mk, mk+1) averaged over internal degrees σk,σk+1. Figure 1: (a) Mapping lattice model of RTPs to an urn model. (b) Effective coarse- grained dynamics: hop rate of a bead u(mk, mk+1) from urn k (with mk particles) to k + 1 (with mk+1 particles) is assumed to be same as the local bead current J(mk, mk+1) averaged over internal degrees σk,σk+1. the right of the particle (as described in Fig. 1(a)). The spin ± of the particle is termed as the internal degree of the urn. Thus we have a beads-in-urn model of N urns indexed by k = 1,2,... N, each carrying an internal degree σk = ± and mk = 0,1,2... beads. The dynamics (1) and (2) now translate to hopping of a bead from urn k to k +1 (k −1) with rate qσk+1 (pσk), and flipping of internal degrees σk →−σk with rate ωδmk,0. The total number of beads PN k=1 mk = L −N ≡M is conserved by the dynamics. Like particle density ρ = N L , the bead density η = M N = 1−ρ ρ is also conserved. ρ Note that in this beads-in-urn model the internal degrees of the urns can flip only when they are empty; this restriction forces k-th urn either to transfer a bead (when mk > 0)or to change the internal degrees (when mk = 0) and help us getting an exact steady state. 3 Mapping of RTM to a beads-in-urn model To calculate ρ(z) we need functional form of J(m1, m2) which can be calculated exactly using matrix product ansatz (MPA) [31] for a system of two urns containing M number of beads (i.e., L = M +2), each one following the dynamics described in Fig. 1(a). For urn models, a matrix product steady state (MPSS) can be obtained following Ref. [32]. We now consider RTM model, which is mapped exactly to the urn model described in Fig. 1(a). The steady state probability of a generic configuration {σkmk}, where kth urn (spin σk) has mk beads, is given by a matrix product ansatz, The bead-current J(mk, mk+1) flowing across the urns can be computed from numerical simulations (will be discussed later), but that does not help us to compute ρ(z) in grand canonical ensemble. To calculate ρ(z) we need functional form of J(m1, m2) which can be calculated exactly using matrix product ansatz (MPA) [31] for a system of two urns containing M number of beads (i.e., L = M +2), each one following the dynamics described in Fig. 1(a). For urn models, a matrix product steady state (MPSS) can be obtained following Ref. [32]. We now consider RTM model, which is mapped exactly to the urn model described in Fig. 1(a). The steady state probability of a generic configuration {σkmk}, where kth urn (spin σk) has mk beads, is given by a matrix product ansatz, P({σkmk}) ∼Tr N Y k=1 Xσk(mk) δ N X k=1 mk −M , (3) (3) where matrix Xσk(mk) represents the kth urn having internal degree σk and mk beads. The δ-function here ensures that the total number of beads M are conserved. These matrices are constrained to follow a matrix algebra so that P({σkmk}) defined above must satisfy the steady state condition dP dt = 0 for the dynamics in Fig 1(a). We find (see Appendix) that for N = 2, matrices Xσ(m) have a 2 × 2 representation (for any ω > 0), X+(m) = 1 0 1 0 , X−(m) = γm 0 1 0 1 ; γ = p+ + q− p−+ q+ . 3 Mapping of RTM to a beads-in-urn model It is easy to see that a left-assisted tumbling dynamics with same rate ω will also map to the same beads-in-urn dynamics when particles are identified as urns containing number of beads same as the consecutive vacancies to their left and the hope rates are p± = q∓. The mapping of RTM to beads-in-urn model is exact but its steady-state could not be ob- tained analytically. We proceed to develop a coarse-grained picture. In the steady state of the urn model, the local bead current J (summed over ± degrees) effectively transports the beads from one urn to its neighbour situated along the direction of total current. Since hop- rates (qσk+1, pσk) in the original beads-in-urn model were dependent on spins of neighbouring urns it is expected that the local bead current must depend on the number of beads present in neighbouring urns, i.e. J ≡J(mk, mk+1). This current can be set as the effective hop-rate of a coarse-grained model where urns lose their internal degrees and a single bead hops from urn k to (k+1) with rate u(mk, mk+1) = J(mk, mk+1); rightward hopping (k to (k+1)) is considered assuming that the current is flowing in +ve x-direction. Thus, in this coarse-grained picture (see Fig 1(b)), all urns are equivalent (as they lose their internal degrees) and the hop-rate depends on the number of beads present in the departure and the arrival urn; such a process is called a misanthrope process (MAP) [23,24]. 4 SciPost Phys. 14, 165 (2023) In fact, mapping of hardcore particle systems to urn model with an exact or effective coarse- grained dynamics, similar to the dynamics of a zero range process (ZRP) [26] are quite reliable and have helped researchers [27] earlier to establish non-existence of phase separation tran- sition in certain lattice models [28] where rigorous numerical simulations have exhibited ap- parent phase separated states. It also helped in predicting true phase separation transition in many other models [27,29,30]. In contrast, mapping to that of misanthrope process, that we introduce here, provides a better coarse-grained picture as steady-state correlation between neighbouring urns are retained here. The bead-current J(mk, mk+1) flowing across the urns can be computed from numerical simulations (will be discussed later), but that does not help us to compute ρ(z) in grand canonical ensemble. 3 Mapping of RTM to a beads-in-urn model To account for that we introduce another fugacity z, so that the new partition function is, In RTM, both N, M = PN k=1 mk vary keeping the system size L fixed. To account for that we introduce another fugacity z, so that the new partition function is, Z(z, y) = ∞ X N=0 QN(y)zN = 1 1 −zF(y) , (8) (8) which gives rise to 〈N〉= z ∂ ∂z ln Z(z, y) and 〈M〉= y ∂ ∂y ln Z(z, y). We now set 〈N〉+ 〈M〉≡L to obtain z in terms of y, z = L (1+L)F(y)+yF′(y). Then, ρ(y) ≡〈N〉 L = F(y) F(y) + yF′(y) = 1 −y . (9) ρ(y) ≡〈N〉 L = F(y) F(y) + yF′(y) = 1 −y . (9) (9) The maximum value of the RTP density, obtained when y →0, ρ∗= 1. Thus the fugacity y can always be tuned to obtain any arbitrary particle density 0 ≤ρ ≤1; thus, irrespective of the value of ρ, the system remains homogeneous and can not phase separate. The above argument is based on a coarse-grained picture where the hop rate u(m, n) ≡u(m + n) is taken same as the average local current of beads. In the following we employ a method to calculate J(.) numerically from Monte Carlo simulations of the model and compare it with Eq. (6). To simulate the dynamics we must set p± = q∓required for the system to have a valid RTP dynamics, which gives γ = p+ p−in Eq. 4. Without loss of generality we can set p−= 1 = q+, by choosing a suitable time unit; then, p+ = q−= γ and the speed of RTPs v = q+ −p+ = 1 −γ. We also consider γ ≤1 (γ > 1 case can be explored directly by using left/right and +/− symmetry). From Eq. (6), J(m) = v Qm (1 −γm), which has an asymptotic form (for large m), J(m) ≡u(m) ≃m 1 −γ m + c ; c = 3 −γ 1 −γ . (10) (10) This implies that u(m)−1 is a linear function of m−1 with slope c(1 −γ)−1 and y-intercept (1 −γ)−1, which we verify from the Monte Carlo simulations of the urn model (Fig 1(a)). For a given value of γ,ρ,ω first we allow the system to relax for a long time starting from a random initial configuration. 3 Mapping of RTM to a beads-in-urn model (4) (4) The steady state probabilities of two urns containing m1, m2 beads are then, Pσ1σ2(m1, m2) The steady state probabilities of two urns containing m1, m2 beads 1 QM Tr[Xσ1(m1)Xσ2(m2)]δ(m1 + m2 −M), where QM = X σ1,σ2 M X m1=0 Tr[Xσ1(m1)Xσ2(M −m1) Explicitly, 1 QM Tr[Xσ1(m1)Xσ2(m2)]δ(m1 + m2 −M), where QM = X σ1,σ2 M X m1=0 Tr[Xσ1(m1)Xσ2(M −m1)]. Explicitly, Explicitly, Pσ1σ2(m1, m2) = 1 QM γ 1 2 (1−σ1)m1+ 1 2 (1−σ2)m2 , (5) (5) (5) with m2 = M −m1. Thus, the average local current carried by the beads when the two urns have (m1, m2) particles is J(m1, m2) = X σ1,σ2 Pσ1σ2(m1, m2)(qσ1 −pσ2) = 1 Qm1+m2 (q+ −p+) + (q+ −p−)γm1 + (q−−p+)γm2 + (q−−p−)γm1+m2 . For RTPs, which need to satisfy the condition p± = q∓, J(m1, m2) = v 1 −γm1+m2 Qm1+m2 , (6) (6) where v = p−−q−= q+ −p+ and γ = p+ p−(as in Eq. (4)). Note that J(m1, m2) depends only on the sum of its arguments, i.e., J(m1, m2) ≡J(m1 + m2). We will now set J(m1 + m2) as where v = p−−q−= q+ −p+ and γ = p+ p−(as in Eq. (4)). Note that J(m1, m2) depends only on the sum of its arguments, i.e., J(m1, m2) ≡J(m1 + m2). We will now set J(m1 + m2) as 5 SciPost Phys. 14, 165 (2023) the hop-rate of beads in the coarse-grained model, i. e., u(mk, mk+1) = J(mk + mk+1). This urn model is a misanthrope process where hop-rate is a function of total number of beads present in the departure and the arrival site. It turns out that the steady state of this specific misanthrope process has a factorized form, P({mk}) ∼ N Y k=1 f (mk), with f (m) = m Y n=1 u(1, n −1) u(n,0) = 1. The grand partition function with a fugacity y that controls the total number of beads M N X k=1 mk is The grand partition function with a fugacity y that controls the total number of beads M N X k=1 mk is is QN(y) = X {mk} P({mk})ymk = F(y)N ; F(y) = X m f (m)ym = 1 1 −y . (7) (7) In RTM, both N, M = PN k=1 mk vary keeping the system size L fixed. 3 Mapping of RTM to a beads-in-urn model 1 2 3 0 0.02 0.04 0.06 0.08 0.1 (a) γ= 0.4 γ= 0 1/u(m) 1/m 1 2 3 0 0.02 0.04 0.06 0.08 0.1 (a) γ= 0.4 γ= 0 1/u(m) 1/m 10-6 10-4 10-2 100 0 20 40 60 80 100 (b) ρ = 0.1 ρ = 0.3 ρ = 0.9 p(m) m 10-6 10-4 10-2 100 0 20 40 60 80 100 (b) ρ = 0.1 ρ = 0.3 ρ = 0.9 p(m) m 10-6 10-4 10-2 100 0 20 40 60 80 100 (b) ρ = 0.1 ρ = 0.3 ρ = 0.9 p(m) m 10-6 10-4 10-2 100 0 400 800 ρ = 0.1 p(m) m Figure 2: Simulation of RTM model with dynamics (1) and (2) (equivalently an Figure 2: Simulation of RTM model with dynamics (1) and (2) (equivalently an urn model described in Fig. 1(a)). (a) Hop rate u(m)−1 obtained from numerical simulations (solid line) for γ = 0,0.4 and ω = 0.005 to 1 (top to bottom) are com- pared with Eq. (10) (dashed line) when ρ = 0.02. All the curves approach linearly to the asymptotic value (1−γ)−1, as predicted. (b) Marginal distribution p(m) of the separation m are compared for γ = 0 and ρ = 0.1,0.3,0.9 in semi-log scale. Solid lines (results from simulations for ω = 0.2 to 10 (right to left) are shown along with dashed lines, ρ(y)m with y = 1 −ρ obtained from coarse-grained description of the model. The inset shows the same for ρ = 0.1 but smaller ω = 0.005 to 1 (right to left). In all cases p(m) shows exponential behaviour; but for small ω, y differs substantially from the predicted value (1 −ρ). Here, p+ = γ = q−, p−= 1 = q+, L = 104. In each case, statistical averaging is done for more than 107 samples. In the coarsening regime we consider a large time interval and calculate (Fr(m1 + m2), Fl(m1 + m2)), the number of times beads move to (right, left) when the de- parture and arrival urns have exactly m1 and m2 beads respectively (internal degree of the urns are ignored). Also, we keep track of F(m1 + m2), the number of jump-events attempted during that interval. Clearly, u(m) = (Fr(m)−Fl(m))/F(m). In Fig. 3 Mapping of RTM to a beads-in-urn model The system may take a very long time to reach a true phase separated state when it exists, but the hoping dynamics in the coarsening regime given by u(m1, m2) = J(m1, m2) can predict, well in advance, if the system is approaching towards a inhomogeneous (MIPS) or a homogeneous state. 6 SciPost Phys. 14, 165 (2023) 1 2 3 0 0.02 0.04 0.06 0.08 0.1 (a) γ= 0.4 γ= 0 1/u(m) 1/m 1 2 3 0 0.02 0.04 0.06 0.08 0.1 (a) γ= 0.4 γ= 0 1/u(m) 1/m 10-6 10-4 10-2 100 0 20 40 60 80 100 (b) ρ = 0.1 ρ = 0.3 ρ = 0.9 p(m) m 10-6 10-4 10-2 100 0 20 40 60 80 100 (b) ρ = 0.1 ρ = 0.3 ρ = 0.9 p(m) m 10-6 10-4 10-2 100 0 20 40 60 80 100 (b) ρ = 0.1 ρ = 0.3 ρ = 0.9 p(m) m 10-6 10-4 10-2 100 0 400 800 ρ = 0.1 p(m) m Figure 2: Simulation of RTM model with dynamics (1) and (2) (equivalently an urn model described in Fig. 1(a)). (a) Hop rate u(m)−1 obtained from numerical simulations (solid line) for γ = 0,0.4 and ω = 0.005 to 1 (top to bottom) are com- pared with Eq. (10) (dashed line) when ρ = 0.02. All the curves approach linearly to the asymptotic value (1−γ)−1, as predicted. (b) Marginal distribution p(m) of the separation m are compared for γ = 0 and ρ = 0.1,0.3,0.9 in semi-log scale. Solid lines (results from simulations for ω = 0.2 to 10 (right to left) are shown along with dashed lines, ρ(y)m with y = 1 −ρ obtained from coarse-grained description of the model. The inset shows the same for ρ = 0.1 but smaller ω = 0.005 to 1 (right to left). In all cases p(m) shows exponential behaviour; but for small ω, y differs substantially from the predicted value (1 −ρ). Here, p+ = γ = q−, p−= 1 = q+, L = 104. In each case, statistical averaging is done for more than 107 samples. 3 Mapping of RTM to a beads-in-urn model 2(a) we plot u(m)−1 versus m−1 for γ = 0,0.4,ρ = 0.02 and ω = 0.005 to 1; in all cases, u(m)−1 is found to be linear for large m as expected from Eq. (10). The y- intercepts also approach to the known value (1−γ)−1 but the slopes differ a bit. Further, in Fig. 2(b) we plot the marginal distribution p(m) of number beads m for γ = 0,ρ = 0.1,0.3,0.9, ω = 0.2 to 10. The dashed line corresponds to the theoretical curve obtained from the coarse-grained picture: p(m) = ym f (m)/F(y) = ρ ym where y = 1 −ρ. In all cases, as shown Fig. 2(b), p(m) exhibits exponential distributions that match very well with the prediction when ω is large. As ω →0 the exponential feature remains persistent but the value of y differs substantially from the theoretical value 1−ρ. This is because ergodicity is broken at ω = 0; the system there falls into one of the fully jammed (or absorbing) configuration and remains there. Essentially, the coarse-grained picture turns out to be a good description of the RTP model as p(m) decays exponentially for large m as predicted - rest of the details are less relevant because an exponential form of p(m) is enough to assure that the fugacity in GCE can always be tuned to secure any desired particle density 0 < ρ < 1. Such a system can not support any stable MIPS phase and settles to form a homogeneous density profile for all ω > 0,γ ≥0. The above conclusion can also be obtained from using an approximate matrix product steady state (MPSS). Matrix representations (4), that provides exact MPSS exclusively for N = 2, are also excellent approximations for larger N (justified in the Appendix). With these matrices, for N > 2, the grand partition function Z(z, y) and density ρ(y) are given by Eqs. 7 SciPost Phys. 14, 165 (2023) (A.6) and (A.7) respectively, (A.6) and (A.7) respectively, (A.6) and (A.7) respectively, Z(z, y) = 1 1 −zF(y); F(y) = 1 1 −y + 1 1 −γy and ρ(y) = (1 −y)(1 −γy)(2 −y −γy) (1 −γy)2 + (1 −y)2 . 4 Summary In this article we show that phase separation of free hardcore-RTPs with constant run and tumble rates is not possible in 1D. One may however add some crucial features which are known to enhance or freshly produce phase separated states of passive particles, like invoking explicit attractive interaction [27] or making tumbling rates to decrease with L (so that it vanishes in the thermodynamic limit) [33] or explicitly forcing the run dynamics to depend on (and reduce substantially with increase of) local particle density [26] or adding impurities [34]. Then a phase separation transition may occur, but will it keep its charm and glory to be identified as the motility induced phase separation, particulary when the transition is anyway expected for similar system of passive particles (without motility)? Recently Kourbane-Houssene et. al. [35] have introduced a RTP model where the differ- ence of run-rates (or effective velocity) are taken proportional to 1 L and the tumbling rate is proportional to 1 L2 (downplayed by a factor 1/L compared to the run rates); using an exact coarse-grained hydrodynamic description they show that a homogeneous phase in 1D loses its stability in certain parameter regimes. Another way might be to use strongly biased tumbling rates where, say, + →−occurs much more frequently than −→+. In this case a phase sep- aration transition occurs [33] when q± = 0, where the dynamics of RTM reduces to that of a two species exclusion process [36]. Its extension to small q± ≃0, is a RTP model (having a good continuum limit) and it is reasonable to assume that the phase separation features may also survive there. Yet another possibility is to introduce defects. Recent studies [37] have shown that a jammed phase does exist in RTM like models with defects. More investigations are required in all these directions to confirm if RTP models in 1D can phase separate. 3 Mapping of RTM to a beads-in-urn model (11) (11) Clearly, the maximum density that can be achieved in GCE by tuning y is ρ∗= 1 (when y = 0) and thus, this RTP model can not undergo a phase separation transition at any ρ < 1. One can safely extend these results for restricted tumbling dynamics to other RTP models where tumbling occurs more frequently; this is because tumbling is generally detrimental to the stability of MIPS. Our conclusions are consistent with the recent results [20–22] that MIPS transition in 2D belongs to the Ising universality class that does not have an one dimensional analogue. A Appendix The dynamics (1) and (2) of RTM can be mapped exactly to an urn model described in Fig. 1(a) where beads hop from site k to site k+1 (or site k−1) with rates qσk+1 (or pσk) respectively. The probability density of a generic configuration {σkmk} evolves following the Master equation, d dt P(...,σk−1mk−1,σkmk,σk+1mk+1,...) = −(pσk + qσk+1)P(...,σk−1mk−1,σkmk,σk+1mk+1,...) + qσk P(...,σk−1mk−1 + 1,σkmk −1,σk+1mk+1,...) + pσk+1P(...,σk−1mk−1,σkmk −1,σk+1mk+1 + 1,...) −ωδmk,0P(...,σk−1mk−1,σkmk,σk+1mk+1,...) + ωδmk,0P(...,σk−1mk−1,−σkmk,σk+1mk+1,...), (A.1) (A.1) where first three terms in the right hand side corresponds to the run dynamics and the rest describes tumbling at a generic site k. In the steady state d dt P({σkmk}) must vanish; this, along with the matrix product ansatz (3) leads to PN k=1 Tr[HR k + H T k ] = 0, where HR k and H T k correspond to the run and the tumble dynamics respectively, HR k = −(pσk + qσk+1)Xσk−1(mk−1)Xσk(mk)Xσk+1(mk+1) + qσkXσk−1(mk−1 + 1)Xσk(mk −1)Xσk+1(mk+1) + pσk+1Xσk−1(mk−1)Xσk(mk −1)Xσk+1(mk+1 + 1) (A.2 (A.2) and H T k = ω[X−σk(0) −Xσk(0)]Xσk+1(mk+1). and H T k = ω[X−σk(0) −Xσk(0)]Xσk+1(mk+1). We now introduce some suitable choice of auxiliary matrices ˜Xσk,σk+1(mk, mk+1), yet to be determined along with Xσk(mk), so that both P k HR k and P k H T k vanish separately; one such cancellation scheme for HR k is, HR k = ˜Xσk−1,σk(mk−1, mk)Xσk+1(mk+1) −Xσk−1(mk−1) ˜Xσk,σk+1(mk, mk+1). (A.3) (A.3) that a choice ˜Xσ,σ′(m, n) = hσσ′Xσ(m)Xσ′(n) with some scalar parameter hσσ′ does e steady state condition with 2 × 2 matrices X+(m) = 1 0 1 0 , X−(m) = γm 0 1 0 1 , (A.4) (A.4) when γ = p++q− p−+q+ , h+−= 0 = h−+ and h++ = h−−= qσ(1 −γσ), m > 0, n > 0, 0, else. (A.5) (A.5) These matrices also satisfy the condition P k Tr[H T k ] = 0 set by the tumbling dynamics because Xσ(0)Xσ′(m) = Xσ′(m) for all σ,σ′, m. The only troubling part is that hσσs depend implicitly on m, n violating the assumption that they are constants. This implicit dependence of h++ and h−−on m, n drops out when (i) q± = 0 (all particles move in the same direction), (ii)γ = 1 (which sets the speed of RTPs v = 1 −γ = 0 when p± = q∓). Acknowledgements PKM acknowledges stimulating discussions with Urna Basu. IM acknowledges the sup- port of Council of Scientific and Industrial Research, India (Research Fellowship, Grant No. 09/921(0335)/2019-EMR-I). 8 SciPost Phys. 14, 165 (2023) A Appendix In both cases we have an exact MPSS, but neither of these cases constitutes the scenario of MIPS. Yet another case is N = 2 where matrices given by Eq. (A.4) leads to an exact MPSS. This is because the cancellation scheme in Eq. (A.2) acts on product of three consecutive matrices which are not present when N = 2; thus, one can make hσσ′ independent of m, n by setting safely hσσ′ = 0 for all σ,σ′. Steady state probabilities for N = 2 is given by Eq. (5). 9 SciPost Phys. 14, 165 (202 0 2 4 6 8 0 0.2 0.4 0.6 0.8 1 (a) η+ γ 0.5 0.6 0.7 0.8 0.9 1 0 0.2 0.4 0.6 0.8 1 (b) ρ+ γ Figure 3: (a) η+, the density of beads in + urn and (b) ρ+, the fraction of + urns are shown as a function of γ for different ρ = 0.1 to 0.9 (top to bottom). Data from Monte Carlo simulations (solid lines) of RTM model described in Fig. 1(a), averaged over 107 samples are compared with Eqs. (A.9) (dashed line). Other parameters are L = 103, p+ = γ = q−, p−= 1 = q+ and ω = 1. SciPost Phys. 14, 165 (2023) 0 2 4 6 8 0 0.2 0.4 0.6 0.8 1 (a) η+ γ 0.5 0.6 0.7 0.8 0.9 1 0 0.2 0.4 0.6 0.8 1 (b) ρ+ γ Figure 3: (a) η+, the density of beads in + urn and (b) ρ+, the fraction of + urns are shown as a function of γ for different ρ = 0.1 to 0.9 (top to bottom). Data from Monte Carlo simulations (solid lines) of RTM model described in Fig. 1(a), averaged over 107 samples are compared with Eqs. (A.9) (dashed line). Other parameters are L = 103, p+ = γ = q−, p−= 1 = q+ and ω = 1. Now we proceed for larger N and get an approximate MPSS while dependence of hσσ′ on m, n are ignored and both h++ and h−−are taken as qσ(1 −γσ) ∀m, n ≥0. We will see that the matrices (A.4) provide a MPSS which are an excellent approximation to the exact ones. A Appendix 3 we plot η+ and ρ+ as a function of γ (dashed lines), for different ρ in the range (0.1,0.9), along with those obtained from the Monte Carlo simulations of the model (solid lines). They match quite well for all γ < 1, indicating that, the approximate MPSS describes the RTP model very well. A Appendix The canonical partition function of the system is QM,N = X {σkmk} Tr N Y k=1 Xσk(mk) δ N X k=1 mk −M , and the grand partition function, with fugacities z, y associated with N, M, is Z(z, y) = ∞ X M=0, N=0 zN y MQM,N = ∞ X N=0 zN F(y)N , F(y) = X σ=± ∞ X m=0 ymTr[Xσ(m)] = 1 1 −y + 1 1 −γy . (A.6) (A.6) Note that F(y)N acts as the partition function of the system when N is fixed. 1 d Note that F(y)N acts as the partition function of the system when N is fixed. From Z(z, y) = 1 1−zF(y) one can calculate 〈N〉= z d dz ln Z(z, y) and 〈M〉= y d d y ln Z(z, y) and set 〈N〉+ 〈M〉to a desired value of L to eliminate z. Particle density ρ(y) = 〈N〉 L in GCE is then, ρ(y) = 1 1 + y F′(y) F(y) = (1 −y)(1 −γy)(2 −y −γy) (1 −γy)2 + (1 −y)2 . (A.7) (A.7) To verify if MPSS obtained here is indeed a good approximation let us calculate and com- pare from Monte Carlo simulations, the steady state values of η+, the average number of beads per + urn and ρ+, the fraction of urns having internal degree +, To verify if MPSS obtained here is indeed a good approximation let us calculate and com- pare from Monte Carlo simulations, the steady state values of η+, the average number of beads per + urn and ρ+, the fraction of urns having internal degree +, η+ = 1 N N X k=1 〈mkδσk,+〉; ρ+ = 1 N N X k=1 〈δσk,+〉. (A.8) (A.8) Since simulations are done at some specific L, N, we can use F(y)N as the partition function of the system; thus p+(m) = ym/F(y) and p−(m) = γm ym/F(y) and, η+ = 1 F(y) ∞ X m=0 mTr[X+(m)]ym = y(1 −γy) (1 −y)(2 −y −γy) ; ρ+ = 1 F(y) ∞ X m=0 Tr[X+(m)]ym = 1 −γy 2 −y −γy . 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https://openalex.org/W2803245016	https://ro.uow.edu.au/cgi/viewcontent.cgi?article=6559&context=smhpapers	English	null	The influence of instrumental line shape degradation on NDACC gas retrievals: total column and profile	Atmospheric measurement techniques	2,018	cc-by	11,329	University of Wollongong University of Wollongong Research Online Research Online University of Wollongong University of Wollongong Research Online Research Online Faculty of Science, Medicine and Health - Papers: part A Faculty of Science, Medicine and Health 1-1-2018 The influence of instrumental line shape degradation on NDACC gas The influence of instrumental line shape degradation on NDACC gas retrievals: Total column and profile retrievals: Total column and profile Youwen Sun Chinese Academy Of Sciences Mathias Palm University of Bremen Cheng Liu University of Science and Technology of China, Chinese Academy of Sciences Frank Hase Karlsruhe Institute of Technology David W. T Griffith University of Wollongong, griffith@uow.edu.au See next page for additional authors Follow this and additional works at: https://ro.uow.edu.au/smhpapers Part of the Medicine and Health Sciences Commons, and the Social and Behavioral Sciences Commons Recommended Citation Recommended Citation Sun, Youwen; Palm, Mathias; Liu, Cheng; Hase, Frank; Griffith, David W. T; Weinzierl, Christine; Petri, Christof; Wang, Wei; and Notholt, Justus, "The influence of instrumental line shape degradation on NDACC gas retrievals: Total column and profile" (2018). Faculty of Science, Medicine and Health - Papers: part A. 5494. https://ro.uow.edu.au/smhpapers/5494 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Recommended Citation Recommended Citation Sun, Youwen; Palm, Mathias; Liu, Cheng; Hase, Frank; Griffith, David W. T; Weinzierl, Christine; Petri, Christof; Wang, Wei; and Notholt, Justus, "The influence of instrumental line shape degradation on NDACC gas retrievals: Total column and profile" (2018). Faculty of Science, Medicine and Health - Papers: part A. 5494. Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au Disciplines Disciplines Medicine and Health Sciences \| Social and Behavioral Sciences Abstract Abstract The sensitivities of the total column, the root mean square (rms) of the fitting residual, the total random uncertainty, the total systematic uncertainty, the total uncertainty, degrees of freedom for signal (DOFs), and the profile with respect to different levels of ILS degradation for all current standard NDACC gases, i.e. O3, HNO3, HCl, HF, ClONO2, CH4, CO, N2O, C2H6, and HCN, were investigated. The Publication Details Publication Details Sun, Y., Palm, M., Liu, C., Hase, F., Griffith, D., Weinzierl, C., Petri, C., Wang, W. & Notholt, J. (2018). The influence of instrumental line shape degradation on NDACC gas retrievals: Total column and profile. Atmospheric Measurement Techniques, 11 (5), 2879-2896. Youwen Sun, Mathias Palm, Cheng Liu, Frank Hase, David W. T Griffith, Christine Weinzierl, Christof Petri, Wei Wang, and Justus Notholt Abstract Abstract We simulated instrumental line shape (ILS) degradations with respect to typical types of misalignment, and compared their influence on each NDACC (Network for Detection of Atmospheric Composition Change) gas. The sensitivities of the total column, the root mean square (rms) of the fitting residual, the total random uncertainty, the total systematic uncertainty, the total uncertainty, degrees of freedom for signal (DOFs), and the profile with respect to different levels of ILS degradation for all current standard NDACC gases, i.e. O3, HNO3, HCl, HF, ClONO2, CH4, CO, N2O, C2H6, and HCN, were investigated. The influence of an imperfect ILS on NDACC gases' retrieval was assessed, and the consistency under different meteorological conditions and solar zenith angles (SZAs) were examined. The study concluded that the influence of ILS degradation can be approximated by the linear sum of individual modulation efficiency (ME) amplitude influence and phase error (PE) influence. The PE influence is of secondary importance compared with the ME amplitude. Generally, the stratospheric gases are more sensitive to ILS degradation than the tropospheric gases, and the positive ME influence is larger than the negative ME. For a typical ILS degradation (10 %), the total columns of stratospheric gases O3, HNO3, HCl, HF, and ClONO2 changed by 1.9, 0.7, 4, 3, and 23 %, respectively, while the columns of tropospheric gases CH4, CO, N2O, C2H6, and HCN changed by 0.04, 2.1, 0.2, 1.1, and 0.75 %, respectively. In order to suppress the fractional difference in the total column for ClONO2 and other NDACC gases within 10 and 1 %, respectively, the maximum positive ME degradations for O3, HNO3, HCl, HF, ClONO2, CO, C2H6, and HCN should be less than 6, 15, 5, 5, 5, 5, 9, and 13 %, respectively; the maximum negative ME degradations for O3, HCl, and HF should be less than 6, 12, and 12 %, respectively; the influence of ILS degradation on CH4 and N2O can be regarded as being negligible We simulated instrumental line shape (ILS) degradations with respect to typical types of misalignment, and compared their influence on each NDACC (Network for Detection of Atmospheric Composition Change) gas. Correspondence: Cheng Liu (chliu81@ustc.edu.cn) Correspondence: Cheng Liu (chliu81@ustc.edu.cn) Received: 24 October 2017 – Discussion started: 10 January 2018 Received: 24 October 2017 – Discussion started: 10 January 2018 Revised: 2 April 2018 – Accepted: 19 April 2018 – Published: 17 May 2018 Received: 24 October 2017 – Discussion started: 10 January 2018 Revised: 2 April 2018 – Accepted: 19 April 2018 – Published: 17 May 2018 Revised: 2 April 2018 – Accepted: 19 April 2018 – Published: 17 May 2018 Abstract. We simulated instrumental line shape (ILS) degra- dations with respect to typical types of misalignment, and compared their inﬂuence on each NDACC (Network for De- tection of Atmospheric Composition Change) gas. The sensi- tivities of the total column, the root mean square (rms) of the ﬁtting residual, the total random uncertainty, the total system- atic uncertainty, the total uncertainty, degrees of freedom for signal (DOFs), and the proﬁle with respect to different levels of ILS degradation for all current standard NDACC gases, i.e. O3, HNO3, HCl, HF, ClONO2, CH4, CO, N2O, C2H6, and HCN, were investigated. The inﬂuence of an imperfect ILS on NDACC gases’ retrieval was assessed, and the con- sistency under different meteorological conditions and solar zenith angles (SZAs) were examined. The study concluded that the inﬂuence of ILS degradation can be approximated by the linear sum of individual modulation efﬁciency (ME) amplitude inﬂuence and phase error (PE) inﬂuence. The PE inﬂuence is of secondary importance compared with the ME amplitude. Generally, the stratospheric gases are more sensi- tive to ILS degradation than the tropospheric gases, and the positive ME inﬂuence is larger than the negative ME. For a typical ILS degradation (10 %), the total columns of strato- spheric gases O3, HNO3, HCl, HF, and ClONO2 changed by 1.9, 0.7, 4, 3, and 23 %, respectively, while the columns of tropospheric gases CH4, CO, N2O, C2H6, and HCN changed by 0.04, 2.1, 0.2, 1.1, and 0.75 %, respectively. Correspondence: Cheng Liu (chliu81@ustc.edu.cn) In order to suppress the fractional difference in the total column for ClONO2 and other NDACC gases within 10 and 1 %, re- spectively, the maximum positive ME degradations for O3, HNO3, HCl, HF, ClONO2, CO, C2H6, and HCN should be less than 6, 15, 5, 5, 5, 5, 9, and 13 %, respectively; the max- imum negative ME degradations for O3, HCl, and HF should be less than 6, 12, and 12 %, respectively; the inﬂuence of ILS degradation on CH4 and N2O can be regarded as being negligible. Authors Authors Youwen Sun, Mathias Palm, Cheng Liu, Frank Hase, David W. T Griffith, Christine Weinzierl, Christof Petri, Wei Wang, and Justus Notholt This journal article is available at Research Online: https://ro.uow.edu.au/smhpapers/5494 Atmos. Meas. Tech., 11, 2879–2896, 2018 https://doi.org/10.5194/amt-11-2879-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Youwen Sun1,3,, Mathias Palm2,, Cheng Liu3,4,1, Frank Hase5, David Grifﬁth6, Christine Weinzierl2, Christof Petri2, Wei Wang1 and Justus Notholt2 1Key Laboratory of Environmental Optics and Technology, Anhui Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Hefei 230031, China 2University of Bremen, Institute of Environmental Physics, P.O. Box 330440, 28334 Bremen, Germany 3Center for Excellence in Urban Atmospheric Environment, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China y University of Science and Technology of China, Hefei, 230026, China y gy 5Karlsruhe Institute of Technology (KIT), Institute for Meteorology and Climate Research (IMK-ASF), Karlsruhe, Germany 6School of Chemistry, University of Wollongong, Northﬁelds Ave, Wollongong, NSW, 2522, Australia These authors contributed equally to this work. 1 Introduction In order to achieve consistent results between different FTIR (Fourier transform infrared) sites, the TCCON (Total Car- bon Column Observing Network; http://www.tccon.caltech. edu/, last access: 11 May 2018) and NDACC (Network for Detection of Atmospheric Composition Change; http: //www.ndacc.org/, last access: 11 May 2018) have devel- oped strict data acquisition and retrieval methods to mini- mize site to site differences (Hase et al., 2012; Wunch et al., 2010, 2011; Washenfelder, 2006; Messerschmidt et al., The inﬂuence of instrumental line shape degradation on NDACC gas retrievals: total column and proﬁle Youwen Sun1,3,, Mathias Palm2,*, Cheng Liu3,4,1, Frank Hase5, David Grifﬁth6, Christine Weinzierl2, Christof Petri2, Wei Wang1, and Justus Notholt2 Published by Copernicus Publications on behalf of the European Geosciences Union. 2010; Kurylo, 1991; Davis et al., 2001; Schneider et al., 2008; Kohlhepp et al., 2011; Hannigan and Coffey, 2009; Vigouroux et al., 2008, 2015). Interferograms are acquired with similar instruments operated with common detectors, acquisition electronics, and/or optical ﬁlters. These interfer- ograms are ﬁrst converted to spectra and then these spec- tra are analysed using dedicated processing algorithms, i.e. GFIT, PROFFIT, or SFIT (Wunch et al., 2010, 2015; Hase et al., 2006; Hannigan and Coffey, 2009). Typically, the TCCON only uses Bruker 125HR instruments (http://www. tccon.caltech.edu/; https://www.bruker.com/, last access: 11 May 2018) with speciﬁed settings (entrance aperture, ampli- ﬁcation of the detected signal). In the NDACC, other instru- ments are used as well, e.g. the Bruker M series, a BOMEM DA8 in Toronto, Canada, and a self-built spectrometer in Pasadena, USA (http://www.ndacc.org/; https://www.bruker. com/). FTIR spectrometers are highly precise and stable de- vices, and if carefully aligned, the instrumental line shape (ILS) might not be far from the theoretical limit. However, their alignment can change abruptly as a consequence of op- erator intervention or drift slowly due to mechanical degra- dation over time (Olsen and Randerson, 2004; Duchatelet et al., 2010; Hase et al., 2012; Feist et al., 2016). Moreover, the NDACC observation may change the entrance ﬁeld stop size if incident radiation changes. This practice may introduce a dependency of the instrument alignment status on the opti- cal settings because the mechanical errors between different The TCCON only operates in the near-infrared region (NIR) and focuses on columns of fewer gases, while the NDACC operates in both the NIR and the mid-infrared re- gion (MIR) and focuses on both columns and proﬁles of many gases. The TCCON assumes an ideal ILS in spectra retrieval, and the maximum ILS degradation is prescribed as 5 % for the modulation efﬁciency (ME) amplitude (Wunch et al., 2011, 2015). This assumption still holds within the required accuracy of the results. In the NDACC gases’ re- trieval, the ILS can be assumed to be ideal if the spectrom- eter is well aligned, or if misalignment exists, described by LINEFIT results derived from dedicated cell measure- ments or retrieved together with the gas proﬁle from an at- mospheric spectrum using a polynomial (Vigouroux et al., 2008, 2015). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2880 Y. Sun et al.: The inﬂuence of instrumental line shape degradation Table 1. Misalignments simulated in the ALIGN60. Type∗ Description Input Output in maximum a No misalignment occurs: interferometer in ideal condition none ME amplitude: 1.00 PE: 0.000 rad. b Decentring of entrance ﬁeld stop deﬁning FOV: causes 0.33 (mrad) ﬁeld stop error ME amplitude: 0.86 a linear increase in misalignment along OPD PE: −0.056 rad. c Decentring of path measuring laser: causes a linear 0.33 (mrad) laser error ME amplitude: 1.00 increase in phase error along OPD PE: −0.152rad. d Constant shear: causes a constant shear offset 0.03 (cm) ME amplitude: 1.00 of ﬁxed retro-reﬂector PE: −0.056 rad. e Decreasing linear shear: causes a linear decrease 0.03–0.00017 × OPD (cm) ME amplitude: 1.16 in misalignment along OPD PE: −0.007 rad. f Increasing linear shear: causes a linear increase 0.00017 × OPD (cm) ME amplitude: 0.86 in misalignment along OPD PE: −0.056 rad. g Cosine bending of scanner bar: causes a cosine 0.03 × cos(π × OPD/360) (cm) ME amplitude: 1.16 decrease in misalignment along OPD PE: −0.013 rad. h Sine bending of scanner bar: causes a sine increase 0.03 × sin(π × OPD/360) (cm) ME amplitude: 0.86 in misalignment along OPD PE: −0.056 rad. i Cosine and sine bending of scanner bar: causes a chord increase 0.073 × (sin(π × OPD/360)+ ME amplitude: 0.86 in misalignment before half-maximum OPD and causes cos(π × OPD/360))−0.073 (cm) PE: −0.029 rad. a chord decrease in misalignment after half-maximum OPD j Constant shear plus cosine and sine bending of scanner bar: −0.073 × (sin(π × OPD/360)+ ME amplitude: 1.16 causes a chordal decrease in misalignment before cos(π × OPD/360))+0.103 (cm) PE: −0.056 rad. half-maximum OPD and causes a chordal increase in misalignment after half-maximum OPD ∗The letters b, f, h, and i refer to increasing misalignment; e, g, and j refer to decreasing misalignment. Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2880 Table 1. Misalignments simulated in the ALIGN60. ﬁeld stops may be non-negligible and inconsistent (Sun et al., 2017). Biases between sites would arise if all these misalign- ments were not properly characterized. ∗The letters b, f, h, and i refer to increasing misalignment; e, g, and j refer to decreasing misalignment. www.atmos-meas-tech.net/11/2879/2018/ Published by Copernicus Publications on behalf of the European Geosciences Union. The answers to how these ILS treatments inﬂu- ence the NDACC gases’ retrieval and how much ILS devia- tion from unity is acceptable for each NDACC gas if an ideal line shape is assumed are still not fully quantiﬁed, and it may be better to assume an ideal ILS. The practice of co-retrieving ILS parameters from atmospheric spectra without dedicated cell measurements is not to be recommended because the ob- served shapes of spectral lines are exploited primarily for in- ferring the vertical distribution of the trace gases; the ILS and the trace gas proﬁles have similar effects on the line shape, www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2881 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2881 Figure 1. Simulated ILS degradation with respect to different types of misalignment. The results are derived from ALIGN60. Panel (a) demonstrates different types of misalignment (a to j) used in the simulation, panel (b) shows the resulting ILS, panel (c) shows the resulting ME amplitude, and panel (d) shows the resulting PE. Descriptions of the misalignments a to j are listed in Table 1. Figure 1. Simulated ILS degradation with respect to different types of misalignment. The results are derived from ALIGN60. Panel (a) demonstrates different types of misalignment (a to j) used in the simulation, panel (b) shows the resulting ILS, panel (c) shows the resulting ME amplitude, and panel (d) shows the resulting PE. Descriptions of the misalignments a to j are listed in Table 1. Figure 2. The Haidinger fringes at maximum OPD for misalignment a to j shown in Fig. 1. Figure 2. The Haidinger fringes at maximum OPD for misalignment a to j shown in Fig. 1. 2 Characteristics of ideal and imperfect ILSs i.e. changing the shape and width of the line. Overlapping lines, i.e. due to interfering gases, may introduce asymmetry in the absorption lines which may be undistinguishable from an ILS phase deviation. The ILS is the Fourier transform of the weighting applied to the interferogram. This weighting consists of two parts: an artiﬁcially applied part to change the calculated spectrum and an unavoidable part which is due to the fact that the inter- ferogram is ﬁnite in length (box car function), the divergence of the beam is non-zero (due to the non-zero entrance aper- ture), and several other effects which are due to misalignment (Davis et al., 2001, chap. 9). The ILS consisting of only the This paper investigates the inﬂuence of ILS degradation on the total column and the proﬁle of current standard NDACC gas retrievals and deduces the maximum ILS deviations al- lowable for suppressing the inﬂuence within a speciﬁed ac- ceptable range. www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 4.1 Retrieval strategy The inﬂuence of ILS degradation on all current standard NDACC gases, i.e. O3, HNO3, HCl, HF, ClONO2, CH4, CO, N2O, C2H6, and HCN, is investigated. Typical atmo- spheric vertical proﬁles of these gases are shown in Fig. 3. There are ﬁve stratospheric gases and ﬁve tropospheric gases. The retrieval settings for all these gases as recommended by the NDACC are listed in Table 2 (https://www2.acom. ucar.edu/irwg/links, last access: 11 May 2018). The latest version of the proﬁle retrieval algorithm SFIT4 v 0.9.4.4 is used (http://www.ndacc.org/, last access: 11 May 2018). The basic principle of SFIT4 is the use of an optimal es- timation technique for ﬁtting calculated to observed spec- tra (Rodgers, 2000; Hannigan and Coffey, 2009). All spec- troscopic line parameters are adopted from HITRAN 2008 (Rothman et al., 2009). This might not be ideal, but we main- tain this to achieve consistent results. A priori proﬁles of pressure, temperature, and water vapour for the measure- ment days are interpolated from the National Centers for Environmental Protection and National Center for Atmo- spheric Research (NCEP/NCAR) reanalysis (Kalnay et al., 1996). A priori proﬁles of the target gases and the inter- fering gases except H2O use the WACCM4 (Whole Atmo- sphere Community Climate Model) model data. We follow the NDACC standard convention with respect to microwin- dows’ (MWs) selection and the interfering gases consider- ation (https://www2.acom.ucar.edu/irwg/links). For the in- terfering molecules that affect the target gas retrieval, H2O should be treated with care as it is almost always present in all MWs, to varying degrees. It has been dealt with differ- ently for different gases. For HNO3 and ClONO2, H2O is treated as the other interfering species: only scaling of a sin- gle a priori proﬁle is done. For other gases, the H2O proﬁle is retrieved simultaneously with the target gas proﬁle. No de- weighting signal-to-noise ratios (SNRs) are used, except for Y. Sun et al.: The inﬂuence of instrumental line shape degradation linear PE deviation. Two types of ILS degradation are evi- dent; one is referred to as a positive ME and has a ME am- plitude of larger than unity. The other one is referred to as a negative ME and has a ME amplitude of less than unity. Typ- ically, the increasing misalignment with increasing OPD (b, f, h, or i) causes negative ME amplitude and the decreasing misalignment with increasing OPD (e, g, or j) causes pos- itive ME amplitude. For the same misalignment amplitude, the decreasing misalignment causes more ME deviation than the increasing misalignment. Regardless of positive or nega- tive ME, the ME deviation shape depends on misalignment type and the same misalignment amplitude causes the same deviation in ME amplitude. The decentring of the entrance ﬁled stop is equivalent to the linear increasing misalignment. where σ is the wavenumber, σ0 is the central wavenumber, L is the optical path difference (OPD), and θ is the angular radius of the circular internal FOV of the spectrometer. For standard NDACC measuring conditions, L ≥180 cm and θ deﬁned by the entrance ﬁeld stop size in the light path. y p g p The LINEFIT software calculates the deviation of the measured ILS from the ideal ILS (Hase et al., 2001, 2012). It retrieves a complex ME as a function of the OPD, which is represented by a ME amplitude and a phase error (PE) (Hase et al., 1999). The ME amplitude is connected to the width of the ILS, while the PE quantiﬁes the degree of ILS asymme- try. For a perfectly aligned spectrometer, it would meet the ideal nominal ILS characteristics if smear and vignetting ef- fects were neglected, and thus have an ME amplitude of unity and a PE of zero along the whole interferogram. However, if a FTIR spectrometer were subject to misalignment, the ME amplitude would deviate from unity and the PE would devi- ate from zero (Hase et al., 2012). This results in an imperfect ILS. Y. Sun et al.: The inﬂuence of instrumental line shape degradation Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2882 Figure 3. Typical proﬁles of 10 NDACC gases. The panels in (b) show the ﬁve tropospheric gases, i.e. CH4, CO, N2O, C2H6, and HCN. The panels in (a) show the ﬁve stratospheric gases, i.e. O3, HNO3, HCl, HF, and ClONO2. Although the CO concentration above 60 km is much higher than that in the troposphere, it is regarded as tropospheric gas because it is an anthropologic pollution gas and shows large variation in the troposphere. Figure 3. Typical proﬁles of 10 NDACC gases. The panels in (b) show the ﬁve tropospheric gases, i.e. CH4, CO, N2O, C2H6, and HCN. The panels in (a) show the ﬁve stratospheric gases, i.e. O3, HNO3, HCl, HF, and ClONO2. Although the CO concentration above 60 km is much higher than that in the troposphere, it is regarded as tropospheric gas because it is an anthropologic pollution gas and shows large variation in the troposphere. Figure 4. Averaging kernels of 10 NDACC gases (coloured ﬁne lines) and their area scaled by a factor of 0.2 (black bold line). They are deduced from the spectra recorded at Hefei on 16 February 2016 with an ideal ILS. Figure 4. Averaging kernels of 10 NDACC gases (coloured ﬁne lines) and their area scaled by a factor of 0.2 (black bold line). They are deduced from the spectra recorded at Hefei on 16 February 2016 with an ideal ILS. of view (FOV) of the spectrometer (Davis et al., 2001). unavoidable parts of the line shape is called the ideal line shape. SINC(σ,L) = 2Lsin(2πσL) 2πσL (1) RECT(σ,σ0,θ) =    2 σ0θ2 if −0.5σ0θ2 ≤σ ≤0 0 otherwise (2) (1) The theoretical ideal ILS, as deﬁned in Eq. (3), when the instrument is well aligned, is a convolution of sinc and rect- angular functions (deﬁned in Eqs. 1 and 2), representing the ﬁnite length of the interferogram and the ﬁnite circular ﬁeld (3) www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 2883 www.atmos-meas-tech.net/11/2879/2018/ Y. Sun et al.: The inﬂuence of instrumental line shape degradation In this study, we assume Sε to be diagonal and its diagonal elements are the inverse square of the SNR. The vertical information content of the retrieved target gas proﬁle can be quantiﬁed by the number of degrees of freedom for signal (DOFs), which is the trace of A, deﬁned in Rodgers (2000) by where Gy is the sensitivity of the retrieval to the measure- ment. Kx is the weighting function matrix or Jacobian ma- trix that links the measurement vector y to the state vector x: 1y = Kx1x. A characterizes the vertical information con- tained in the FTIR retrievals. In this study, we assume Sε to be diagonal and its diagonal elements are the inverse square of the SNR. The vertical information content of the retrieved target gas proﬁle can be quantiﬁed by the number of degrees of freedom for signal (DOFs), which is the trace of A, deﬁned in Rodgers (2000) by ds = tr(A) = tr KT x S−1 ε KT x + S−1 a −1 KT x S−1 ε KT x . (5) (5) The diagonal elements of Sa represent the assumed variabil- ity of the target gas volume mixing ratio (VMR) at a given altitude, and the off-diagonal elements represent the correla- tion between the VMR at different altitudes. We can see in Table 3 that, except CO and HCN, the target gases are using an a priori covariance matrix with diagonal elements con- stant, with altitudes corresponding to 10, 20, 50, or 100 % variability; the largest variabilities are for HNO3, HCl, and ClONO2. For CO, the diagonal elements of Sa correspond to 27 % from the ground up to 34 km and decrease down to 11 % at the top of the atmosphere. For HCN, the diagonal elements of Sa correspond to 79 % from the ground up to 5 km and de- crease down to 21 % at the top of atmosphere. No correlation of off-diagonal matrix elements is assumed in all retrievals except for ClONO2, which assumes exponential correlation with a HWHM (half-width at half-maximum) of 8 km. The SNR values for all retrievals are the real values taken from each individual spectrum. The ILSs for all retrievals are ob- tained using the simulations in Sect. 3. Y. Sun et al.: The inﬂuence of instrumental line shape degradation Gases O3 HNO3 HCl HF ClONO2 CH4 CO N2O C2H6 HCN MW for proﬁle 1000–1004.5 867.5–870 2727.73–2727.83 4109.4–4110.2 779.85–780.45 2613.7–2615.4 2057.7–2058 2441.8–2444.6 2976–2978 3268–3268.38 retrievals 2775.7–2775.8 782.55–782.87 2835.5–2835.8 2069.56–2069.76 2481.2–2482.5 2982.6–2984.5 3287–3287.48 2925.8–2926.0 2921.0–2921.6 2157.5–2159.15 Retrieved interfering H2O, CO2, C2H4, H2O, OCS, NH3 CH4, NO2, O3, H2O, HDO, CH4 O3, HNO3, CO2, NO2, O3, N2O, CO2, CO2, CH4 H2O, CH4, H2O, O3, C2H2, gases O3668, O3686 N2O, HDO H2O, CO2 H2O, HDO OCS, H2O O3CH4 H2O treatment Proﬁle retrieval Scaling retrieval Proﬁle retrieval Proﬁle retrieval Scaling retrieval Proﬁle retrieval Proﬁle retrieval Proﬁle retrieval Proﬁle retrieval Proﬁle retrieval SNR for de-weighting None None 300 None None None 500 None None None Sa Diagonal: 20 % Diagonal: 50 % Diagonal: 50 % Diagonal: 10 % Diagonal: 100 % Diagonal: 10 % Diagonal: 11 % Diagonal: 10 % Diagonal: 10 % Diagonal: 21 % No correlation No correlation No correlation No correlation Exponential correlation No correlation ∼27 % ∼79 % HWHM: 8 km No correlation No correlation No correlation No correlation Error analysis Systematic error: – smoothing error – errors from parameters not retrieved by sﬁt4∗: background curvature, optical path difference, ﬁeld of view, solar line strength, background slope, solar line shift, phase, solar zenith angle, line temperature broadening, line pressure broadening, line intensity Random error: – interference errors: retrieval parameters, interfering species – measurement error – errors from parameters not retrieved by sﬁt4∗: temperature, zero level ∗The input uncertainties of all these items are the same and are included in the error analysis if they are not retrieved. Otherwise, the corresponding uncertainties are not included. The selection of the regularization (a priori covariance ma- trix Sa and SNR) cannot be easily standardized because it depends on the real variability for each gas. In optimal esti- mation, the selection of Sa is very important in the inversion process and, together with the measurement noise error co- variance matrix Sε, will lead to the following averaging ker- nel matrix A (Rodgers, 2000): A = GyKx = (KT x S−1 ε KT x + S−1 a )−1KT x S−1 ε Kx, (4) (4) where Gy is the sensitivity of the retrieval to the measure- ment. Kx is the weighting function matrix or Jacobian ma- trix that links the measurement vector y to the state vector x: 1y = Kx1x. A characterizes the vertical information con- tained in the FTIR retrievals. 3 Simulation of ILS degradation We use the program ALIGN60 to simulate ILS degradation in a high-resolution FTIR spectrometer typically used in the NDACC. As an auxiliary tool of LINEFIT, ALIGN60 is a ray-tracing model for FTIR spectrometers following the clas- sical Michelson design, assuming one ﬁxed and one mov- able arm, and using cube corners instead of plane mirrors. It calculates the resulting phase distortions in the recom- bined beam and from these deduces the variable intensity observed by the detector. ALIGN60 takes into account the lateral shear error of the movable retro-reﬂector as a func- tion of the OPD, a decentring of the ﬁeld stop with respect to the optical axis, an unsharp boundary line or deformation of the ﬁeld stop image (as possibly caused by a defocused col- limator), and vignetting effects with increasing OPD. It can generate trustworthy results with respect to all types of mis- alignment (Hase et al., 1999). In this simulation, the entrance beam section was assumed to be circular with a diameter of 8.0 cm. The ILS was only calculated from the positive side of the interferogram. The smear and vignetting effects were not taken into account. The misalignment of a FTIR spectrome- ter can be expressed via two perpendicular axes perpendic- ular to the beam direction. For a circular entrance beam, the same misalignment in either direction results in a similar ILS. Thus, this work only considers misalignment in one axis. The misalignments as inputs of ALIGN60 are listed in Ta- ble 1, the resulting ILSs are shown in Fig. 1, and the corre- sponding Haidinger fringes at the maximum OPD are shown in Fig. 2. The ME deviation, the decentring of Haidinger fringes, and the ILS deterioration varying over misalignment are evident. All types of misalignment cause non-linear ME deviations except the decentring of measuring laser (c) and the constant shear (d), which mainly affect PE and result in www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 2884 Y. Sun et al.: The inﬂuence of instrumental line shape degradation Table 2. Summary of the retrieval parameters used for all NDACC gases. All microwindows (MWs) are given in cm−1. CO and HCl which utilize a de-weighting SNR of 500 and 300, respectively. CO and HCl which utilize a de-weighting SNR of 500 and 300, respectively. Table 2. Summary of the retrieval parameters used for all NDACC gases. All microwindows (MWs) are given in cm−1. Gases O3 HNO3 HCl HF ClONO2 CH4 CO N2O C2H6 HCN MW for proﬁle 1000–1004.5 867.5–870 2727.73–2727.83 4109.4–4110.2 779.85–780.45 2613.7–2615.4 2057.7–2058 2441.8–2444.6 2976–2978 3268–3268.38 retrievals 2775.7–2775.8 782.55–782.87 2835.5–2835.8 2069.56–2069.76 2481.2–2482.5 2982.6–2984.5 3287–3287.48 2925.8–2926.0 2921.0–2921.6 2157.5–2159.15 Retrieved interfering H2O, CO2, C2H4, H2O, OCS, NH3 CH4, NO2, O3, H2O, HDO, CH4 O3, HNO3, CO2, NO2, O3, N2O, CO2, CO2, CH4 H2O, CH4, H2O, O3, C2H2, gases O3668, O3686 N2O, HDO H2O, CO2 H2O, HDO OCS, H2O O3CH4 H2O treatment Proﬁle retrieval Scaling retrieval Proﬁle retrieval Proﬁle retrieval Scaling retrieval Proﬁle retrieval Proﬁle retrieval Proﬁle retrieval Proﬁle retrieval Proﬁle retrieval SNR for de-weighting None None 300 None None None 500 None None None Sa Diagonal: 20 % Diagonal: 50 % Diagonal: 50 % Diagonal: 10 % Diagonal: 100 % Diagonal: 10 % Diagonal: 11 % Diagonal: 10 % Diagonal: 10 % Diagonal: 21 % No correlation No correlation No correlation No correlation Exponential correlation No correlation ∼27 % ∼79 % HWHM: 8 km No correlation No correlation No correlation No correlation Error analysis Systematic error: – smoothing error – errors from parameters not retrieved by sﬁt4∗: background curvature, optical path difference, ﬁeld of view, solar line strength, background slope, solar line shift, phase, solar zenith angle, line temperature broadening, line pressure broadening, line intensity Random error: – interference errors: retrieval parameters, interfering species – measurement error – errors from parameters not retrieved by sﬁt4∗: temperature, zero level ∗The input uncertainties of all these items are the same and are included in the error analysis if they are not retrieved. Otherwise, the corresponding uncertainties are not included. Table 2. Summary of the retrieval parameters used for all NDACC gases. All microwindows (MWs) are given in cm−1. 4.2 Averaging kernels The rows of A are the so-called averaging kernels and they represent the sensitivity of the retrieved proﬁle to the real proﬁle. Their full-width at half-maximum (FWHM) is a mea- sure of the vertical resolution of the retrieval at a given alti- tude. The area of averaging kernels represents sensitivity of the retrievals to the measurement. This sensitivity at altitude k is calculated as the sum of the elements of the correspond- ing averaging kernels, P iAki. It indicates the fraction of the HCN Atmos. Meas. Tech., 11, 2879–2896, 2018 www.atmos-meas-tech.net/11/2879/2018/ Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2885 Table 3. Altitude ranges with sensitivities larger than 0.5 for all NDACC gases. Items O3 HNO3 HCl HF ClONO2 CH4 CO N2O C2H6 HCN Altitude Ground–44 17–28 18–42 18–44 20–28 Ground–31 Ground–27 Ground–31 Ground–13.5 4.5–18 ranges (km) Total 5.2 1.4 1.5 1.3 0.55 3.5 3.8 4.0 1.2 1.1 DOFs Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2885 5 ILS inﬂuence study retrieval at each altitude that comes from the measurement rather than from the a priori information (Rodgers, 2000). A value close to zero at a certain altitude indicates that the retrieved proﬁle at that altitude is nearly independent of the measurement and is therefore approaching the a priori pro- ﬁle. This section presents the ILS inﬂuence study, whereby the degraded ILSs that are simulated by ALIGN60 are used in the SFIT forward model, and the fractional differences (D %) in various quantities for each gas relative to the retrieval with an ideal ILS are computed. For each gas, in Sect. 5.1 and 5.2 only one typical spectrum is selected for study. In order to retrieve these 10 gases, 5 spectra with different wavenum- ber coverage are used. All of them are randomly selected from the routine measurements on a clear day at Hefei on 16 February 2016. The consistency of the resulting deduction is evaluated in Sect. 5.3, for which 1 year of measurements from August 2015 to August 2016 was used. The Hefei site has run NDACC observations with the Bruker 125HR for more than 3 years. We regularly use a low-pressure HBr cell to diagnose the misalignment of the spectrometer and to re- align the instrument when indicated. As shown in Fig. 5, all actual ILS degradations of the FTIR spectrometer within this selected period are less than 2 % and can be regarded as be- ing ideal. For all spectra used in this study, the retrievals with all levels of ILS degradation fulﬁl the following ﬁlter criteria. The averaging kernels and their areas for these 10 NDACC gases are shown in Fig. 4. The altitude ranges with sensi- tivity larger than 0.5 and the corresponding total DOFs are summarized in Table 3. These sensitivity ranges indicate that the retrieved proﬁle information comes, by more than 50 %, from the measurement, or, in other words, that the a pri- ori information inﬂuences the retrieval by less than 50 %. Each gas has a different sensitivity range. The sensitivity range for HCN, CO, and C2H6 is mainly tropospheric, and for ClONO2, HCl, and HF, it is mainly stratospheric. O3, CH4, and N2O have high retrieval sensitivity in both the tro- posphere and the stratosphere. The HNO3 has high retrieval sensitivity in the stratosphere and in the atmospheric bound- ary layer below 1.5 km. 4.3 Error analysis 1. The root mean square (rms) of the residual (difference between measured and calculated spectra after the ﬁt) in all ﬁtting windows has to be less than 3 %. As listed in Table 2, we classiﬁed errors as systematic or ran- dom according to whether they are constant between con- secutive measurements or whether they vary randomly. For comparison, the error items considered in error analysis are the same for the retrieval of all gases. The smoothing error Es is calculated via Eq. (6), the measurement error Em is calcu- lated via Eq. (7), and all other error items Evar are calculated via Eq. (8) (Rodgers, 2000). 2. The retrievals should converge for all levels of ILS degradation. 3. The concentrations of the target and interfering gases at each sublayer should be positive. 4. The solar intensity variation (SIV) should be less than 10 %. The SIV within the duration of a spectrum is the ratio of the standard deviation to the average of the mea- sured solar intensities. Es = (A −I)Sa(A −I)T (6) Em = GySεGT y (7) Evar = GyKvarSvarKT varGT y , (8) (8) These criteria are used to remove those spectra that have sam- pling errors or are contaminated by aerosols, clouds, hazes, or other unpredictable objects which cause a low SNR or a large detecting intensity variation. In the following calcula- tions, we have taken the retrievals with an ideal ILS as the reference. The fractional difference is deﬁned here as where Svar is the error covariance matrix of var. Kvar is the weighting function matrix of var. Here var refers to any one of the error items in Table 2, except smoothing error and mea- surement error. In this study, the a priori error covariance for all non-retrieval parameters is set the same for all gases’ re- trieval. D % = X −Xref Xref × 100, (9) (9) (9) 5.1 ME amplitude and PE inﬂuence In order to determine how the ILS degradation affects the NDACC gas retrievals, the results deduced from ILS consid- ering both ME amplitude and PE are compared to those only considering ME amplitude or PE. All types of ILS degrada- tion in Sect. 3 are used in this study. Figure 6 exempliﬁes the case of ILS j, where the differences in the total column, the rms, the random uncertainty, the systematic uncertainty, the total uncertainty, and DOFs for each gas relative to the retrieval are compared with an ideal ILS. Figure 7 shows the fractional difference in proﬁle of each gas for ILS j. The re- sults show that the inﬂuence of ILS degradation on the total column, the rms, the random uncertainty, the systematic un- certainty, the total uncertainty, DOFs, and the proﬁle can be approximated by the linear sum of individual ME amplitude inﬂuence and PE inﬂuence. The PE inﬂuence is of secondary importance compared with the ME amplitude inﬂuence. The comparisons for the results retrieved with ILS a to i come to the same conclusions. With respect to the total column, the inﬂuence of ILS degradation on the stratospheric gases is generally larger than the tropospheric gases. For a typical ILS degradation (10 %), the total columns of stratospheric gases O3, HNO3, HCl, HF, and ClONO2 changed by 1.9, 0.7, 4, 3, and 23 %, respec- tively, while the total columns of tropospheric gases CH4, CO, N2O, C2H6, and HCN changed by 0.04, 2.1, 0.2, 1.1, and 0.75 %, respectively. For O3 and HNO3, positive ME causes an overestimated total column and negative ME causes an underestimated total column. For other gases, negative ME causes an overestimated total column and positive ME causes an underestimated total column. For all gases except O3 and CH4, the positive ME inﬂuence is larger than the negative ME inﬂuence. For CH4, the negative ME inﬂuence is larger than the positive ME inﬂuence. For O3, the level of the pos- Figures 8 and 9 show the inﬂuence of ILS a to j on the total column and the proﬁle of all NDACC gases. The result- ing inﬂuence amounts depend on the deviation amount and the deviation shape of ME. For positive MEs, in most cases, the ILS j causes the maximum inﬂuence, and for negative MEs, the ILS i causes the maximum inﬂuence. 5.2 Sensitivity study We simulated seven levels of negative ME i and positive ME j with ALIGN60, incorporated them in the SFIT forward model, and then calculated the fractional difference in var- ious quantities for each gas relative to the retrieval with an ideal ILS. The misalignments as inputs of ALIGN60 and the resulting ILSs are shown in Figs. 10 and 12. The correspond- ing Haidinger fringes at the maximum misalignment position are shown in Figs. 11 and 13. The ME deviation, the decen- tring of Haidinger fringes, and the ILS deterioration varying over misalignment are evident. Figure 14 is the sensitivity of the total column with respect to different levels of ILS degradation. Figures 15–18 are the same as Fig. 14 but for DOFs, the rms, the uncertainty, and the proﬁle. The results show that the ILS degradation affected the total column, the rms, DOFs, the retrieval uncertainty, and the proﬁle. Gen- erally, the larger the ME deviation, the larger the inﬂuence. The positive and negative ME have opposite inﬂuence on to- tal column, DOFs, the total uncertainty and proﬁle. www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 2886 Y. Sun et al.: The inﬂuence of instrumental line shape degradation Figure 5. ME amplitudes (a) and phase errors (b) along with the OPD deduced from HBr cell measurements at Hefei. Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2886 Figure 5. ME amplitudes (a) and phase errors (b) along with the OPD deduced from HBr cell measurements the following, misalignment i and j are selected on behalf of negative and positive ME, respectively, to investigate how the ILS degradation inﬂuences the NDACC gas retrievals. where X is a vector which can include multiple elements such as the gas proﬁle or only one element such as DOFs, the rms, the total column, the total random uncertainty, the total systematic uncertainty, or the total uncertainty. The to- tal random uncertainty and the systematic uncertainty are the sum in quadrature of each individual uncertainty listed in Ta- ble 2, and the total uncertainty is the sum in quadrature of the total random uncertainty and the total systematic uncertainty. Xref is the same as X but for the nominal ideal ILS. 5.1 ME amplitude and PE inﬂuence In a real in- strument, the misalignment is a combination of misalignment a to j. In principle, for the same misalignment amplitude, it should not cause inﬂuence exceeding misalignment i or j. In www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2887 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2887 Figure 6. Fractional difference in the total column, the rms, the total random uncertainty, the total systematic uncertainty, the total uncertainty, and DOFs for misalignment j. “ME amplitude” represents the ILS which only took ME amplitude deviation into account. “PE” represents the ILS which only took PE deviation into account. “ME amplitude & PE” represents the ILS which took both the ME amplitude and PE deviations into account. The fractional difference of each item is the “linear sum” of the ME amplitude and PE. The ME amplitude and PE are obtained from ALIGN60 with misalignment j in Fig. 1. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 6. Fractional difference in the total column, the rms, the total random uncertainty, the total systematic uncertainty, the total uncertainty, and DOFs for misalignment j. “ME amplitude” represents the ILS which only took ME amplitude deviation into account. “PE” represents the ILS which only took PE deviation into account. “ME amplitude & PE” represents the ILS which took both the ME amplitude and PE deviations into account. The fractional difference of each item is the “linear sum” of the ME amplitude and PE. The ME amplitude and PE are obtained from ALIGN60 with misalignment j in Fig. 1. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 7. Fractional difference in the proﬁle for misalignment j. The nomenclature in the plot legend is the same as Fig. 6. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 7. Fractional difference in the proﬁle for misalignment j. The nomenclature in the plot legend is the same as Fig. 6. The results are deduced from the spectra recorded at Hefei on 16 February 2016. itive ME inﬂuence and the negative ME inﬂuence is very close. tive ME increases rms, while negative ME decreases rms. For CH4, positive ME decreases rms and negative ME increases rms. 5.1 ME amplitude and PE inﬂuence For all gases, positive ME increases the DOFs and negative ME decreases DOFs. For all gases except HF and CH4, both positive ME and negative ME increase rms. For HF, posi- The inﬂuence on the systematic uncertainty and the ran- dom uncertainty depends on the ME deviation type and the www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2888 Figure 8. Sensitivity of the total column to different types of ILS degradation. The ILSs a to j correspond to misalignment a to j in Table 1. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 8. Sensitivity of the total column to different types of ILS degradation. The ILSs a to j correspond to misalignment a to j in Table 1. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 9. Sensitivity of the proﬁle to different types of ILS degradation. The ILSs a to j correspond to misalignment a to j in Table 1. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 9. Sensitivity of the proﬁle to different types of ILS degradation. The ILSs a to j correspond to misa results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 9. Sensitivity of the proﬁle to different types of ILS degradation. The ILSs a to j correspond to misalignment a to j in Table 1. The results are deduced from the spectra recorded at Hefei on 16 February 2016. The ILS degradation causes an evident difference in the proﬁle within the altitude ranges that show high retrieval sen- sitivity in Fig. 4, or in other words, the sensitive ranges listed in Table 3. Generally, the proﬁle is more sensitive to positive ME than negative PE, and the inﬂuence of ILS degradation on the stratospheric gases is larger than on the tropospheric gases. gas type. The inﬂuence on the total uncertainty is the com- bination of the inﬂuence on the total systematic uncertainty and the total random uncertainty. For all gases except O3, positive ME decreases the total uncertainty and negative ME increases the total uncertainty. For O3, positive ME increases the total uncertainty and negative ME decreases the total un- certainty. Atmos. Meas. Tech., 11, 2879–2896, 2018 www.atmos-meas-tech.net/11/2879/2018/ Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2889 Y. Sun et al.: The inﬂuence of instrumental line shape degradation Y. Sun et al.: The inﬂuence of instrumental line shape degradation Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2889 Figure 10. Simulated positive ME deviations along with the OPD. Panel (a) demonstrates the misalignment, panel (b) shows the resulting ILS, panel (c) shows the resulting ME amplitude, and panel (d) shows the resulting PE. Figure 10. Simulated positive ME deviations along with the OPD. Panel (a) demonstrates the misalignment, panel (b) shows the resulting ILS, panel (c) shows the resulting ME amplitude, and panel (d) shows the resulting PE. Figure 11. The Haidinger fringes at maximum OPD (the maximum misalignment position) for Fig. 10. Figure 11. The Haidinger fringes at maximum OPD (the maximum misalignment position) for Fig. 10. Atmos. Meas. Tech., 11, 2879–2896, 2018 Atmos. Meas. Tech., 11, 2879–2896, 2018 www.atmos-meas-tech.net/11/2879/2018/ www.atmos-meas-tech.net/11/2879/2018/ Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2890 2890 Y. Sun et al.: The inﬂuence of instrumental line shape degradation Figure 12. Simulated negative ME deviations along with the OPD. Panel (a) demonstrates the misalignment, panel (b) shows the resulting ILS, panel (c) shows the resulting ME amplitude, and panel (d) shows the resulting PE. Figure 12. Simulated negative ME deviations along with the OPD. Panel (a) demonstrates the misalignment, panel (b) shows the resulting ILS, panel (c) shows the resulting ME amplitude, and panel (d) shows the resulting PE. Figure 13. The Haidinger fringes at half-maximum OPD (the maximum misalignment position) for Fig. 12. 12. Figure 13. The Haidinger fringes at half-maximum OPD (the maximum misalignment position) for Fig. 12. 5.3 Consistency evaluation retrievals that fulﬁl the above ﬁlter criteria are included in this study. A simulated ILS j with a maximum ME ampli- tude deviation of 5 % is used in the retrieval. The results are compared to the retrievals deduced from an ideal ILS. This section uses the spectra recorded at Hefei from August 2015 to August 2016 to evaluate the consistency of the above study. These spectra span a large range of atmospheric water vapour, solar zenith angles (SZAs), surface pressures, surface temperatures, wind speeds, and wind directions (Fig. 19). All Figure 20 exempliﬁes the fractional differences in the to- tal column, the rms, the total uncertainty, and DOFs for each gas as a function of the SZA. The results show that the frac- www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. Sun et al.: The inﬂuence of instrumental line shape degradation Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2891 Figure 14. Sensitivity of the total column with respect to ME deviation. “P_Tclmn” represents the sensitivity of the total column with respect to positive ME deviation and “N_Tclmn” represents the sensitivity of the total column with respect to negative ME deviation. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 14. Sensitivity of the total column with respect to ME deviation. “P_Tclmn” represents the sensitivity of the total column with respect to positive ME deviation and “N_Tclmn” represents the sensitivity of the total column with respect to negative ME deviation. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 15. The same as Fig. 14 but for DOFs and rms of the ﬁtting residual. The acronyms in the legend are similar to those in Fig. 14. Figure 15. The same as Fig. 14 but for DOFs and rms of the ﬁtting residual. The acronyms in the legend are similar to those in Fig. 14. Figure 15. The same as Fig. 14 but for DOFs and rms of the ﬁtting residual. The acronyms in the legend are Fig. 14 but for DOFs and rms of the ﬁtting residual. The acronyms in the legend are similar to those in Fig. 14 der different SZAs. The fractional differences as a function of the humidity, the pressure, the SZA, the temperature, the wind direction, and the wind speed show the same conclu- sions. tional differences in the total column, the total uncertainty, and DOFs for all gases are consistent under different SZAs. For most gases, the fractional difference in rms exhibits more scatter than the total column, the total uncertainty, and DOFs. However, they are independent of the SZA, and most of them are less than 10 %. In general, the inﬂuence of ILS degrada- tion on NDACC gases’ retrieval shows good consistency un- Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2892 Figure 16. The same as Fig.14 but for the total random uncertainty, the total systematic uncertainty, and the total uncertainty. The acronyms in the legend are similar to those in Fig. 14. “Trnd”, “Tsys”, and “Tstd” represent the total random uncertainty, the total systematic uncertainty, and the total uncertainty, respectively. Figure 16. The same as Fig.14 but for the total random uncertainty, the total systematic uncertainty, and the total uncertainty. The acronyms in the legend are similar to those in Fig. 14. “Trnd”, “Tsys”, and “Tstd” represent the total random uncertainty, the total systematic uncertainty, and the total uncertainty, respectively. Figure 17. Sensitivity of the proﬁle with respect to ME deviation. “4 %” denotes a ME amplitude deviation of 4 %. The nomenclature for other plot labels is straightforward. The results are deduced from the spectra recorded at Hefei on 16 February 2016. Figure 17. Sensitivity of the proﬁle with respect to ME deviation. “4 %” denotes a ME amplitude deviation of 4 %. The nomenclature for other plot labels is straightforward. The results are deduced from the spectra recorded at Hefei on 16 February 2016. www.atmos-meas-tech.net/11/2879/2018/ Y. Sun et al.: The inﬂuence of instrumental line shape degradatio Y. Sun et al.: The inﬂuence of instrumental line shape degradation Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2893 Figure 18. The same as Fig. 17 but for negative ME deviation. Figure 18. The same as Fig. 17 but for negative ME deviation. gure 19. The meteorological data and SZA record at Hefei. Large span of all these parameters is shown for the period from August 20 Figure 19. The meteorological data and SZA record at Hefei. Large span of all these parameters is shown for the period from August 2015 to August 2016 (black dotted squares). Figure 19. The meteorological data and SZA record at Hefei. Large span of all these parameters is shown for the period from August 2015 to August 2016 (black dotted squares). cal data and SZA record at Hefei. Large span of all these parameters is shown for the period from August 201 d squares). aggravates the mismatch between the calculated spectra and the measured spectra. for ClONO2 and other NDACC gases as 10 and 1 %, respec- tively. Considering that an excessively large ME degradation (e.g. > 20 %) seldom occurred within NDACC because of the regular alignment at each site, the maximum permitted ILS degradation for each gas is given in Table 4 and is deduced by the following points. The stratospheric gases are more sensitive to ILS degra- dation than the tropospheric gases, and ClONO2 exhibits the largest sensitivity. This is because the absorption structure in the stratosphere is narrower than that in the troposphere and is therefore more easily affected by ILS degradation. We set the acceptable fractional difference in the total column Atmos. Meas. Tech., 11, 2879–2896, 2018 6 Discussion and recommendations quantities such as the total column, the rms, the random un- certainty, the systematic uncertainty, the total uncertainty, DOFs, and the proﬁle. The change of Kx is attributed to the fact that the ILS degradation alters the gas absorption line shape, and hence alters the structure of calculated spectra and For each gas, the a priori covariance matrices of Sa, Sε, and Svar are the same in the aforementioned study. According to Eqs. (6–8), we conclude that the ILS degradation altered the weighting function matrix Kx and eventually altered the www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. Sun et al.: The inﬂuence of instrumental line shape degradation 28 Figure 18. The same as Fig. 17 but for negative ME deviation. Figure 19. The meteorological data and SZA record at Hefei. Large span of all these parameters is shown for the period from August 20 to August 2016 (black dotted squares). aggravates the mismatch between the calculated spectra and the measured spectra. The stratospheric gases are more sensitive to ILS degra for ClONO2 and other NDACC gases as 10 and 1 %, resp tively. Considering that an excessively large ME degradat (e g > 20 %) seldom occurred within NDACC because of Y. Sun et al.: The inﬂuence of instrumental line shape degradation 2894 Figure 20. Fractional difference in the total column, the rms, the total uncertainty, and DOFs as a function of the SZA from August 2015 to August 2016, for which the ILS j with a maximum ME deviation of 5 % is used. Figure 20. Fractional difference in the total column, the rms, the total uncertainty, and DOFs as a function of the SZA from August 2015 to August 2016, for which the ILS j with a maximum ME deviation of 5 % is used. Table 4. Recommendations for suppressing the fractional difference in the total column for ClONO2 and other NDACC gases within 10 and 1 %, respectively. le 4. Recommendations for suppressing the fractional difference in the total column for ClONO2 and other NDACC gases within 10 and respectively. Items O3 HNO3 HCl HF ClONO2 CH4 CO N2O C2H6 HCN Positive ME < 6 % < 15 % < 5 % < 5 % < 5 % ∗ < 5 % ∗ < 9 % < 13 % Negative ME < 6 % ∗ < 12 % < 12 % ∗ ∗ ∗ ∗ ∗ ∗ ∗The inﬂuence on ClONO2 is less than 10 % and the inﬂuences on all other NDACC gases are less than 1 %. Even an excessively large ILS degradation of 28 % can normally be regarded as being negligible. ∗The inﬂuence on ClONO2 is less than 10 % and the inﬂuences on all other NDACC gases are less than 1 %. Even an excessively large ILS degradation of 28 % can normally be regarded as being negligible. 1. The inﬂuence of ILS degradation on CH4 and N2O can be regarded as being negligible. www.atmos-meas-tech.net/11/2879/2018/ Y. Sun et al.: The inﬂuence of instrumental line shape degradatio Y. Sun et al.: The inﬂuence of instrumental line shape degradation www.atmos-meas-tech.net/11/2879/2018/ Y. Sun et al.: The inﬂuence of instrumental line shape degradation satellite and model data, J. Geophys. Res., 115, D22306, https://doi.org/10.1029/2010JD014677, 2010. spheric gases O3, HNO3, HCl, HF, and ClONO2 changed by 1.9, 0.7, 4, 3, and 23 %, respectively, while the columns of tropospheric gases CH4, CO, N2O, C2H6, and HCN changed by 0.04, 2.1, 0.2, 1.1, and 0.75 %, respectively. The inﬂuence of instrumental line shape degradation on NDACC gas re- trievals shows good consistency under different meteorolog- ical conditions and solar zenith angle. In order to suppress the fractional difference in the total column for ClONO2 and other NDACC gases within 10 and 1 %, respectively, the maximum positive modulation efﬁciency degradations for O3, HNO3, HCl, HF, ClONO2, CO, C2H6, and HCN should be less than 6, 15, 5, 5, 5, 5, 9, and 13 %, respectively. The maximum negative modulation efﬁciency degradations for O3, HCl, and HF should be less than 6, 12, and 12 %, respec- tively; the inﬂuence of ILS degradation on CH4 and N2O can be regarded as being negligible. Feist, D. G., Arnold, S. G., Hase, F., and Ponge, D.: Rugged optical mirrors for Fourier transform spectrometers operated in harsh environments, Atmos. Meas. Tech., 9, 2381–2391, https://doi.org/10.5194/amt-9-2381-2016, 2016. Hannigan, J. and Coffey, M.: semiautonomous FTS ob- servation system for remote sensing of stratospheric and tropospheric gases, J. Atmos. Ocean. Tech., 26, https://doi.org/10.1175/2009JTECHA1230.1, 2009. Hase, F.: Improved instrumental line shape monitoring for the ground-based, high-resolution FTIR spectrometers of the Net- work for the Detection of Atmospheric Composition Change, Atmos. Meas. Tech., 5, 603–610, https://doi.org/10.5194/amt-5- 603-2012, 2012. Hase, F., Blumenstock, T., and Paton-Walsh, C.: Analysis of the instrumental line shape of high-resolution Fourier transform IR spectrometers with gas cell measurements and new retrieval software, Appl. Opt., 38, 3417–3422, https://doi.org/10.1364/AO.38.003417, 1999 (data available at: http://www.imk-asf.kit.edu/english/897.php, last access: 11 May 2018). Data availability. The ALIGN60 software is obtained on request from KIT Karlsruhe (http://www.imk-asf.kit.edu/english/897.php) (Hase et al., 1999). The SFIT4 software can be found via https: //www2.acom.ucar.edu/irwg/links (last access: 11 May 2018). The input ﬁles used for ALIGN60 and SFIT4 are attached in the Sup- plement. Hase, F., Demoulin, P., Sauval, A., Toon, G. C., Bernath, P., Gold- man, A., Hannigan, J., and Rinsland C.: An empirical line-by- line model for the infrared solar transmittance spectrum from 700 to 5000 cm−1, J. Quant. Spectrosc. Ra., 102, 450–463, 2006. Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. This work is jointly supported by the National High Technology Research and Development Program of China (no. 2016YFC0200800, 2016YFC0203302, 2016YFC0200404, and 2017YFC0210002), the National Science Foundation of China (no. 41605018, 41775025, 41405134, 41575021, 51778596, 91544212, and 41722501), the Anhui Province Natural Science Foundation of China (no. 1608085MD79), and the German Federal Ministry of Education and Research (BMBF) (grant no. 01LG1214A). The processing environment of SFIT4 and some plot programs are provided by the National Center for Atmo- spheric Research (NCAR), Boulder, Colorado, USA. The NDACC is acknowledged for supplying the SFIT software and giving advice. Kohlhepp, R., Barthlott, S., Blumenstock, T., Hase, F., Kaiser, I., Raffalski, U., and Ruhnke, R.: Trends of HCl, ClONO2, and HF column abundances from ground-based FTIR mea- surements in Kiruna (Sweden) in comparison with KASIMA model calculations, Atmos. Chem. Phys., 11, 4669–4677, https://doi.org/10.5194/acp-11-4669-2011, 2011. Kurylo, M. J.: Network for the detection of stratospheric change (NDSC), SPIE Proceedings 1991, P. Soc. Photo-Opt. Ins., 1491, 168–174, 1991. Messerschmidt, J., Macatangay, R., Notholt, J., Petri, C., Warneke, T., and Weinzierl, C.: Side by side measurements of CO2 by ground-based Fourier transform spectrometry (FTS), Tellus B, 62, 749–758, https://doi.org/10.1111/j.1600-0889.2010.00491.x, 2010. Edited by: Gabriele Stiller Reviewed by: two anonymous referees Reviewed by: two anonymous referees Olsen, S. C. and Randerson, J. T.: Differences between sur- face and column atmospheric CO2 and implications for car- bon cycle research, J. Geophys. Res.-Atmos., 109, D02301, https://doi.org/10.1029/2003JD003968, 2004. References Rodgers, C. D.: Inverse methods for atmospheric sounding: The- ory and Practice, Series on Atmospheric, Oceanic and Plane- tary Physics, vol. 2, World Scientiﬁc Publishing Co., Singapore, 2000. Davis, S. P., Abrams, M. C., and Brault, J. W.: Fourier transform spectrometry, Academic Press, ISBN: 0-12-042510-6, 2001. Davis, S. P., Abrams, M. C., and Brault, J. W.: Fourier transform spectrometry, Academic Press, ISBN: 0-12-042510-6, 2001. Duchatelet P., Demoulin P., Hase F., Ruhnke R., Feng W., Chip- perﬁeld M. P., Bernath P. F., Boone C. D., Walker K. A., and Mahieu E.: Hydrogen ﬂuoride total and partial column time series above the Jungfraujoch from long term FTIR mea- surements: Impact of the line shape model, characterization of the error budget and seasonal cycle, and comparison with Duchatelet P., Demoulin P., Hase F., Ruhnke R., Feng W., Chip- perﬁeld M. P., Bernath P. F., Boone C. D., Walker K. A., and Mahieu E.: Hydrogen ﬂuoride total and partial column time series above the Jungfraujoch from long term FTIR mea- surements: Impact of the line shape model, characterization of the error budget and seasonal cycle, and comparison with Rothman, L. S., Gordon, I. E., Barbe, A., Benner, D. C., Bernath, P. F., Birk, M., Boudon, V., Brown, L. R., Campargue, A., Champion, J.-P., Chance, K., Coudert, L. H., Danaj, V., Devi, V. M., Fally, S., Flaud, J.-M., Gamache, R. R., Goldmanm, A., Jacquemart, D., Kleiner, I., Lacome, N., Lafferty, W. J., Y. Sun et al.: The inﬂuence of instrumental line shape degradation Kalnay, E., Kanamitsu, M., Kistler, R., Collins, W., Deaven, D., Gandin, L., Iredell, M., Saha, S., White, G., Woollen, J., Zhu Y., Chelliah, M., Ebisuzaki, W., Higgins, W., Janowiak, J., Mo, K. C., Ropelewski, C., Wang, J., Leetmaa, A., Reynolds, R., Jenne, R., and Joseph, D. : The NCEP/NCAR 40-year reanalysis project, B. Am. Meteorol. Soc., 77, 437–472, 1996. 7 Conclusions We assessed the inﬂuence of instrumental line shape degra- dation on all current NDACC gases’ retrieval via investiga- tion of sensitivities of the total column, the root mean square of the ﬁtting residual, the total random uncertainty, the total systematic uncertainty, the total uncertainty, degrees of free- dom, and the proﬁle with respect to modulation efﬁciency degradations. The study concluded that the inﬂuence of in- strumental line shape degradation can be approximated by the linear sum of the individual modulation efﬁciency am- plitude inﬂuence and the phase error inﬂuence. The phase error inﬂuence is of secondary importance compared with the modulation efﬁciency amplitude inﬂuence. The inﬂuence amounts depend on the deviation amount and the deviation shape of the modulation efﬁciency. 2. If a misalignment causes positive ME degradation, the maximum degradations for O3, HNO3, HCl, HF, ClONO2, CO, C2H6, and HCN should be less than 6, 15, 5, 5, 5, 5, 9, and 13 %, respectively. 3. If a misalignment causes negative ME degradation, the maximum degradations for O3, HCl, and HF should be less than 6, 12, and 12 %, respectively. 3. If a misalignment causes negative ME degradation, the maximum degradations for O3, HCl, and HF should be less than 6, 12, and 12 %, respectively. Note that the retrievals of certain gases, e.g. O3, CH4, CO, and N2O, can be divided into multiple independent sublayers depending on total DOFs. The above deductions do not apply to the partial column integrated over each sublayer because, as Figs. 17 and 18 show, the sensitivity of the proﬁle to ILS degradation is altitude-dependent. The answers to how ILS degradation inﬂuences the partial column of each NDACC gas and how much ILS deviation from unity is acceptable if an ideal line shape is assumed are beyond the scope of this paper and will be published elsewhere. The stratospheric gases are more sensitive to instrumental line shape degradation than the tropospheric gases, and the positive modulation efﬁciency has more inﬂuence on the total column or the proﬁle than the negative modulation efﬁciency. For a typical ILS degradation (10 %), the columns of strato- www.atmos-meas-tech.net/11/2879/2018/ Atmos. Meas. Tech., 11, 2879–2896, 2018 Y. 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Sun, Y., Palm, M., Weinzierl, C., Petri, C., Notholt, J., Wang, Y., and Liu, C.: Technical note: Sensitivity of instrumental line shape monitoring for the ground-based high-resolution FTIR spectrometer with respect to different optical attenuators, Atmos. Meas. Tech., 10, 989–997, https://doi.org/10.5194/amt-10-989- 2017, 2017. Wunch, D., Toon, G. C., Blavier, J.-F. L.,Washenfelder, R., Notholt, J., Connor, B. J., Grifﬁth, D. W. T., Sherlock, V., and Wennberg, P. O.: The Total Carbon Column Ob- serving Network, Philos. T. Roy. Soc. A, 369, 2087–2112, https://doi.org/10.1098/rsta.2010.0240, 2011. Vigouroux, C., De Mazière, M., Demoulin, P., Servais, C., Hase, F., Blumenstock, T., Kramer, I., Schneider, M., Mellqvist, J., Strand- berg, A., Velazco, V., Notholt, J., Sussmann, R., Stremme, W., Rockmann, A., Gardiner, T., Coleman, M., and Woods, P.: Evalu- ation of tropospheric and stratospheric ozone trends over Western Europe from ground-based FTIR network observations, Atmos. Chem. 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https://openalex.org/W2084435824	https://zenodo.org/records/1909155/files/article.pdf	English	null	THE PREVENTION OF PULMONARY TUBERCULOSIS IN PREDISPOSED CHILDREN.	JAMA	1,901	public-domain	7,006	Certainly it is far easier to take it that just as this woman had four brothers, only one of whom was a bleeder, so of hei three sons two were bleeders and the third was not; even with the most hereditary complaints some members of a family escape. It was a coincidence and nothing more that antenatal treatment was instituted in the case in which the hereditary influence was going to fail ! But, there are some circumstances which en- courage me to express the opinion that after all there is a chance that the treatment in this case may have something more than a coincidental relation to the healthy state of the third infant. It is claimed that cases of tuberculosis occurring during childhood are due to direct or indirect infection in nearly all cases, statisticians claiming the infection occurs in an increasing ratio during the first and second years of life from 9 to 38 per cent. y p The pendulum has been swinging toward the bacillary origin of the disease so long that there is danger of losing sight of the other important causal factor of infectious disease, the condition of the tissues which permit and favor the growth of infectious diseases. It is to protest against neglecting this phase of the subject that urges me to discuss the question in this paper. As physicians whose aim is not only to cure, but prevent disease, we must not neglect to consider the predisposing factors. I stoutly maintain that we must give equal consideration to the two factors—heredity and the bacillus. y In the first place it may be taken, from what is known of the physiology of the fetus and more particu- larly of placental transmission, that the chlorid of calcium given to the mother reached the fetal tissues ; there is no reason to doubt that the iron, arsenic, and strychnia did also. In the second place there is evidence that chlorid of calcium is beneficial in hem- ophilia after birth, and there is also evidence that hemophilia, if persistently treated in postnatal life, shows a certain amelioration. In the third place there is in the extraordinary power of recovery possessed by the fetus, a factor which must not be left out of ac- count in dealing with all questions of antenatal treat- ment. When we remember the marvellous power of growth and tissue-building which the fetus displays, a power so great that in one month of intra-uterine life the body weight is quadrupled, we are led to ask our- selves whether this wonder of construction may not be accompanied by an equally great wonder of rep- arative energy? If there be a greatly exaggerated vis medicatrix naturœ in the fetus, is it not possible that even the hereditary maladies may, if properly influ- enced, show a tendency to cure during antenatal life. May it not be that medicines acting upon the organs and tissues, while these are still in the stage of construc- tion, may be more efficacious than when they act upon structures which are, as it were, set either for health or disease. In the meantime the case and these com- ments on it are placed before the profession. In judi- cando criminosa est celeritas. M l ill Authorities differ as to the percentage of predisposi- tion, but average is about 38 per cent. When we con- sider that 38 per cent, of the consumptives give an hered- itary history of the disease, there need be no apology for presenting the subject for your consideration. p g j As that master of the subject of the pedigree of dis- ease, Jonathan Hutchinson, says : "We must take cog- nizance not only of the bacillus itself but also upon the state of the tissues upon which it is implanted. The possibilities of inheritance are twofold. It may be that the bacillus itself may pass bodily or potentially with the sperm or germ from parent to child, or it may be that a condition of tissues liable to its attacks, but for the time free from its presence, may be the result of transmission. If we are permitted to name the tissue condition, which is prone to favor the development of the' tubercle bacil- lus, the name 'scrofula' will perhaps be convenient for the present. A child may then inherit 'scrofula' without the bacillus, or the bacillus without 'scrofula,' or, what probably is most common, both may be present together." p y p g If this view of the heredity of disease is correct, we may take it for granted that the following proposi- tions are admitted: 1. Read at the Fifty-second Annual Meeting of the American Medical Association, in the Section on Diseases of Children, and approved for publication by the Executive Committee of the Section: H. E. Tuley, Edwin Rosenthal and Samuel W. Kelley. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/11/2015 gists and phthisiologists, who believe more in the direct contagion theory. Norbury states that "in the light of modern scientific inquiry infection has supplanted direct heredity as the etiological factor par excellence in tuberculosis." Direct transmission of the bacillus is claimed to be extremely rare, although authorities agree that this rarity will be less when the observations have been more extensive. tary maladies, to accept the conclusion that it was a co- incidence. If we accept the other view, that the healthy, non-hemophilic state of this woman's third son was due to chlorid of calcium administered during the third trimester of pregnancy, we are face to face with the conclusion that it is possible by medicinal substances given to the mother in the last three months of gesta- tion to cure the unborn infant of a malady which no medicines in after-life are capable of curing. Here I am tempted to leave the question. Certainly it is far easier to take it that just as this woman had four brothers, only one of whom was a bleeder, so of hei three sons two were bleeders and the third was not; even with the most hereditary complaints some members of a family escape. It was a coincidence and nothing more that antenatal treatment was instituted in the case in which the hereditary influence was going to fail ! But, there are some circumstances which en- courage me to express the opinion that after all there is a chance that the treatment in this case may have something more than a coincidental relation to the healthy state of the third infant. tary maladies, to accept the conclusion that it was a co- incidence. If we accept the other view, that the healthy, non-hemophilic state of this woman's third son was due to chlorid of calcium administered during the third trimester of pregnancy, we are face to face with the conclusion that it is possible by medicinal substances given to the mother in the last three months of gesta- tion to cure the unborn infant of a malady which no medicines in after-life are capable of curing. Here I am tempted to leave the question. Healthy individuals possess certain degrees of immunity to tuberculosis, as well as other infectious diseases, and this immunity is trans- mitted to the offspring. 2. If one parent is tuberculous the immunity is weakened; if both parents are tuber- culous, the immunity is lessened to a greater degree, and there is engrafted on the progeny a cellular nutritional weakness which permits the invasion of the infective germs. Perhaps it would be more nearly correct if we were to say that such individuals have an "infective predisposition," for they are especially liable to other forms of infection, such as influenza, etc., and their children inherit the same susceptibility. The reason they become tuberculous is because the tubercle bacillus is so omnipresent. 3. Children of tuberculous parents are easily infected, directly or indirectly. 24 Melville Street. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. JOHN A. ROBISON, A.M., M.D. Attending Physician to Cook County and Presbyterian Hospitals; President of Chicago Society of Internal Medicine, Etc. CHICAGO. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. JOHN A. ROBISON, A.M., M.D. Attending Physician to Cook County and Presbyterian Hospitals; President of Chicago Society of Internal Medicine, Etc. CHICAGO. oaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/11/2015 THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. p q p y p After the period of education comes the choosing of a vocation. The tuberculous inclined should be advised to choose occupations which would necessitate them to be out of doors the greater part of the time. The open- air occupation, if such that it requires the individual to be active, is the best form of work. Avoid dusty work, and factories, crowded assemblies and violent physical exercises. Newsholme, in his work on vital sta- tistics, states the lowest mortality is in clergymen, rail- way engine stokers and engineers, farmers, brick-burners,_ coal merchants, and coal miners. Contrary to what would be believed, coal miners seem to be quite exempt from tuberculosis. , physical Infancy.—A babe should never be suckled by a tu- berculous mother, but by a healthy wet-nurse, or it should be fed with food which is absolutely free from suspicion of being contaminated by the tubercle bacillus. As the babe grows the dietary should contain more fat, and as the salivary glands develop a very slight amount of starchy food should be added. Early a taste for hydro- carbons should be cultivated, so that the patient will not have a repugnance for fatty food as is commonly the case in persons who are inclined to tuberculosis. In a large experience it has been found that consumptives consume an insufficient amount of fat. During the time the physician and parent have been guiding the development oí the predisposed patient into more perfect maturity, it is necessary to keep in mind that the clothing is a question of importance, especially if the patient be a girl. The whole subject may be com- pressed in two words : the patient should be warmly and loosely clad. By warmly clad is meant sufficient cloth- ing to prevent chilling, and meeting the weather changes by different weights of outer clothing. The clothing should be loose enough to permit free exercise and un- embarrassed respiration. A child with a predisposition to tuberculosis should not wear a corset, and the mother who attempts to control the outline of her child's figure by the use of the corset is laying the foundation of tu- bercular infection by interfering with the processes of nutrition and the development of the respiratory func- tions of the lungs. Thin shoes, insufficient head cover- ing, or deficient underclothing are the stepping-stones to tuberculosis. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. But on the other hand excessive swaddling of the body, or the neck and chest should be avoided, as it tends to a susceptibility to cold catching, and sudden changes of temperature can not be so well withstood. As the period of childhood approaches, the diet should contain more carbohydrates, and the inordinate cravings of children for sweets should not be curbed too strictly. In my opinion, pure candy in moderation is beneficial to growing children, if given after and not between meals. A point of not minor importance is the giving of water freely to children. As a rule, children do not drink enough water, as it is a great aid to nutrition, dissolving the food so that it can be more easily digested and assimilated, and acts as a solvent for effete material in the body so that it can be better eliminated. During the period of childhood the diet should con- tain a greater proportion of nitrogenous food. Nitrogen supplies muscular force, and the demands upon the muscular system increases with the increasing age of the patient and the entering an occupation of some kind. The period of puberty is a critical one. The physi- cian should supervise the proper development of the physico-moral nature during this period. Enforced ignorance of the sexual laws prevents youth from seeing through the vista of years and perceiving the woes which follow the abuse of the body at a period when nature is attempting to reinforce it in every way and strengthen it against the future invasion of disease germs. Edu- cate the youth in such necessary physiological laws as will tend to preserve the integrity of all the tissues, and maintain nutrition at its highest point. g p Cleanliness is the great preventer of germ infection. A clean consumptive will not spread the infection, and a clean body does not afford so good a lodging-place for the germs. Therefore, the predisposed must avoid con- tact so far as possible with persons who are consumptive or should not use any articles used by such. y y Hydrotherapy is of great value. It stimulates the nutritive processes, steadies the vasomotor nerves so that they are not so susceptible to sudden changes of tem- perature, and hardens the individual. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. Before the discovery of the bacillus tuberculosis hered- ity was supposed to be of great importance as a causal factor of tuberculosis, but its importance is rapidly fading into the distance in the opinion of bacteriolo- y y y Granted, therefore, that these statements are true, what can be done to prevent the subsequent develop- ment of tuberculosis? As Holmes has remarked, the preventive treatment should commence with the an- cestors. Stirpiculture, or the improvement of the race, and hygienic home surroundings. It is encouraging to note the tendency of modern school boards to install gymnasia, and employ competent teachers of physical development. Two-thirds of the period of youth is spent in education, and physicians should see that the parents and teachers give each child who has a predis- position to tuberculosis such thought and care in its mental and physical training as will insure health in- stead of disease. A harmonious and dual development of the mind and body must be secured. Our school- teachers, as a rule, care more to send the parents reports of the rapid mental development of the children than to supervise the equal mental and physical development. is the most potent means to be used to eventually eradi- cate tuberculosis. The education of the people in stirpi- culture would mean the improvement of the coming gen- erations, but of course has nothing to do with the gen- eration already born. But the knowledge that children born of invalid parents are susceptible of improvement is of great value to the present generation. It is our duty to educate parents how to rear children who are predisposed to tubercular disease. is the most potent means to be used to eventually eradi- cate tuberculosis. The education of the people in stirpi- culture would mean the improvement of the coming gen- erations, but of course has nothing to do with the gen- eration already born. But the knowledge that children born of invalid parents are susceptible of improvement is of great value to the present generation. It is our duty to educate parents how to rear children who are predisposed to tubercular disease. p p The period of development must be divided into sub- divisions : infancy, childhood, youth and maturity. The great aim must be to promote nutrition, to develop the body symmetrically, to educate the mind judiciously, and to inculcate correct habits, physical and moral. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. It is impossible to enter into this subject in detail, but its benefit is so great to the consumptively inclined, that it is the duty of the physician to give his clients the necessary instruc- tion. g po t Now is the time for education, and it is the time when the mind is apt to receive more training than the body. Mens sana in corpore sano is a motto which is forgotten not only by educators but by parents as well. We boast of our public school system, yet there is no method whereby so many budding into maturity can be physically wrecked so readily as by the class method of teaching. The strong and weak are given the same tasks, and the hours are unbroken by physical training, to say nothing of the lack of supervision of the attitude of the scholars in the school room, the want of proper ventilation, and the housing together of children whose vital forces are already lowered by lack of proper food nutr they pera to e grea of th tion It of th ven and air disp It goes without saying that the hygienic surroundings of the patients must be as good as possible. Clean, well- ventilated, comfortable houses, built upon proper soil, and so arranged as to receive the maximum of fresh air and sunshine., afford the best chances for the pre- disposed. already by proper disposed. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/11/2015 harm about the age of puberty. It seems to me that children thus instructed in school would be taught how to prevent the diseases in their own children when they become parents. This can be done by teaching them in the public schools that such persons should not marry. This would be better, in my opinion, than legislation, which will surely increase il- legitimacy. And here the value of the open-air treatment is made manifest. The fresh-air sanatoria of our larger cities, and the country vacation clubs are doing a great deal of good in lessening the susceptibility of the poor in our midst. The sad part of it is that these provisions are necessarily of short duration. There should be established throughout the land fresh-air sanatoria for the children of tuberculous parents, and for the orphans of tuberculous origin. y DISCUSSION. Dr. T. F. Wood, Angola, Ind.—The subject has been handled in a way to be commended. I wish, however, the essayist had said something about the prevention of the marriage of indi- viduals who are not only predisposed, but are really consump- tives. I believe this is one of the great sources of tuberculous disease, and yet our hands are tied, as it were, regarding that phase of the subject. Many physicians dare not discuss this subject, lest they become unpopular and lose business; never- theless, it is an important matter, and we should speak with- out fear or favor on this vital point. To-day the statutes of almost every state allow any person, even in the last stages of consumption, to marry and rear children, and in this way transmit the disease to those yet unborn. y y y Dr. C. A. Kelset, Minneapolis—I have been exceedingly in- terested in this paper, and was very glad to see the position taken by the author regarding the necessity for fats. It is a point which we should bear in mind, for I think the ma- jority of persons having children with a tendency to tuber- cular troubles seem to have the erroneous impression that these children must not have fat. In regard to the valuable remarks of the last speaker, I would say that, if I understand the germ theory, none of us can hope to escape these ubiquit- ous germs. I do not mean to say that I would discourage taking proper care of the sputa, but does it follow that the diminution in the mortality in the State of Michigan is due wholly to the care of the sputa? Can we not claim something for improved hygiene in fortifying the system against the attack of the germ. In tubercular wards, nurses of good con- stitution can remain constantly without becoming infected with tuberculosis. Let us not, however, lose sight of the im- portance of building up and fortifying the system against the attacks of these germs, developing a physical system in the rising generation which can withstand these noxious organ- isms. y Dr. Wadsworth Warren, Detroit—One point in the latter part of the paper appealed to me especially, and I desire to give it additional emphasis. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. At the children's hospital at Ormesson, under the auspices of the French society for the care of tuberculous children, 34 per cent, of the cases were cured, and at Villiers the per cent, was 34.4 cured, or nearly cured. France beats the world in car- ing for children who have consumption, or have the ten- dency, having over twenty seaside and mountain resorts. But if we have not the sanatoria we can instruct our patients in sanatoria! methods. These instructions should include dietetic, hygienic, balneological, physi- cal and therapeutic methods of the well-known sana- toria. Dr. Columbus G. Slag le, Minneapolis—I desire to lay stress on malnutrition. I have been in the habit of telling my patrons and friends that consumption does not begin in the lungs but in the stomach. This may not sound very scientific, but it appeals to the popular mind, and 1 think does good by impressing upon them the importance of a proper dietary. Another point is in regard to fats. One of the most practical lessons 1 ever learned was in the Univeisity of Louisville when I was a student. Professor J. B. Flint always asked any one suspected to be tuberculous if he liked fat, and such persons usually replied in the negative. At one time he was examining a patient far advanced in tuberculosis, and this question about fats was, to the surprise of the professor, answered in the affirmative. So surprised was he that the question was repeated and was answered again in the affirma- tive. The professor then turned to the students, and ex- pressed his belief that the patient had lied. He then went on to impress upon them the importance of tuberculous in- dividuals eating fats, and particularly those persons who had an aversion to fats because ordinarily they were the very ones most likely to fall victims to tuberculosis. Finally, it is the province of the physician to warn parents of the danger of neglecting the so-called benign children's diseases, such as measles, whooping-cough, in- fluenza, tonsillitis, adenoids, etc. The feeble-minded are especially liable to tubercular disease. The acute diseases may be the preparers of the soil for tubercular infection, and they should not be neglected. Nutrition is the foundation stone on which to rear the preventive treatment. y Dr. Collins H. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. The one most certain method for the prevention of tuberculosis not only in children but in the community at large is to prevent the dissemination of the tubercle bacillus. This is a matter which rests largely in the hands of the medical profession and state boards of health. The State Board of Health of Michigan in 1891 began a campaign against tuberculosis by disseminating throughout the state literature dealing with this subject. Whenever a case of tuberculosis is reported to the local board of health, it is immediately sent to the secretary of the State Board of Health, and this officer sends literature to the family, explaining in detail the proper methods of caring for sputum. In this way, in the last ten years, the death-rate from pul- monary tuberculosis has been very materially reduced. It is impossible in the time allotted to me to crystallize all the thoughts on this subject, but if my theme shall be discussed and its importance magnified, and some practical method of instituting preventive measures throughout the United States whereby the increasing army of consumptives may be lessened, no apology need be offered for the mediocrity of this communication. THE PREVENTION OF PULMONARY TUBER- CULOSIS IN PREDISPOSED CHILDREN. Johnston, Grand Rapids, Mich.—In this connection I desire to speak of a paper published in Virchow's Archiv, in May, 1900, in which it is maintained, on the basis of 500 autopsies performed at Zurich, that 99 per cent, of the human race who attain the age of 30 show, on postmortem, lesions of tuberculosis. From this it would seem that almost every one is infected with tuberculosis at some time or other. I wish to insist that one can not have tuberculosis without the tubercle bacilli. Osier maintains that there are only 20 cases on record of persons who have been born with the dis- ease. Undoubtedly there is a predisposition in certain fam- ilies to tuberculosis, the same as there is to scarlatina, diph- theria, and other infectious diseases. The one most certain method for the prevention of tuberculosis not only in children but in the community at large is to prevent the dissemination of the tubercle bacillus. This is a matter which rests largely in the hands of the medical profession and state boards of health. The State Board of Health of Michigan in 1891 began a campaign against tuberculosis by disseminating throughout the state literature dealing with this subject. Whenever a case of tuberculosis is reported to the local board of health, it is immediately sent to the secretary of the State Board of Health, and this officer sends literature to the family, explaining in detail the proper methods of caring for sputum. In this way, in the last ten years, the death-rate from pul- monary tuberculosis has been very materially reduced. y Dr. Collins H. Johnston, Grand Rapids, Mich.—In this connection I desire to speak of a paper published in Virchow's Archiv, in May, 1900, in which it is maintained, on the basis of 500 autopsies performed at Zurich, that 99 per cent, of the human race who attain the age of 30 show, on postmortem, lesions of tuberculosis. From this it would seem that almost every one is infected with tuberculosis at some time or other. I wish to insist that one can not have tuberculosis without the tubercle bacilli. Osier maintains that there are only 20 cases on record of persons who have been born with the dis- ease. Undoubtedly there is a predisposition in certain fam- ilies to tuberculosis, the same as there is to scarlatina, diph- theria, and other infectious diseases. y DISCUSSION. I have dwelt upon these facts with some detail because this tremendous reduction of the death-rate from one of the most serious diseases of mankind was accom- plished by a study of one of the diseases of animals and the application of the knowledge thus obtained to human sanitation. This great advance in medical science and in the ability to prevent disease demonstrates the prac- tical value of a knowledge of comparative pathology, and of a sincere effort to apply this knowledge in the formulation of measures for the control of the diseases of man. Taking up the problem of tuberculosis, we find that the disease is caused by a bacillus so exacting in its conditions of growth that it can hardly be expected, in its virulent form, to multiply elsewhere than in the animal body or under the artificial environment sup- plied by the laboratory. The source of virulent tubercle bacilli is, therefore, either a person affected with tuber- culosis, or one of the lower animals affected with that disease. When we consider the facts which have been demonstrated by the studies of comparative pathology, we can not but be impressed by the conclusion that there is a great culture ground and reservoir for tubercle bacilli outside of the human body, and existing prin- cipally in the bodies of the domesticated animals which supply some of the principal articles of human food. This well-known condition of affairs is, it seems to me, sufficient justification for a review of our knowledge of animal tuberculosis and the relation which it bears to the human form of the disease. We must go abroad for most of the statistics of tuber- culosis in animals, and while it is more frequent in European countries than here, we can learn from them as to the conditions which are likely to develop here as the animal population becomes more dense, and providing the spread of the infection remains practically unrestricted. The slaughter-house statistics of Prussia show 14.6 per cent, of the cattle and 2.14 per cent, of the swine to be tuberculous; in Saxony the percentage is 29.13 with cattle and 3.10 with swine; in the city of Leipsic the figures are 36.4 per cent, with cattle and 2.17 per cent, with swine (Siedamgrotzky). Of 20,850 animals in Belgium tested with tuberculin in 1896, 48.88 per cent, reacted (Stubbe). y DISCUSSION. Robison, in reply—I am glad that this subject has brought out such a full discussion, and that the paper has re- ceived your sanction on most of the points presented. Some of the states have already framed laws regarding the preven- tion of marriage of the unfit. Most of these laws have been directed towards preventing the marriage of those infected with venereal disease. In time, by the process of education, the people will learn to dread this disease. Within the last five years the newspapers have helped us to educate» the public; yet even lawyers and judges and public men know almost nothing about tuberculosis. I think the subject of infectious diseases should be taught in the public schools just as much as botany is taught. Malnutrition is certainly the starting point of tuberculosis. The case of the woman who liked fats, mentioned by Dr. Scott, seems to me to be the ex- ception which proves the rule. The giving of fat by aiding nutrition prevents the disease. Osier's statistics regarding the transmission of tuberculosis should not be taken as authori- tative; for, in the first place, the observations have not ex- tended over a sufficient length of time, and the opportunities for carrying on such investigations are very limited. If you will consult the records of the veterinary surgeons, you will find that the percentage of cases of direct transmission in animals is much larger than 2 per cent., and it is probably true also of the human. The Brompton Hospital for Con- sumptives has been in existence for two centuries and a half, yet it has been found that the disease is rarely taken by the nurses. In our general hospitals in this country I am sorry to say the internes and nurses do frequently develop tuber- culosis. In a hospital in Chicago we have had three internes who have died of tuberculosis in one form or another, although it is quite possible that they may have had the disease before entering the hospital. 1 will say, however, that the methods of prevention in a general hospital are not so strict as in special hospitals. It is the custom, though obviously wrong, to use a broom in cleaning out the wards of our general hos- pitals. y DISCUSSION. The death\x=req-\ rate from tuberculosis, and particularly from phthisis, has been greatly decreased in England and Wales since the date mentioned, but in 1896 the deaths from all forms of tuberculosis still equaled 10.5 out of every 100 total deaths. At the beginning of the 20th century we find the mor- tality from smallpox insignificant compared with what it was at the beginning of the 19th century. In England and Wales in 1896 it was as low as 1.8 per million living. That is, there were only as many deaths from smallpox in 1896 among 2,000,000 people as there were among 1000 people in 1796. In other words, there was but one death in 1896 from smallpox where there were 2000 in 1796. I have dwelt upon these facts with some detail because this tremendous reduction of the death-rate from one of the most serious diseases of mankind was accom- plished by a study of one of the diseases of animals and the application of the knowledge thus obtained to human sanitation. This great advance in medical science and in the ability to prevent disease demonstrates the prac- tical value of a knowledge of comparative pathology, and of a sincere effort to apply this knowledge in the formulation of measures for the control of the diseases of man. Taking up the problem of tuberculosis, we find that the disease is caused by a bacillus so exacting in its conditions of growth that it can hardly be expected, in its virulent form, to multiply elsewhere than in the animal body or under the artificial environment sup- plied by the laboratory. The source of virulent tubercle bacilli is, therefore, either a person affected with tuber- culosis, or one of the lower animals affected with that disease. When we consider the facts which have been demonstrated by the studies of comparative pathology, we can not but be impressed by the conclusion that there is a great culture ground and reservoir for tubercle bacilli outside of the human body, and existing prin- cipally in the bodies of the domesticated animals which supply some of the principal articles of human food. This well-known condition of affairs is, it seems to me, sufficient justification for a review of our knowledge of animal tuberculosis and the relation which it bears to the human form of the disease. f th i i f b Dr. I. J. K. y DISCUSSION. Of 25,439 tested in Denmark from 1893 to 1895, 49.3 per cent, reacted, and of 67,263 tested from 1896 to 1898, 32.8 per cent, reacted (Bang). B i i h y DISCUSSION. Golden, Chicago—The main thing in the pre- vention of tuberculosis among children is to educate them not to be afraid of water. The parents of such children are usually afraid to have their children bathe lest they may catch cold. For the same reason these children are very warmly clad. I am of the opinion that this practice tends rather to favor than to prevent tuberculosis. Tuberculous children should be made to bathe daily, and if not too warmly clad they may not be so susceptible to tuberculosis. 1 am op- posed to the present craze for woolen clothing. If they catch cold, no matter ; let them develop a little antitoxin in the system. If laws were passed to prevent the marriage of tuberculous individuals or of those predisposed to this disease, I am afraid there would be nothing but bachelors, and the population of the world would soon vanish. in 1838, when 17 deaths out of every 100 total deaths were caused by phthisis,2 and if the deaths from other forms of tuberculosis could be added the maximum small- pox death-rate of 1796 would be surpassed. The death\x=req-\ rate from tuberculosis, and particularly from phthisis, has been greatly decreased in England and Wales since the date mentioned, but in 1896 the deaths from all forms of tuberculosis still equaled 10.5 out of every 100 total deaths. in 1838, when 17 deaths out of every 100 total deaths were caused by phthisis,2 and if the deaths from other forms of tuberculosis could be added the maximum small- pox death-rate of 1796 would be surpassed. The death\x=req-\ rate from tuberculosis, and particularly from phthisis, has been greatly decreased in England and Wales since the date mentioned, but in 1896 the deaths from all forms of tuberculosis still equaled 10.5 out of every 100 total deaths. At the beginning of the 20th century we find the mor- tality from smallpox insignificant compared with what it was at the beginning of the 19th century. In England and Wales in 1896 it was as low as 1.8 per million living. That is, there were only as many deaths from smallpox in 1896 among 2,000,000 people as there were among 1000 people in 1796. In other words, there was but one death in 1896 from smallpox where there were 2000 in 1796. population Dr. Read at the Fifty-second Annual Meeting of the American Medical Association, in the Section on Hygiene and Sanitary Science, and approved for publication by the Executive Committee of the Section. 1. Vital Statistics, 3d Ed., p. 211. of the Section. 1. Vital Statistics, 3d Ed., p. 211. 2. Dr. Ransome, Lancet, July 11, 1896. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/11/2015 , , Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/11/2015 1. Vital Statistics, 3d Ed., p. 211. 2. Dr. Ransome, Lancet, July 11, 1896. Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/11/2015 , , Downloaded From: http://jama.jamanetwork.com/ by a Univer y DISCUSSION. I refer to the necessity of warning the parents of these children regarding the care of these little ones during an acute infectious disease, or even when they are inflicted only with a mild laryngitis or pharyn- gitis. Very often these apparently innocent disorders lower the child's powers of resistance so much as to afford a suit- able culture medium for the tubercle bacilli ; attention to the mouth, throat and pharynx, and to the removal of adenoids and enlarged and patulous tonsils is of great importance. g p great po ta ce Dr. James A. Work, Elkhart, lnd.—With reference to clothing, I would say that 1 believe we should instruct parents more with reference to the changing of the clothing in ac- cordance with the changes in the weather. We know how great and sudden are the changes of the weather in this country, and I think the clothing should be adapted to meet these changes. changes. Dr. Clifton Scott, Des Moines—The author emphasized the importance of teaching young children in the public schools certain things in regard to abuses which do them g ega d d From: http://jama.jamanetwork.com/ by a University of Manitoba User on 06/11/2015 g g Downloaded From: http://jama.jamanetwork.com/ by a University of Manitoba User o Dr. I. J. K. Golden, Chicago—The main thing in the pre- vention of tuberculosis among children is to educate them not to be afraid of water. The parents of such children are usually afraid to have their children bathe lest they may catch cold. For the same reason these children are very warmly clad. I am of the opinion that this practice tends rather to favor than to prevent tuberculosis. Tuberculous children should be made to bathe daily, and if not too warmly clad they may not be so susceptible to tuberculosis. 1 am op- posed to the present craze for woolen clothing. If they catch cold, no matter ; let them develop a little antitoxin in the system. If laws were passed to prevent the marriage of tuberculous individuals or of those predisposed to this disease, I am afraid there would be nothing but bachelors, and the population of the world would soon vanish. Dr. Robison, in reply—I am glad that this subject has brought out such a full discussion, and that the paper has re- ceived your sanction on most of the points presented. y DISCUSSION. Some of the states have already framed laws regarding the preven- tion of marriage of the unfit. Most of these laws have been directed towards preventing the marriage of those infected with venereal disease. In time, by the process of education, the people will learn to dread this disease. Within the last five years the newspapers have helped us to educate» the public; yet even lawyers and judges and public men know almost nothing about tuberculosis. I think the subject of infectious diseases should be taught in the public schools just as much as botany is taught. Malnutrition is certainly the starting point of tuberculosis. The case of the woman who liked fats, mentioned by Dr. Scott, seems to me to be the ex- ception which proves the rule. The giving of fat by aiding nutrition prevents the disease. Osier's statistics regarding the transmission of tuberculosis should not be taken as authori- tative; for, in the first place, the observations have not ex- tended over a sufficient length of time, and the opportunities for carrying on such investigations are very limited. If you will consult the records of the veterinary surgeons, you will find that the percentage of cases of direct transmission in animals is much larger than 2 per cent., and it is probably true also of the human. The Brompton Hospital for Con- sumptives has been in existence for two centuries and a half, yet it has been found that the disease is rarely taken by the nurses. In our general hospitals in this country I am sorry to say the internes and nurses do frequently develop tuber- culosis. In a hospital in Chicago we have had three internes who have died of tuberculosis in one form or another, although it is quite possible that they may have had the disease before entering the hospital. 1 will say, however, that the methods of prevention in a general hospital are not so strict as in special hospitals. It is the custom, though obviously wrong, to use a broom in cleaning out the wards of our general hos- pitals. in 1838, when 17 deaths out of every 100 total deaths were caused by phthisis,2 and if the deaths from other forms of tuberculosis could be added the maximum small- pox death-rate of 1796 would be surpassed. TUBERCULOSIS OF ANIMALS IN SOME OF ITS RELATIONS TO HUMAN TUBERCULOSIS. D. E. SALMON, D.V.M. WASHINGTON, D. C. It is not necessary to consume time in demonstrating the importance of a thorough consideration, from every point of view, of the subject of tuberculosis. The im- portance of such a study is now generally recognized. As smallpox was the great medical problem at the close of the 18th century, so tuberculosis was the most urgent problem at the end of the 19th century. According to Newsholme,1 smallpox reached its highest point in England in 1796, when 18.5 deaths out of every 100 total deaths were caused by that disease. Tuberculosis was apparently at its maximum in England and Wales (Bang). The British "Royal Commission appointed to inquire into the effect of food derived from tuberculous ani-
https://openalex.org/W4311888769	https://www.researchsquare.com/article/rs-1688449/latest.pdf	English	null	Efficacy and Safety of First-line Carboplatin-paclitaxel and Carboplatin-gemcitabine in Patients With Advanced Triple-negative Breast Cancer: A Monocentric, Retrospective Comparison	Clinical breast cancer	2,023	cc-by	10,277	Efficacy and safety of first and carboplatin-gemcitab advanced triple-negative b monocentric, retrospectiv Riccardo Lobefaro Fondazione IRCCS Istituto Nazionale dei Tumori Luigi Mariani Fondazione IRCCS Istituto Nazionale dei Tumori Giorgia Peverelli Fondazione IRCCS Istituto Nazionale dei Tumori Francesca Ligorio Fondazione IRCCS Istituto Nazionale dei Tumori Giovanni Fucà Fondazione IRCCS Istituto Nazionale dei Tumori Alessandro Rametta Fondazione IRCCS Istituto Nazionale dei Tumori Emma Zattarin Fondazione IRCCS Istituto Nazionale dei Tumori Rita Leporati Fondazione IRCCS Istituto Nazionale dei Tumori Beatrice Cantarelli IFOM, the FIRC Institute of Molecular Oncology Catherine Depretto Fondazione IRCCS Istituto Nazionale dei Tumori Andrea Vingiani Fondazione IRCCS Istituto Nazionale dei Tumori Siranoush Manoukian Fondazione IRCCS Istituto Nazionale dei Tumori Gianfranco Scaperrotta Fondazione IRCCS Istituto Nazionale dei Tumori Giulia Valeria Bianchi Fondazione IRCCS Istituto Nazionale dei Tumori Giuseppe Capri Fondazione IRCCS Istituto Nazionale dei Tumori Riccardo Lobefaro Alessandro Rametta Fondazione IRCCS Istituto Nazionale dei Tumori Emma Zattarin Fondazione IRCCS Istituto Nazionale dei Tumori Rita Leporati Fondazione IRCCS Istituto Nazionale dei Tumori Beatrice Cantarelli IFOM, the FIRC Institute of Molecular Oncology Catherine Depretto Fondazione IRCCS Istituto Nazionale dei Tumori Andrea Vingiani Fondazione IRCCS Istituto Nazionale dei Tumori Siranoush Manoukian Fondazione IRCCS Istituto Nazionale dei Tumori Gianfranco Scaperrotta Fondazione IRCCS Istituto Nazionale dei Tumori Page 1/28 Page 1/28 Page 1/28 Research Article Posted Date: May 31st, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/28 Abstract Background: Platinum-based chemotherapy is widely used in patients with advanced triple-negative breast cancer (TNBC). However, the most effective platinum-based chemotherapy combination in the first-line treatment setting remains unclear. Methods: We evaluated the efficacy of first-line carboplatin-paclitaxel (CP) or carboplatin-gemcitabine (CG) combinations in advanced TNBC patients treated at “Fondazione IRCCS Istituto Nazionale dei Tumori” between April 2007 and April 2021. CP and CG were compared in terms of progression-free survival (PFS), overall survival (OS) and incidence of adverse events (AEs). Multivariable Cox Models were used to adjust the efficacy of CP vs. CG for clinically relevant covariates. Results: Of 418 consecutive advanced breast cancer patients receiving carboplatin-based doublet chemotherapy, 88 patients had advanced TNBC and were treated in the first-line setting. Of these, 56 (63.6%) and 32 (36.4%) patients received CP and CG, respectively. Clinically relevant variables were well- balanced in the two treatment cohorts, except for a higher percentage of patients with shorter disease- free interval in the CG group (59.4% vs. 44.6%; p value 0.038). After adjusting for clinically relevant variables, patients receiving CG had significantly better PFS when compared to CP-treated patients [aHR: 0.49 (95%CI: 0.27-0.87), p value 0.014]. Of note, CG was associated with better PFS only among patients previously treated with taxanes in the (neo)adjuvant setting (aHR: 0.39; 95%CI: 0.21-0.75), but not in patients not exposed to taxanes (aHR: 1.20; 95%CI: 0.37-3.88). CG was also independently associated with better OS when compared to CP [aHR: 0.31 (95%CI: 0.15-0.64), p value 0.002]. Overall, grade 3-4 AEs were more common in patients treated with CG than in patients treated with CP (68.8% vs. 21.4%, p value 0.009). Conclusions: CG and CP are effective and well tolerated first-line platinum doublets in advanced TNBC patients. CG could be more effective than CP in patients previous exposed to taxanes despite worse toxicity profile. Introduction Despite these progresses, chemotherapy remains the mainstay of treatment for advanced TNBC patients Several chemotherapeutical agents, such as taxanes (paclitaxel, nab-paclitaxel, docetaxel), anthracyclines (doxorubicin, liposomal doxorubicin), anti-metabolites (capecitabine, gemcitabine), microtubule inhibitors (eribulin, vinorelbine) and platinum agents (carboplatin, cisplatin), are potentially effective anti-TNBC treatments. Unfortunately, the duration of tumor control with these agents, used either alone or in combination, is sub-optimal in the majority of patients, and advanced TNBC cells becomes progressively more chemo-resistant during the course of subsequent lines of therapy. Therefore, using the most effective therapies in the first- or second-line treatment settings is a clinical priority, potentially impacting on long-term clinical outcomes. anthracyclines (doxorubicin, liposomal doxorubicin), anti-metabolites (capecitabine, gemcitabine), microtubule inhibitors (eribulin, vinorelbine) and platinum agents (carboplatin, cisplatin), are potentially effective anti-TNBC treatments. Unfortunately, the duration of tumor control with these agents, used either alone or in combination, is sub-optimal in the majority of patients, and advanced TNBC cells becomes progressively more chemo-resistant during the course of subsequent lines of therapy. Therefore, using the most effective therapies in the first- or second-line treatment settings is a clinical priority, potentially impacting on long-term clinical outcomes. Different studies support the use of platinum-based combinations in advanced TNBC patients. In a retrospective analysis including 379 metastatic TNBC patients, platinum-based chemotherapy was associated with longer PFS compared to non-platinum-based chemotherapy (7.8 vs. 4.9 months respectively), without statistically significant OS differences in the two patient cohorts [13]. TNBCs arising in patients harboring germline BRCA1/2 gene mutations are exquisitely sensitive to platinum compounds, which induce the formation of DNA inter-stand and DNA-protein crosslinks [14–16]. In the phase III, randomized trial TNT, carboplatin monotherapy was associated with higher tumor overall response rates (ORR) when compared with docetaxel monotherapy in advanced TNBC patients bearing germline BRCA1/2 mutations; on the other hand, in the whole patient cohort carboplatin and docetaxel showed similar antitumor activity/efficacy, with carboplatin being associated with a better safety profile [17]. To date, different platinum-based doublets, including gemcitabine-, vinorelbine-, paclitaxel- and nab- paclitaxel-based combinations, have been evaluated in prospective studies, and higher response rates have been reported with chemotherapy doublets (ranging between 28% and 69.2%) as compared to single-agent chemotherapy [7, 18–27]. However, the long-term benefit associated with platinum doublets, as well as the most effective chemotherapy partner to be used in combination with platinum salts, remain unclear. Introduction On these grounds, we conducted a retrospective analysis to evaluate the efficacy and the safety profiles of first-line carboplatin-gemcitabine (CG) and carboplatin-paclitaxel (CP) combinations in advanced TNBC patients treated in our Institution between 2007 and 2021. Introduction Breast cancer (BC) is the primary cause of cancer-related death in women, with 684,996 deaths in 2020 worldwide [1]. Approximately 15–20% of all BCs are classified as triple-negative BC (TNBC), as defined by absent or minimal (< 1%) expression of estrogen receptor and progesterone receptor by immunohistochemistry (IHC) analysis, and by absence of Human Epidermal growth factor Receptor 2 (HER2) overexpression by IHC and/or HER2 gene amplification by in situ hybridization (ISH). TNBC is the most clinically aggressive and deadly BC subtype, and its clinical course is characterized by a higher risk of distant metastases after curative surgery and by shorter survival in patients with advanced disease [2– 4]. Indeed, median overall survival (OS) in the advanced stage does not go beyond two years even with the most effective systemic treatments [5–8]. Page 3/28 Recently, the advent of new biomarkers and pharmacological targets has remarkably expanded the therapeutic options for patients with advanced TNBC [9–10]. In particular, PolyAdenosine diphosphate- Ribose Polymerase inhibitors (PARPi) in patients harboring germline BRCA1/2 mutations, or first-line chemo-immunotherapy combinations in patients with programmed death-ligand 1 (PD-L1)-positive neoplasms, have significantly prolonged patient progression free survival (PFS) and/or OS [6, 11, 12]. Despite these progresses, chemotherapy remains the mainstay of treatment for advanced TNBC patients. Several chemotherapeutical agents, such as taxanes (paclitaxel, nab-paclitaxel, docetaxel), anthracyclines (doxorubicin, liposomal doxorubicin), anti-metabolites (capecitabine, gemcitabine), microtubule inhibitors (eribulin, vinorelbine) and platinum agents (carboplatin, cisplatin), are potentially effective anti-TNBC treatments. Unfortunately, the duration of tumor control with these agents, used either alone or in combination, is sub-optimal in the majority of patients, and advanced TNBC cells becomes progressively more chemo-resistant during the course of subsequent lines of therapy. Therefore, using the most effective therapies in the first- or second-line treatment settings is a clinical priority, potentially impacting on long-term clinical outcomes. Recently, the advent of new biomarkers and pharmacological targets has remarkably expanded the therapeutic options for patients with advanced TNBC [9–10]. In particular, PolyAdenosine diphosphate- Ribose Polymerase inhibitors (PARPi) in patients harboring germline BRCA1/2 mutations, or first-line chemo-immunotherapy combinations in patients with programmed death-ligand 1 (PD-L1)-positive neoplasms, have significantly prolonged patient progression free survival (PFS) and/or OS [6, 11, 12]. Methods For the definition of TNBC, the most recently collected tumor specimen (i.e., surgical tumor specimen for patients not undergoing tumor re-biopsy for metastatic disease, or biopsy of a metastatic lesion for patients undergoing tumor re-biopsy at disease relapse after surgery) was used; (b) advanced disease, as defined as the presence of distant metastases or locally advanced, inoperable disease; (c) age ≥ 18 years; (d) treatment with at least one cycle of one of the following first-line regimens: CG (carboplatin at an area under the curve (AUC) of 2 plus gemcitabine 800 mg/m2, both administered i.v. on days 1 and 8 every 3 weeks; or carboplatin at an AUC of 5 on day 1 every 3 weeks plus gemcitabine 800 mg/m2 on days 1 and 8 every 3 weeks), or CP (carboplatin AUC 2 plus paclitaxel 80 mg/m2, both administered i.v. on days 1 and 8 every 3 weeks); (e) availability of data regarding clinical outcomes, including patient PFS and OS; (f) available data about previous therapies received (i.e., surgery, radiotherapy and (neo)adjuvant therapy); (g) available data about number and sites of metastatic lesions; (h) available data about treatment-related adverse events (AEs). Objectives of the study The objectives of this study were to compare the antitumor efficacy, activity and safety profiles of CG and CP as a first-line treatment in patients with advanced TNBC. The primary study endpoint was PFS, as defined as the time between treatment initiation and disease progression or patient death from any cause, whichever occurred first. OS, ORR, disease control rate (DCR) and duration of response (DOR) were secondary activity/efficacy endpoints. ORR was defined as the percentage of patients achieving partial response (PR) or complete response (CR) as their best response. DCR was defined as the percentage of patients achieving PR, CR, or stable disease (SD) as their best response. OS was defined as the time between chemotherapy initiation and patient death from any cause. Disease-free interval (DFI) was defined as the time between surgical resection of the primary tumor and the detection of disease recurrence. The DOR was defined as the time between the first documentation of PR or CR and disease progression or patient death from any cause. Patients who had not undergone disease progression or death at the time of data cut-off and analysis were censored at the time of their last disease evaluation. Methods Page 4/28 Page 4/28 This is a retrospective, monocentric study that included advanced TNBC patients treated with first-line platinum-based chemotherapy at Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, Italy) between April 2007 and April 2021. Inclusion criteria were: (a) women with pathologically/cytologically confirmed diagnosis of TNBC, as defined as estrogen receptor (ER) and progesterone receptor (PgR) expression in less than 1% of cancer cells by IHC analysis, and an IHC score for HER2 expression of 0/1+, or an IHC score of 2 + in the absence of HER2 gene amplification by ISH analysis. For the definition of TNBC, the most recently collected tumor specimen (i.e., surgical tumor specimen for patients not undergoing tumor re-biopsy for metastatic disease, or biopsy of a metastatic lesion for patients undergoing tumor re-biopsy at disease relapse after surgery) was used; (b) advanced disease, as defined as the presence of distant metastases or locally advanced, inoperable disease; (c) age ≥ 18 years; (d) treatment with at least one cycle of one of the following first-line regimens: CG (carboplatin at an area under the curve (AUC) of 2 plus gemcitabine 800 mg/m2, both administered i.v. on days 1 and 8 every 3 weeks; or carboplatin at an AUC of 5 on day 1 every 3 weeks plus gemcitabine 800 mg/m2 on days 1 and 8 every 3 weeks), or CP (carboplatin AUC 2 plus paclitaxel 80 mg/m2, both administered i.v. on days 1 and 8 every 3 weeks); (e) availability of data regarding clinical outcomes, including patient PFS and OS; (f) available data about previous therapies received (i.e., surgery, radiotherapy and (neo)adjuvant therapy); (g) available data about number and sites of metastatic lesions; (h) available data about treatment-related adverse events (AEs). This is a retrospective, monocentric study that included advanced TNBC patients treated with first-line platinum-based chemotherapy at Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, Italy) between April 2007 and April 2021. Inclusion criteria were: (a) women with pathologically/cytologically confirmed diagnosis of TNBC, as defined as estrogen receptor (ER) and progesterone receptor (PgR) expression in less than 1% of cancer cells by IHC analysis, and an IHC score for HER2 expression of 0/1+, or an IHC score of 2 + in the absence of HER2 gene amplification by ISH analysis. Assessment of treatment efficacy and safety Tumor response was assessed every three chemotherapy cycles (~ every 2 months) through computed tomography (CT) or magnetic resonance imaging (MRI) using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Tumor radiological assessment was performed by central review by a radiologist. Data were collected at the time of patient enrollment. AEs were classified according to common terminology criteria for adverse events (CTCAE) version 4.03 of June 14, 2010 of National Institutes of Health, National Cancer Institute. Hematological toxicities were collected from computerized blood sample data. Non-hematological toxicities were retrieved from medical records, where they had been regularly annotated during patient visits. Exploratory evaluations We performed an exploratory evaluation to study the potential impact of previous exposure to taxanes on the efficacy (in terms of PFS or OS) of CG vs. CP. For this evaluation, we analyzed the interaction between previous exposure to taxanes (yes vs. no) and the efficacy of CG vs. CP (see Statistical analyses below). In addition, since 1st line platinum chemotherapy has been shown to be superior to taxane-based chemotherapy in advanced TNBC patients who are carries of pathogenic BRCA1 or BRCA2 mutations, we also performed an exploratory analysis to evaluate the potential impact of germline BRCA1 or BRCA2 Page 5/28 Page 5/28 mutations on the PFS of patients included in this study (regardless of the treatment cohort) [17]. For this analysis, patients were divided in three different cohorts: a) patients undergoing genetic evaluations and found to be carriers of pathogenetic germline BRCA1 or BRCA2 alterations; b) patients undergoing genetic testing and not found to be carries of pathogenetic germline BRCA1 or BRCA2 alterations; c) patients not undergoing genetic testing. We reported results of PFS evaluations in these three cohorts. Sensitivity analysis excluding patients with de novo metastatic disease In addition to including the presence of de novo metastatic disease as a covariate in all multivariable analyses performed in this study, we also performed a sensitivity analysis in which we evaluated the impact of CG and CP on patient PFS and OS after removing patients diagnosed with de novo metastatic disease. In these multivariable Cox models, we included the same covariates included in the main models (see Statistical analyses below), with the only exception of presence/absence of de novo metastatic disease. Statistical analyses The χ2 test was used to study the distribution of dichotomous patient- or tumor-related variables in CG vs. CP groups, whereas the Welch Two Sample t test was used to compare the distribution of continuous variables in the two patient groups. Median follow-up was calculated using reverse Kaplan Meier method. The impact of the type of platinum doublet (CG vs. CP) on PFS and OS was evaluated through Cox proportional hazard models. Clinically relevant variables previously associated with clinical outcomes in advanced TNBC patients, namely age, Eastern Cooperative Oncology Group (ECOG) Performance Status (PS), body mass index (BMI), previous taxane exposure, previous anthracycline exposure, DFI, de novo metastatic (stage IV) disease, number of metastatic sites, presence of lung, liver, brain or bone metastases at treatment initiation, were included in multivariable models [6, 7, 19, 27]. To avoid the exclusion of patients for whom data on specific covariates were not available, missing data were imputed, and Cox models for the main study analyses were fitted by including imputed data. Single imputation technique using mean value of the available data was performed [28]. Results of the main analyses were confirmed in Cox models in which we only included original data (i.e., without imputation). Adjusted PFS and OS estimates based on multivariable Cox proportional hazard regression models were Page 6/28 Page 6/28 calculated by using the “conditional method”, and represented as adjusted survival curves [29, 30]. Bootstrap resampling method (1000 times) provided robust estimates of adjusted median PFS/OS and 95% confidence intervals (CI), as well as estimates of the median differences between the two treatment groups with the corresponding 95% CIs [31]. To test the impact of the interaction between type of treatment (CG vs. CP) and previous taxane exposure, we also fitted multivariable Cox models in which we included a product term accounting for the interaction between these variables. A threshold of significance (p value) of 0.10 was set for the interaction term analyses, while other statistical evaluations were considered as statistically significant if the p value was lower than 0.05. All statistical analyses were performed using the software R (version 4.0.2 (2020-06-22)). Availability of data and materials The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Patient population With a total number of 83 progression events and 68 death events, median PFS and OS in the whole patient population were 6.49 months (95% CI: 5.26 – 8.94) and 18.0 months (95% CI: 15.5 – 27.6), respectively. At multivariable analysis, CG was independently associated with significantly better PFS when compared to CP [Hazard Ratio (HR): 0.49; 95% CI: 0.27-0.87; p value 0.014], while not being diagnosed with ab initio metastatic disease (no vs. yes) and a higher number of metastatic sites (>3 vs. 1-3) were associated with worse PFS (HR: 3.93; 95% CI: 1.18-13.11; p value = 0.026 and HR: 2.70; 95% CI: 1.09-6.66; p value = 0.032, respectively) (Table 2). After adjustment for the same covariates, CG was also associated with significantly better patient OS (HR: 0.31; 95% CI: 0.15-0.64; p value = 0.002), along with longer DFI after surgery, here evaluated as a continuous variable (HR: 0.99; 95% CI: 0.986-0.999; p value = 0.028). By contrast, factors associated with worse OS were: not being diagnosed with metastatic disease ab initio (HR: 6.33; 95% CI: 1.20-33.42; p value = 0.030), worse ECOG PS (HR: 5.11; 95% CI: 2.34-11.18; p value < 0.001), presence of lung metastases (HR: 2.05; 95% CI: 1.07-3.92; p value = 0.031), presence of liver metastases (HR: 2.37; 95% CI: 1.20-4.68; p value = 0.013) (Table 3). PFS and OS estimates in the two treatment groups, adjusted for the covariates included in the Cox multivariable models, are shown as adjusted survival curves in Figure 1A and 1B, respectively. Patients treated with CG had longer adjusted PFS and OS when compared to patients treated with CP combination (8.98 vs. 5.36 months for adjusted PFS and 31.59 vs. 15.68 months for adjusted OS). These results were confirmed by bootstrap analyses performed on adjusted survival curves, which showed statistically significant survival advantage in patients treated with CG vs. CP in terms of both PFS [median PFS: 9.01 months (95% CI: 6.35 – 10.59) vs. 5.29 months (95% CI: 4.47 – 6.71), absolute PFS difference: 3.45 months (95% CI: 0.07 – 5.53)] and OS [median OS: 30.05 months (95% CI: 18.21 – 48.33) vs. 15.48 months (95% CI: 12.30 – 20.35), absolute OS difference: 14.17 months (95% CI: 2.30 – 32.65)]. Patient population We evaluated 418 advanced BC patients treated with platinum-based doublets in our Institution between April 2007 and April 2021 (Additional file 1: Figure S1). Of these, 88 patients had advanced TNBC and received carboplatin-based combinations as a first-line treatment for advanced disease; therefore, they were selected for the current study. Patient and tumor characteristics are reported in Table 1. Median patient age in the whole patient cohort was 55.9 years (range 34-80). Of 88 patients included, 56 (63.6%) were treated with CP, while 32 (36.4%) received CG. The majority of patients had previously received taxane- and anthracycline-based chemotherapy in the (neo)adjuvant setting (n = 62, 70.5% and n = 66, 75.0%, respectively), while only three patients (3.4%) had previously received (neo)adjuvant carboplatin. Nine patients were diagnosed with de novo metastatic disease (n = 3 in the CP subgroup and n = 6 in the CG subgroup, p-value = 0.103). The majority of patients had visceral involvement (n = 56, 63.6%) and an ECOG PS of 0 (n = 55, 62.5%). Baseline characteristics were well balanced in the two treatment groups, with the exception of a higher proportion of patients with shorter DFI in the CG group when compared to the CP group (59.4% vs. 44.6%; χ2 test p value = 0.038). Out of the 88 patients included in the study, 79 patients were diagnosed with limited-stage disease and had undergone basal evaluation of ER/PgR/HER2 status. Of these 79 patients, 60 (75.9%) had a diagnosis of TNBC from the beginning of their clinical history (i.e., on the basis of basal ER/PgR/HER2 status evaluation in the primary tumor), while 16 (20.3%) and 2 (2.5%) patients were initially diagnosed with HR+HER2- BC and HER2+ BC, respectively, and their tumors underwent biological switch to TNBC upon disease recurrence and biological tumor re-characterization of disease recurrence (Additional file 1: Table S1). The proportion of patients undergoing a HR+HER2- BC/HER2+ BC to TNBC conversion was numerically lower in the CG group as compared to the CP group (Additional file 1: Table S1). Page 7/28 Page 7/28 Treatment efficacy and antitumor activity At data cut-off and analysis (April 1, 2021), median patient follow-up was 41.19 months (IQR 28.73 – 54.05). Impact of germline BRCA1/BRCA2 status on the efficacy of CG/CP doublets The presence of germline BRCA1 or BRCA2 mutations has been associated with an increased sensitivity to platinum-based chemotherapy in advanced TNBC patients [17]. In our patient cohort, 30 patients (34.1%) underwent genetic testing for the evaluation of germinal BRCA1 and BRCA2 status. Of these patients, 13 (43.3%) were found to be carries of pathologic germline BRCA1/BRCA2 mutations, while 17 patients (56.7%) had wild type BRCA1/BRCA2 genes. Germline BRCA1/BRCA2 mutation carriers were equally distributed in the CG and CP groups (Additional file 1: Table S7). Although median PFS was numerically longer in BRCA1/2 mutation carries as compared to patients with wild type BRCA1/2 status or patients not evaluated for the presence of germline BRCA1/2 mutations (8.98 vs. 6.36 vs. 6.4 months, respectively), this difference did not reach statistically significant differences, likely due to the low number of patients included in these sub-cohorts (Additional file 1: Figure S2). Patient population Results of this exploratory analysis suggest that the observed association between CP and worse PFS might be driven by the subset of patients previously exposed to taxane-based chemotherapy in the (neo)adjuvant setting. Adjusted PFS curves stratified on the basis of previous taxane exposure and type of platinum-based doublet are shown in Figure 2A. By contrast, CG was associated with better OS regardless of previous exposure to taxanes (p value of interaction term: 0.772) (Table 5). In this model, higher ECOG PS scores retained a strong association with worse OS (HR 4.95; 95% CI: 2.20 – 11.12; p value <0.001). Adjusted OS curves stratified on previous taxane exposure in CP and CG subgroups are shown in Figure 2B. Patient population Of note, CG treatment maintained an independent association with significantly better patient PFS and OS when multivariable analysis was repeated by using original data (i.e., without imputation of missing data) (Additional file 1: Table S2 and Table S3, respectively), and also at a sensitivity multivariable analysis that excluded patients with de novo metastatic disease [HR of 0.37 (95% CI: 0.20-0.72) and 0.33 (95% CI: 0.16-0.69) for PFS and OS] (Additional file 1: Table S4 and Table S5, respectively). Tumor response was evaluated in 87 patients according to RECIST 1.1 criteria. Overall, we detected 9 CR (10.3%), 41 PR (47.1%), 18 SD (20.7%) and 19 disease progression (PD) (21.8%) as best tumor responses, resulting in an ORR of 57.5% (95% CI: 46.4 – 68.0) and in a DCR of 78.2% (95% CI: 68.0 – 86.3). We found no statistically significant ORR differences between patients treated with CG or CP [58.9% (95% CI: 45.0-71.9) vs. 54.8% (95% CI: 36.0-72.7), respectively; p-value = 0.70]. The DCR was also similar in the two treatment groups [83.3% for CG (95% CI: 65.3-94.4) vs. 75.4% (95% CI: 62.2-85.9) for CP, p value = 0.53]. Although DOR was numerically higher in patients treated with CG as compared to patients receiving CP, this difference did not reach statistical significance (Additional file 1: Table S6). Page 8/28 Impact of previous taxane exposure on treatment efficacy To investigate if worse PFS in CP-treated patients could be in part justified by the fact that some patients had previously received taxane-based (neo)adjuvant therapy, possibly resulting in more taxane-resistant disease at the time of first-line treatment initiation, we performed an exploratory analysis to investigate the potential impact of the interaction between previous taxane exposure (yes vs. no) and the type of platinum doublet (CG vs. CP) on patient PFS. In a multivariable model that also included a product term accounting for the interaction between these two variables, CG was associated with significantly better PFS (HR of 0.39; 95% CI: 0.21 – 0.75) in patients previously exposed to taxanes, but not in patients not pre-treated with taxanes (HR: 1.20; 95% CI: 0.37-3.88) (Table 4). Not being diagnosed with de novo metastatic disease and a higher number of metastatic sites maintained their significant association with worse PFS [HR 4.86 (95% CI: 1.48-15.98; p value = 0.009) and HR 2.90 (95% CI: 1.18-7.15; p value = 0.021)]. Treatment Safety and Tolerability Page 9/28 Treatment-related AEs are described in Table 6. The incidence of any grade AEs was 85.7% in CP subgroup and 100% in CG subgroup (p value = 0.063). Hematologic AEs were the most common any- grade AEs in patients treated with CG, and we found a statistically higher incidence of neutropenia (90.6% vs. 66.1%, p value = 0.021) and thrombocytopenia (59.4% vs. 14.3%, p value < 0.001) in CG vs. CP groups. Other AEs more commonly occurring in patients treated with CG than in patients treated with CP were any grade fatigue (59.4% vs. 32.1%, p value = 0.024) and increased blood ALT levels (56.2% vs. 19.6%, p-value Page 9/28 Page 9/28 = 0.001). On the other hand, CP was associated with significantly higher rates of peripheral neuropathy (26.8% vs. 3.1%, p value = 0.013). Considering the whole patient population, 34 out of 88 patients (38.6%) experienced AEs graded as 3 or 4. Overall, CG was associated with higher rates of G3-G4 AEs as compared to CP [22 out of 32 patients (68.8%) vs. 12 out of 56 patients (21.4%), p-value = 0.009). In the whole patient cohort, the most common G3-G4 AE was neutropenia, and it occurred significantly more frequently in patients treated with CG (59.4% vs. 12.5%, p-value < 0.001). Moreover, 6 patients in the CG subgroup experienced G3-G4 thrombocytopenia compared to one thrombocytopenia event in the CP subgroup (18.7% vs. 1.8%, p value = 0.016). While the median number of doublet chemotherapy cycles and the proportion of patients switching to single-agent chemotherapy was not statistically significantly different in the CG vs. CP cohorts, a higher proportion of patients in the CG arm underwent dose reduction or omission of one or more treatment administrations as compared to the CP arm. In addition, the absolute number and proportion of chemotherapy cycles that were omitted (over the total number of cycles) was significantly higher in the cohort of patients treated with CG (Additional file 1: Table S8). Discussion The use of platinum-based chemotherapy combinations as a standard first-line treatment for advanced TNBC patients is a debated topic. On the one hand, single-agent carboplatin has demonstrated similar antitumor activity when compared to single-agent docetaxel as a first-line therapy for advanced TNBC, and it is more active than docetaxel in the subset of advanced TNBC patients bearing germline BRCA1 or BRCA2 mutations [17]. On the other hand, combination chemotherapy, including platinum-based doublets, has not demonstrated to improve patient OS when compared to single-agent chemotherapy despite longer PFS and higher ORRs, and it also results in higher toxicity rates [32,33]. In addition, the increasing use of carboplatin as part of standard neoadjuvant chemotherapy regimens might contribute to the relatively low use of platinum doublets as first-line treatments in advanced TNBC patients [34,35]. Even when platinum-based doublets are considered for advanced TNBC treatment, the most effective drug to be combined with carboplatin/cisplatin is still unclear. Here, we found that CG, when a first-line chemotherapy combination in advanced TNBC patients, is independently associated with significantly better PFS and OS when compared to CP. These results are especially relevant if we take into account that CG-treated patients: a) had significantly shorter DFI, which is indicative of more aggressive and/or chemo-resistant disease; b) more frequently underwent treatment dose reduction/omissions when compared to patients treated with CP. To explain the observed superiority of CG over CP, we reasoned that previous tumor exposure to taxanes, which are part of the standard-of- care therapy in the (neo)adjuvant setting, might determine a more taxane-resistant disease, thus resulting in shorter PFS in patients treated with CP when compared to patients treated with CG. Consistent with this hypothesis, CG was associated with better PFS (when compared to CP) only in patients previously exposed to taxanes, while CG and CP were similarly effective in patients who had not received prior taxane-based therapy. Although these findings are of potential clinical interest, they should be interpreted with caution due to the limited number of patients included in the taxane-pre-treated or taxane-not- pretreated CG or CP cohorts. Future prospective trials including a larger number of patients are needed to Page 10/28 Page 10/28 investigate if carboplatin-gemcitabine might be a more effective 1st line treatment option as compared to CP in taxane-pretreated, advanced TNBC patients. Discussion In addition to prior patient exposure to taxanes, the following two factors could contribute to explain the more favorable clinical outcomes observed in the CG cohort as compared to the CP cohort: 1) while most of patients had previously received taxanes as a (neo)adjuvant therapy, only 3 of them had previously received carboplatin, and none of them had been exposed to gemcitabine; therefore, patients receiving CG in our clinical cohort were exposed concomitantly to two new chemotherapeutical agents that they had never received during their clinical course, thus potentially resulting in higher tumor sensitivity to the treatment and better PFS; 2) gemcitabine modulates antitumor immunity, and in particular it reduces myeloid-derived suppressive cells, regulatory T cells (Tregs) and B-cells, while concomitantly enhancing T- cell mediated anti-tumor immune effects [36,37]. In this regard, the recently published phase III trial Keynote-355, which showed that the addition of pembrolizumab to platinum-based doublets (carboplatin- nab-paclitaxel, carboplatin-paclitaxel or carboplatin-gemcitabine) prolongs patient PFS especially in the case of tumors expressing PD-L1 and with high combined positive score (CPS), failed to reveal a positive impact of pembrolizumab in the subset of patients receiving carboplatin-gemcitabine [27]. These results could reflect the fact that gemcitabine is sufficiently potent to modulate systemic immunity, thus reducing the therapeutic impact of adding pembrolizumab to platinum-gemcitabine doublets. An ongoing phase III study, namely the IMpassion132 trial, is evaluating the combination of atezolizumab with capecitabine or carboplatin plus gemcitabine in patients with advanced TNBC recurring ≤ 12 months after completing standard (neo)adjuvant anthracycline and taxane chemotherapy. The primary endpoint of this study is OS, and this trial may provide further evidence on the efficacy of combining immune-checkpoint inhibitors with platinum-gemcitabine doublets [38]. In the recently published phase II trial TnAcity, first-line carboplatin-nab-paclitaxel resulted in longer PFS when compared to nab-paclitaxel-gemcitabine (8.3 vs. 5.5 months, respectively) or carboplatin-gemcitabine (8.3 vs. 6.0 months, respectively) combinations, thus suggesting that nab-paclitaxel could be the best partner to combine with carboplatin in this setting [7]. The main limitations of TnAcity consist in the relatively low number of patients included, and in the fact that nab-paclitaxel is not approved as a first-line therapy for TNBC treatment in Europe; therefore, since the TnAcity study did not compare the efficacy of CG and CP, the conclusions of this trial are poorly applicable to the clinical practice in Europe and Italy. Discussion In a Chinese, phase III trial that randomized 240 advanced TNBC patients to gemcitabine plus either cisplatin or paclitaxel, the cisplatin-gemcitabine combination was associated with significantly better PFS when compared to the paclitaxel-gemcitabine combination (HR for PFS 0.69; 95% CI: 0.523-0.915) [24]. However, in this study the absence of a cisplatin-paclitaxel arm prevents any evaluation on the most effective platinum-based combination (i.e. cisplatin-gemcitabine or cisplatin-paclitaxel). Several variables, including previous exposure to carboplatin and taxanes, or the presence of BRCA1/2 mutations, could affect the clinical efficacy of different first-line platinum-based combinations. For instance, in the recently published phase III, randomized BROCADE3 trial, carboplatin-paclitaxel, alone or combined with the PARP inhibitor veliparib, was associated with excellent PFS (median PFS of 12.6 Page 11/28 Page 11/28 Page 11/28 and 14.5 months in the carboplatin-paclitaxel and carboplatin-paclitaxel-veliparib, respectively) in advanced TNBC patients bearing germline BRCA1/2 mutations [39]. In our study, although PFS was numerically longer in patients carrying germline BRCA1/BRCA2 mutations, these differences did not reach statistical significance, likely due to the low number of patients included. Future prospective studies are needed to investigate if platinum-based doublets are more effective in BRCA1/BRCA2 mutations carries than in BRCA1/BRCA2 wt patients, as well as to determine the most effective platinum doublets in advanced TNBC patients carrying pathogenic BRCA1/BRCA2 mutations. and 14.5 months in the carboplatin-paclitaxel and carboplatin-paclitaxel-veliparib, respectively) in advanced TNBC patients bearing germline BRCA1/2 mutations [39]. In our study, although PFS was numerically longer in patients carrying germline BRCA1/BRCA2 mutations, these differences did not reach statistical significance, likely due to the low number of patients included. Future prospective studies are needed to investigate if platinum-based doublets are more effective in BRCA1/BRCA2 mutations carries than in BRCA1/BRCA2 wt patients, as well as to determine the most effective platinum doublets in advanced TNBC patients carrying pathogenic BRCA1/BRCA2 mutations. The main limitations of our study consist in its retrospective design and in the relatively low number of patients included. Discussion Strengths of the study consist in: 1) the homogeneity of the clinical cohort, which included advanced TNBC patients treated with first-line platinum-based doublets; 2) its monocentric nature, which guarantees reproducible collection of data, tumor response assessment and homogeneous patient management; 3) finally, tumor ORR, median PFS and median OS in the whole patient cohort were in line with data previously reported in the literature, thus indicating that our study cohort is representative of the population of advanced TNBC patients receiving first-line chemotherapy. Conclusions To the best of our knowledge, this is the first study to compare the efficacy and safety of two commonly used platinum doublets, namely CG and CP, as first-line chemotherapy options in advanced TNBC patients. Although our findings need prospective validation in larger patient cohorts, they suggest that CG and CP are valuable treatment options in this poor-prognosis patient population, with an acceptable toxicity profile. In particular, future prospective studies should investigate if CG might be a preferred treatment option for patients previously exposed to taxanes in the (neo)adjuvant treatment setting. Abbreviations Page 12/28 Page 12/28 DCR: Disease Control Rate DFI: Disease-Free Interval DOR: Duration Of Response ECOG: Eastern Cooperative Oncology Group ER: Estrogen Receptor HER2: Human Epidermal Growth Factor Receptor 2 HR: Hazard Ratio i.v.: Intravenous IHC: ImmunoHistoChemistry ISH: In Situ Hybridization ORR: Overall Response Rate OS: Overall Survival PARPi: Polyadenosine Diphosphate-Ribose Polymerase Inhibitors PD-L1: Programmed Death-Ligand 1 PFS: Progression Free Survival PgR: Progesterone Receptor PR: Partial Response PS: Performance Status SD: Stable Disease TNBC: Triple Negative Breast Cancer Declarations Competing interests The authors declare no conflicts of interest related to this work Acknowledgments We would like to thank the “Associazione Italiana per la Ricerca sul Cancro” (AIRC) and the Scientific Directorate of Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, Italy) for funding our research (MFAG 2019 -22977 P.I Dr. Claudio Vernieri) Funding Our research is supported by the “Associazione Italiana per la Ricerca sul Cancro” (AIRC) for funding our research (MFAG 2019 -22977 P.I Dr. Claudio Vernieri) and the Scientific Directorate of Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, Italy) Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The study protocol was approved by the Internal Review Board (IRB) and the Local Ethics Committee of the “Fondazione IRCCS Istituto Nazionale dei Tumori” (INT 79/17). Patient data were collected according to the ethical principles for medical research involving human subjects adopted in the Declaration of Helsinki. Patients who were alive at the time of data collection and/or analyses signed an informed consent for the use of their data for research purposes. Authors’ contributions Conceptualization: CV and RL. Data curation: RL, LM, GP, AR and SM. Formal analysis: LM, RL and CV. Software: LM. Investigation: RL, CV, LM, GP, FL, GF, AR, EZ, RLe, BC, CD, AV, SM, GS, GVB, GC, GP and FdB. Conceptualization: CV and RL. Data curation: RL, LM, GP, AR and SM. Formal analysis: LM, RL and CV. Software: LM. Investigation: RL, CV, LM, GP, FL, GF, AR, EZ, RLe, BC, CD, AV, SM, GS, GVB, GC, GP and FdB. Conceptualization: CV and RL. Data curation: RL, LM, GP, AR and SM. Formal analysis: LM, RL and CV. Software: LM. Investigation: RL, CV, LM, GP, FL, GF, AR, EZ, RLe, BC, CD, AV, SM, GS, GVB, GC, GP and FdB. Page 13/28 Validation: RLe. Writing – original draft: RL and CV. Writing – review & editing: LM, GP, FL, GF, AR, EZ, RLe, BC, CD, AV, SM, GS, GVB, GC, GP and FdB. Supervision: CV, GS, GVB, GC, GP and FdB. Validation: RLe. Writing – original draft: RL and CV. Writing – review & editing: LM, GP, FL, GF, AR, EZ, RLe, BC, CD, AV, SM, GS, GVB, GC, GP and FdB. Supervision: CV, GS, GVB, GC, GP and FdB. Validation: RLe. Writing – original draft: RL and CV. Writing – review & editing: LM, GP, FL, GF, AR, EZ, RLe, BC, CD, AV, SM, GS, GVB, GC, GP and FdB. Supervision: CV, GS, GVB, GC, GP and FdB. References 1. 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Patient and disease characteristics Page 17/28 Overall N= 88 CP N= 56 CG N= 32 P value Age, median (range) 55.9 (34-80) 56.5 (34-79) 55.0 (39-80) 0.792 ECOG PS 0 1 NA 55 (62.5) 18 (20.5) 15 (17.0) 35 (62.5) 8 (14.3) 13 (23.2) 20 (62.5) 10 (31.2) 2 (6.3) 0.246 BMI (Kg/m2) < 25 ≥ 25 41 (46.6) 47 (53.4) 25 (44.6) 31 (55.4) 16 (50.0) 16 (50.0) 0.793 DFI (years) < 3 ≥ 3 NA 44 (50.0) 33 (37.5) 11 (12.5) 25 (44.6) 27 (48.3) 4 (7.1) 19 (59.4) 6 (18.7) 7 (21.9) 0.038 Previous Taxanes No Yes 26 (29.5) 62 (70.5) 18 (32.1) 38 (67.9) 8 (25.0) 24 (75.0) 0.643 Previous Anthracyclines No Yes 22 (25.0) 66 (75.0) 11 (19.6) 45 (80.4) 11 (34.4) 21 (65.6) 0.201 Previous Carboplatin No Yes 85 (96.6) 3 (3.4) 55 (98.2) 1 (1.8) 30 (93.7) 2 (6.3) 0.617 De novo metastatic disease No Yes 79 (89.8) 9 (10.2) 53 (94.6) 3 (5.4) 26 (81.3) 6 (18.7) 0.103 N. metastatic sites 1-3 74 (84.1) 46 (82.1) 28 (87.5) 0.720 No Yes 32 (36.4) 56 (63.6) 20 (35.7) 36 (64.3) 12 (37.5) 20 (62.5) 1.000 Liver metastasis No Yes 66 (75.0) 22 (25.0) 44 (78.6) 12 (21.4) 22 (68.8) 10 (31.2) 0.443 Lung metastasis No Yes 43 (48.9) 45 (51.1) 25 (44.6) 31 (55.4) 18 (56.2) 14 (43.8) 0.409 Bone metastasis No Yes 42 (47.7) 46 (52.3) 25 (44.6) 31 (55.4) 17 (53.1) 15 (46.9) 0.586 Brain metastasis No Yes 80 (90.9) 8 (9.1) 51 (91.1) 5 (8.9) 29 (90.6) 3 (9.4) 1.000 ata are presented as n (%) except where otherwise specified. The p value of the unpaired t-test (age) or 2 test (other variables) is indicated in bold numbers when statistically significant. In case of not vailable (NA) information for specific variables, the p value refers to the χ2 test performed after xcluding patients with NA data. bbreviations: BMI: body mass index; CG: Carboplatin plus Gemcitabine; CP: Carboplatin plus Paclitaxel FI: Disease Free Interval; ECOG: Eastern Cooperative Oncology Group; PS: Performance Status. Table 2. Multivariable analysis for PFS. Tables No Yes 32 (36.4) 56 (63.6) 20 (35.7) 36 (64.3) 12 (37.5) 20 (62.5) 1.000 Liver metastasis No Yes 66 (75.0) 22 (25.0) 44 (78.6) 12 (21.4) 22 (68.8) 10 (31.2) 0.443 Lung metastasis No Yes 43 (48.9) 45 (51.1) 25 (44.6) 31 (55.4) 18 (56.2) 14 (43.8) 0.409 Bone metastasis No Yes 42 (47.7) 46 (52.3) 25 (44.6) 31 (55.4) 17 (53.1) 15 (46.9) 0.586 Brain metastasis No Yes 80 (90.9) 8 (9.1) 51 (91.1) 5 (8.9) 29 (90.6) 3 (9.4) 1.000 ata are presented as n (%) except where otherwise specified. The p value of Data are presented as n (%) except where otherwise specified. The p value of the unpaired t-test (age) or χ2 test (other variables) is indicated in bold numbers when statistically significant. In case of not available (NA) information for specific variables, the p value refers to the χ2 test performed after excluding patients with NA data. Abbreviations: BMI: body mass index; CG: Carboplatin plus Gemcitabine; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG: Eastern Cooperative Oncology Group; PS: Performance Status. Abbreviations: BMI: body mass index; CG: Carboplatin plus Gemcitabine; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG: Eastern Cooperative Oncology Group; PS: Performance Status. Table 2. Multivariable analysis for PFS. Page 19/28 Page 19/28 Page 19/28 Variables HR 95% CI P value Type of treatment CG vs. CP 0.49 0.27 – 0.87 0.014 Age (years) ≤ 65 vs. > 65 1.20 0.58 – 0.87 0.624 ECOG PS* 1.36 0.72 – 2.51 0.342 BMI (kg/m2) ≥ 25 vs. < 25 0.60 0.34 – 1.07 0.083 Previous taxanes Yes vs. No 1.99 0.92 – 4.31 0.079 Previous anthracyclines Yes vs. No 0.51 0.25 – 1.04 0.064 DFI (years)* 0.99 0.98 – 1.00 0.073 De novo metastatic disease No vs. Yes 3.93 1.18 – 13.11 0.026 N° of metastatic sites > 3 vs. ≤ 3 2.70 1.09 – 6.66 0.032 Lung metastasis Yes vs. No 0.78 0.45 – 1.36 0.380 Liver metastasis Yes vs. No 1.57 0.85 – 2.90 0.147 Brain metastasis Yes vs. No 1.46 0.62 – 3.46 0.391 Bone metastasis Yes vs. No 0.86 0.52 – 1.44 0.564 mputed data are evaluated as continuous variables. The p value is ind tistically significant Covariates with imputed data are evaluated as continuous variables. The p value is indicated in bold numbers when statistically significant. Covariates with imputed data are evaluated as continuous variables. Tables The p value is indicated in bold numbers when statistically significant. Abbreviations: BMI: Body Mass Index; CG: Carboplatin plus Gemcitabine; CI: Confidence Interval; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG PS: Eastern Cooperative Oncology Group Page 20/28 Page 20/28 Page 20/28 Performance Status; HR: Hazard Ratio. Table 3. Multivariable analysis for OS. Variables HR 95% CI P value Type of treatment CG vs. CP 0.31 0.15 – 0.64 0.002 Age (years) ≤ 65 vs. > 65 1.63 0.68 – 3.91 0.270 ECOG PS* 5.11 2.34 – 11.18 <0.001 BMI (kg/m2) ≥ 25 vs. < 25 0.72 0.37 – 1.39 0.324 Previous taxanes Yes vs. No 2.07 0.93 – 4.62 0.075 Previous anthracyclines Yes vs. No 0.67 0.30 – 1.48 0.319 DFI (years)* 0.99 0.98 – 0.99 0.028 De novo metastatic disease No vs Yes 6.33 1.20 – 33.42 0.030 N° of metastatic sites > 3 vs. ≤ 3 0.80 0.31 – 2.07 0.641 Lung metastasis Yes vs. No 2.05 1.07 – 3.92 0.031 Liver metastasis 2.37 1.20 – 4.68 0.013 Table 3. Multivariable analysis for OS. Variables HR 95% CI P value Type of treatment CG vs. CP 0.31 0.15 – 0.64 0.002 Age (years) ≤ 65 vs. > 65 1.63 0.68 – 3.91 0.270 ECOG PS* 5.11 2.34 – 11.18 <0.001 BMI (kg/m2) ≥ 25 vs. < 25 0.72 0.37 – 1.39 0.324 Previous taxanes Yes vs. No 2.07 0.93 – 4.62 0.075 Previous anthracyclines Yes vs. No 0.67 0.30 – 1.48 0.319 DFI (years)* 0.99 0.98 – 0.99 0.028 De novo metastatic disease No vs Yes 6.33 1.20 – 33.42 0.030 N° of metastatic sites > 3 vs. ≤ 3 0.80 0.31 – 2.07 0.641 Lung metastasis Yes vs. No 2.05 1.07 – 3.92 0.031 Liver metastasis Yes vs. No 2.37 1.20 – 4.68 0.013 Brain metastasis Yes vs. No 1.61 0.60 – 4.31 0.344 Bone metastasis Yes vs. No 1.40 0.79 – 2.50 0.249 Page 21/28 Covariates with imputed data are evaluated as continuous variables. The p value is indicated in bold numbers when statistically significant. Abbreviations: BMI: Body Mass Index; CG: Carboplatin plus Gemcitabine; CI: Confidence Interval; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG PS: Eastern Cooperative Oncology Group Performance Status; HR: Hazard Ratio. Table 4. Tables Multivariable model for PFS including the interaction between previous taxane exposure and type of platinum doublet Page 22/28 Variables HR 95% CI P value Type of treatment x Previous taxanes CG vs. CP - Previous taxanes - No previous taxanes 0.39 1.20 0.21 – 0.75 0.37 - 3.88 0.091# Age (years) ≤ 65 vs. > 65 1.21 0.58 – 2.52 0.615 ECOG PS 1.70 0.85 – 3.40 0.132 BMI (kg/m2) ≥ 25 vs. < 25 0.59 0.33 – 1.05 0.073 Previous anthracyclines Yes vs. No 0.56 0.27 – 1.16 0.119 DFI (years)* 0.99 0.99 – 1.00 0.110 De novo metastatic disease No vs Yes 4.86 1.48 – 15.98 0.009 N° of metastatic sites > 3 vs. ≤ 3 2.90 1.18 – 7.15 0.021 Lung metastasis Yes vs. No 0.80 0.46 – 1.40 0.438 Liver metastasis Yes vs. No 1.67 0.90 – 3.09 0.101 Brain metastasis Yes vs. No 1.42 0.60 – 3.36 0.421 Bone metastasis Yes vs. No 0.81 0.48 – 1.37 0.433 with imputed data are evaluated as continuous variables. # A threshold of signifi i t ti t t Th l i i di t d i b ld b h t ti ti ll i Covariates with imputed data are evaluated as continuous variables. # A threshold of significance of 0.1 was set for the interaction test. The p value is indicated in bold numbers when statistically significant. Covariates with imputed data are evaluated as continuous variables. # A threshold of significance of 0.1 was set for the interaction test. The p value is indicated in bold numbers when statistically significant. Abbreviations: BMI: Body Mass Index; CG: Carboplatin plus Gemcitabine; CI: Confidence Interval; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG PS: Eastern Cooperative Oncology Group Page 23/28 Page 23/28 Performance Status; HR: Hazard Ratio. Table 5. Multivariable model for OS analyzing the interaction between previous taxane exposure and type of platinum doublet Table 5. Multivariable model for OS analyzing the interaction between previous taxane exposure and type of platinum doublet r OS analyzing the interaction between previous taxane exposure and type Page 24/28 oublet Variables HR 95% CI P value Type of treatment x Previous taxanes CG vs. CP - Previous taxanes - No previous taxanes 0.32 0.22 0.15 – 0.69 0.02 – 2.05 0.772# Age (years) ≤ 65 vs. able 6. Treatment-related adverse events in the CG and CP cohorts. Tables > 65 1.61 0.67 – 3.88 0.286 ECOG PS* 4.95 2.20 – 11.12 <0.001 BMI (kg/m2) ≥ 25 vs. < 25 0.73 0.37 – 1.42 0.349 Previous anthracyclines Yes vs. No 0.65 0.29 – 1.46 0.300 DFI (years)* 0.99 0.98 – 0.99 0.028 De novo metastatic disease No vs Yes 6.25 1.17 – 33.33 0.032 N° of metastatic sites > 3 vs. ≤ 3 0.80 0.31 – 2.06 0.639 Lung metastasis Yes vs. No 2.01 1.04 – 3.89 0.040 Liver metastasis Yes vs. No 2.35 1.19 – 4.65 0.014 Brain metastasis Yes vs. No 1.60 0.60 – 4.29 0.350 Bone metastasis Yes vs. No 1.41 0.79 – 2.52 0.244 Covariates with imputed data are evaluated as continuous variables. # A threshold of significance of 0.1 was set for the interaction test. The p value is indicated in bold numbers when statistically significant. Covariates with imputed data are evaluated as continuous variables. # A threshold of significance of 0.1 was set for the interaction test. The p value is indicated in bold numbers when statistically significant. Abbreviations: BMI: Body Mass Index; CG: Carboplatin plus Gemcitabine; CI: Confidence Interval; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG PS: Eastern Cooperative Oncology Group *Covariates with imputed data are evaluated as continuous variables. # A threshold of significance of 0.1 was set for the interaction test. The p value is indicated in bold numbers when statistically significant. Abbreviations: BMI: Body Mass Index; CG: Carboplatin plus Gemcitabine; CI: Confidence Interval; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG PS: Eastern Cooperative Oncology Group Performance Status; HR: Hazard Ratio. Abbreviations: BMI: Body Mass Index; CG: Carboplatin plus Gemcitabine; CI: Confidence Interval; CP: Carboplatin plus Paclitaxel; DFI: Disease Free Interval; ECOG PS: Eastern Cooperative Oncology Group Performance Status; HR: Hazard Ratio. Table 6. Treatment-related adverse events in the CG and CP cohorts. Table 6. Treatment-related adverse events in the CG and CP cohorts. Tables Abbreviations: AE: Adverse Event; CG: Carboplatin plus Gemcitabine; CP: Carboplatin plus Paclitaxel. Tables Page 25/28 AEs Any Grade P value Grade ≥ 3 P value CP N= 56 CG N= 32 CP N= 56 CG N= 32 Anemia No Yes 13 (23.2) 43 (76.8) 4 (12.5) 28 (87.5) 0.345 51 (91.1) 5 (8.9) 30 (93.8) 2 (6.2) 0.970 Neutropenia No Yes 19 (33.9) 37 (66.1) 3 (9.4) 29 (90.6) 0.021 49 (87.5) 7 (12.5) 13 (40.6) 19 (59.4) <0.001 Thrombocytopenia No Yes 48 (85.7) 8 (14.3) 13 (40.6) 19 (59.4) <0.001 55 (98.2) 1 (1.8) 26 (81.3) 6 (18.7) 0.016 Peripheral neuropathy No Yes 41 (73.2) 15 (26.8) 31 (96.9) 1 (3.1) 0.013 - - - Diarrhea No Yes 51 (91.1) 5 (8.9) 30 (93.8) 2 (6.2) 0.970 - - - Constipation No Yes 51 (91.1) 5 (8.9) 30 (93.8) 2 (6.2) 0.970 - - - Nausea No Yes 41 (73.2) 15 (26.8) 19 (59.4) 13 (40.6) 0.270 - - - Vomiting No Yes 51 (91.1) 5 (8.9) 28 (87.5) 4 (12.5) 0.868 - - - Mucositis 0.617 Fatigue No Yes 38 (67.9) 18 (32.1) 13 (40.6) 19 (59.4) 0.024 55 (98.2) 1 (1.8) 31 (96.9) 1 (3.1) 1.000 AST increase No Yes 46 (82.1) 10 (17.9) 20 (62.5) 12 (37.5) 0.073 - - - ALT increase No Yes 45 (80.4) 11 (19.6) 14 (43.8) 18 (56.2) 0.001 56 (100) 0 (0) 30 (93.8) 2 (6.2) 0.251 Infusion-related reaction No Yes - - - 55 (98.2) 1 (1.8) 32 (100) 0 (0) 1.000 ata are presented as n (%) except where otherwise specified. The p value of the χ2 test assessing the ssociation between each AE and the type of treatment received is indicated in the right column of the able. The p value is indicated in bold numbers when statistically significant. Data are presented as n (%) except where otherwise specified. The p value of the χ2 test assessing the association between each AE and the type of treatment received is indicated in the right column of the table. The p value is indicated in bold numbers when statistically significant. Data are presented as n (%) except where otherwise specified. The p value of the χ2 test assessing the association between each AE and the type of treatment received is indicated in the right column of the table. The p value is indicated in bold numbers when statistically significant. Abbreviations: AE: Adverse Event; CG: Carboplatin plus Gemcitabine; CP: Carboplatin plus Paclitaxel. Figure 2 Progression free survival (PFS) and overall survival (OS) curves adjusted for multivariable Cox model, stratified on type of first line platinum-based chemotherapy (Carboplatin plus Paclitaxel (CP) vs. Carboplatin plus Gemcitabine (CG)) and previous taxane exposure (preTax vs. no preTax). Progression free survival (PFS) and overall survival (OS) curves adjusted for multivariable Cox model, stratified on type of first line platinum-based chemotherapy (Carboplatin plus Paclitaxel (CP) vs. Carboplatin plus Gemcitabine (CG)) and previous taxane exposure (preTax vs. no preTax). A: Adjusted PFS curves; B: Adjusted OS curves. Additionalfile1.docx Figures Page 27/28 Figure 1 Figure 1 Progression free survival (PFS) and overall survival (OS) curves according to first line chemotherapy (Carboplatin plus Paclitaxel vs. Carboplatin plus Gemcitabine) and adjusted for multivariable Cox model. A: Adjusted PFS curves; B: Adjusted OS curves. Figure 2 Progression free survival (PFS) and overall survival (OS) curves adjusted for multivariable Cox model, stratified on type of first line platinum-based chemotherapy (Carboplatin plus Paclitaxel (CP) vs. Carboplatin plus Gemcitabine (CG)) and previous taxane exposure (preTax vs. no preTax). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 28/28 Page 28/28
https://openalex.org/W4388042782	https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/654051f4c573f893f179c513/original/li-ion-batteries-an-overview-on-commercial-electrode-advances.pdf	English	null	Li-ion Batteries: An Overview on Commercial Electrode Advances	null	2,023	cc-by	11,832	Li-ion Batteries: An Overview on Commercial Electrode Advances Sarthak Patnaik Masters in Technology, Indian Institute of Technology Bombay, Mumbai ABSTRACT This review paper presents a comprehensive analysis of the electrode materials used for Li-ion batteries. We explore key areas of electrode materials for Li-ion batteries and discuss the advancements and challenges associated with them. Through an extensive literature review, we identify the current state of research, methodologies, and applications related to Li-ion battery electrodes. Our study covers a wide range of subtopics, including the theoretical aspects of the basic functioning of lithium-ion batteries and the crystal structures of different electrode materials. Additionally, we discuss emerging trends and future directions in the development of high- performance commercial battery electrodes, providing insights into promising avenues for further research. By synthesizing existing knowledge and analyzing the latest research, this review aims to provide a valuable resource for researchers, practitioners, and stakeholders interested in developing state-of-the-art high-performance Li-ion batteries. The findings and perspectives presented in this paper contribute to a deeper understanding of the electrode materials for Li-ion batteries and their advantages and disadvantages, ultimately fostering advancements and innovation in the commercial electrode technology. Keywords: Lithium-ion battery, electrode, cathode, anode, future trends 1. INTRODUCTION The primary issues confronting the globe in this millennium are that of pollution and sustainability. There is a global push towards the use of renewable energy to reduce the traditional dependence on fossil fuels this in turn helps in reducing emissions. This push has led to alternative green energy sources being explored to enable a green energy transition. This energy transition has in turn resulted in considerable interest in the development of portable energy storage devices. Lithium- ion batteries (LiBs) are a promising candidate for reducing our emissions[1] and fulfilling our energy storage needs. The lithium-ion batteries were first conceptualized by Prof. Wittingham 1970s with the discovery of Li-TiS2[2] but the very low potential generated resulted in limiting its energy density and subsequently its practical applications. Lithium-ion batteries were commercialized due to the discovery of cathode chemistries like Lithium cobalt oxide (LiCoO2)[3], Lithium manganese oxide https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 (LiMn2O4)[4] and Lithium iron phosphate (LiFePO4)[5] by Professor Goodenough in the 1980s and 1990s. Li-ion batteries also have certain fundamental advantages over traditional battery chemistries like Ni-Cd, Ni-Mh and Pb-acid. Lithium has the lowest reduction potential among all of the elements, this in turn results in Li-ion batteries having the highest cell potential. Lithium is the third lightest element and has a small ionic radius. This results in Li-ion batteries having high gravimetric and volumetric capacity and power density. Other advantages include a slow self-discharge rate, high columbic efficiency, fast charging and discharging, and a long-life cycle. Figure 1- Ragone plot for battery chemistries [6] Figure 1- Ragone plot for battery chemistries [6] Figure 1 shows that LIBs have the highest energy density (150-200Wh/kg) and high-power density (10-104 W/kg) among all the available battery chemistries. Today imagining a world without Li- ion batteries is almost impossible. Li-ion batteries are currently used in multiple domains including mobility applications such as electric vehicles and storage applications like electrical grids and energy harvesting from renewable sources like wind, solar and geothermal. They also find applications in niche domains such as portable electronics and wireless communications. Development in Li-ion battery technology will not only improve their performance and cost but will have a positive feedback effect on the development of new technologies which are dependent on energy storage. Li-ion battery research has significantly been focused on the research and development of high-performance electrode materials. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 1. INTRODUCTION Electrodes that have characteristics like high charge capacity, rate capability and high voltage (consideration for cathodes) can improve the power and energy densities of Li-ion batteries. The objective of this review is to provide a simple yet comprehensive understanding of LiBs and its electrodes. The scope of the following review is restricted to electrode materials that are either commercially available and have a significant market share in the global market or ones that are expected to be commercially available in the coming decades. Electrode chemistries that are purely in the nascent research and development stage and do not have a significant industrial and commercial presence have not been extensively covered. The comprehensive review begins by providing a theoretical understanding of the working of a Li-ion battery along with all the major LiB parts. The theoretical considerations involved during selection of electrode materials are then described with the help of the orbital picture of the battery electrodes. The next two sections comprehensively discuss the various electrode chemistries which are commonly used in Li-ion batteries commercially. Finally, the review concludes by discussing in detail the advances in commercial electrode technology. 2. PARTS OF A LI-ION BATTERY & THEORETICAL UNDERSTANDING OF ELECTRODES of A Li-ion Battery Figure 2- Schematic Illustration of a Li-ion Battery[7] 2.1 Parts of A Li-ion Battery Figure 2- Schematic Illustration of a Li-ion Battery[7] A standard Li-ion battery has a cathode (conventionally the positive electrode), anode (conventionally the negative electrode) and a separator dipped in an electrolyte. During the charging cycle Li ions deintercalate from the cathode and travel through the electrolyte to get intercalated in the anode. During the discharging cycle the opposite motion of Li-ions take place (i.e from anode to cathode) via the electrolyte. Electrons on the other hand travel via the external circuit and thus can be used for driving our desired devices as shown in Figure 2. The intercalation and deintercalation of the Li-ions happen due to the redox reactions happening at the electrode electrolyte interface. During the charging cycle the positive electrode undergoes oxidation and releases electrons to the external circuit while the negative electrode undergoes reduction by accepting the released electrons. During the discharging cycle the negative electrode undergoes oxidation releasing electrons while the positive electrode undergoes reduction by accepting the released electrons. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 2.1.1 CATHODE LiCoO2 is the Cathode active material, and it has alternating layers of Cobalt, Oxygen and Lithium ions. During the discharging process the Lithium ions deintercalated from the structure and release and electron. Thus, oxidizing Co3+ to Co4+ During the discharging cycle the Lithium ions shuttle back into the lattice and Co4+ is reduced to Co3+ by gaining one electron. Aluminium is used as the current collector for the cathode[9]. The major commercially available cathodes for LIB include Lithium Cobalt Oxide[10], Lithium Manganese Oxide[11], Lithium Nickel Manganese Cobalt Oxide[12], Lithium Nickel Cobalt Aluminium Oxide[13] and Lithium Iron Phosphate[14]. 1. INTRODUCTION Figure 3- Schematic Illustration of a LCO Battery[8] Figure 3- Schematic Illustration of a LCO Battery[8] Figure 3- Schematic Illustration of a LCO Battery[8] The half-reaction at the cathode in an LCO battery is as follows LiCoO2 ⇌ Li+ + CoO2 + e- (1) The half-reaction at the anode in an LCO battery is as follows C6 (graphite) + Li+ + e- ⇌ LiC6 (2) The half-reaction at the cathode in an LCO battery is as follows LiCoO2 ⇌ Li+ + CoO2 + e- (1) The half-reaction at the anode in an LCO battery is as follows C6 (graphite) + Li+ + e- ⇌ LiC6 (2) The half-reaction at the cathode in an LCO battery is as follows LiCoO2 ⇌ Li+ + CoO2 + e- (1) The half-reaction at the anode in an LCO battery is as follows C6 (graphite) + Li+ + e- ⇌ LiC6 (2) (1) (2) The net reaction is the sum of the above two half-reactions (right to left: discharging, left to right: charging) The net reaction is the sum of the above two half-reactions (right to left: discharging, left to right: charging) C6 (graphite) + LiCoO2 ⇌ LiC6 + CoO2 (3) (3) (3) https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 1. INTRODUCTION It can be thus noted that both the electrodes undergo oxidation and reduction depending upon the charging or the discharging cycle. The motion of Lithium ions inside an LIB can be described in the following four steps- (i) Lithium ions and the electron dissociate from the Cathode and move concurrently towards the electrolyte and the current collector respectively. This process is driven by solid state diffusion. (ii) The Lithium ions have to then pass through the electrode electrolyte interface and reach the electrolyte. Further the lithium ions move via the electrolyte to the anode and this process is driven by ionic diffusion in the liquid electrolyte. (ii) The Lithium ions have to then pass through the electrode electrolyte interface and reach the electrolyte. Further the lithium ions move via the electrolyte to the anode and this process is driven by ionic diffusion in the liquid electrolyte. (iii) The electrons are driven forward by the presence of potential difference between the positive and negative electrodes. They pass through the Cathode, conductive additions, and the Cathode current interface. Finally migrating in the external circuit. Thus, reaching the other electrode. (iv) In the meantime, the Lithium ions pass through the SEI layer at the A node and thus the electron and the Lithium ions enter the anode material simultaneously. This process is driven by solid state diffusion. The above description presents the picture of the charging cycle of Lithium-ion battery. It is to be noted that during the discharging cycle the reverse movement of ions and electrons take place. Let us take the example of Lithium Cobalt Oxide (LCO) battery to understand the various parts of LIB as shown in Figure 3. The charge and discharge cycles of a lithium-ion battery (LCO) are described below in Equations 1-3. 2.1.4 SEPARATOR The separator acts as a safety feature in LiBs. It acts as a physical barrier keeping the cathode and the anode apart. It prevents contact between the electrodes, which might lead to a short circuit. It only allows ions to pass freely through its internal microscopic holes which is essential for the functioning of the battery. Various separator materials used for liquid electrolyte-based Li-ion batteries have been reviewed in the following article[20]. Synthetic membranes like polyethene (PE) and polypropylene (PP) are generally used as separators for LiBs. 2.1.3 ELECTROLYTE The electrolyte acts as the medium that allows the movement of lithium ions between the cathode and anode. High ionic conductivity is to be taken into consideration as it would ensure easier movement of lithium ions back and forth during the charge and discharge cycle. The electrolyte is made up of two components the solvent and the Li+ conducting salt. The solvents used are aprotic and highly polar. Commonly used LIB electrolyte solvents include ethylene carbonate (EC) dimethyl carbonate (DMC), diethyl carbonate (DEC), and ethyl methyl carbonate (EMC). The conducting salts have high solubility in the solvent. It should also be compatible with battery components like the current collector and the separator. Lithium hexafluorophosphate (LiPF6), Lithium-bis(trifluoro methyl sulfonyl)imide (LiTFSI) and lithium-bis(fluoro sulfonyl) imide (LiFSI) are commonly used conducting salts in LiBs. The electrolyte plays a major role in determining the relative ease with which lithium ions can move through it. Various electrolytes used in LiBs have been extensively reviewed[19]. 2.1.2 ANODE The traditional anode in a LIB is Graphite. During the charging process Lithium ions get intercalated into the interlayers of Graphite. An electron is sourced from the external circuit to convert the Li ions into elemental Lithium. During the discharging process the Lithium in the atomic state loses an electron to get converted to Li ions. Thus, getting deintercalated from the Graphitic layers. This motion of Li ions between the cathode and anode leads to LIB being called a rocking chair battery or shuttle cock battery. Copper is used as the current collector for the anode[15]. Graphite[16], Lithium-Titanium-Oxide (LTO)[17] and Silicon[18] are the major commercial anode materials for Li-ion batteries. 2.2 THEORETICAL UNDERSTANDING OF ELECTRODES As previously discussed, the negative electrode of the battery, which is also known as the anode, is made up of carbon graphite, while the positive electrode or the cathode is made up of layered LMO (Lithium metal oxide) where M signifies a transition metal. The definition of the anode and cathode depends on whether the battery is charging or discharging. In the most generic sense, the electrode which undergoes oxidation is termed as the anode and the one which undergoes reduction is termed as the cathode. The properties of the cathode and the anode material (active material A.M) to be selected for Li- ion batteries have been tabulated in Table-1. The properties of the cathode and the anode material (active material A.M) to be selected for Li- ion batteries have been tabulated in Table-1. Negative Electrode AM Positive electrode AM AM should have a reversible reaction with Li, with the minimum possible structural changes on insertion/extraction The insertion/extraction of Li should be carried out as rapidly as possible to achieve high power densities AM should insert/react with a maximum of Li to reach a high capacity Weak potential close to that of Li+ /Li to obtain maximum tension in the Li-ion battery High potential compared to that of Li+ /Li to obtain maximum tension in the Li-ion battery AM should have good compatibility with the electrolyte AM should have good electronic and ionic conductivity AM should be abundant; synthesis should be simple and not expensive AM should be thermally and chemically stable and non-toxic Negative Electrode AM Positive electrode AM AM should have a reversible reaction with Li, with the minimum possible structural changes on insertion/extraction The insertion/extraction of Li should be carried out as rapidly as possible to achieve high power densities AM should insert/react with a maximum of Li to reach a high capacity Weak potential close to that of Li+ /Li to obtain maximum tension in the Li-ion battery High potential compared to that of Li+ /Li to obtain maximum tension in the Li-ion battery AM should have good compatibility with the electrolyte AM should have good electronic and ionic conductivity AM should be abundant; synthesis should be simple and not expensive AM should be thermally and chemically stable and non-toxic Table 1- Active Material Properties for Electrodes [21] The richness of a material to be suitable for battery application is evaluated by two major theoretical parameters. 1. Volumetric energy density (Wh/L) 1. Volumetric energy density (Wh/L) 2. Gravimetric energy density (Wh/kg) 2. Gravimetric energy density (Wh/kg) The richness of a pair of materials is evaluated by the product of the capacity and the potential difference between the pair of electrodes. Theoretically, one may think that the selection of a given pair of materials with the highest gravimetric or volumetric capacities with the highest possible potential in the cathode and lowest possible potential in the anode must be done to get the best pair of materials for cathode applications. However, in real life, this is not the case because other complexities are involved such as the negative electrode should have a potential higher than that of graphite to facilitate increasing safety. The power density, which is defined as W/kg or W/L, is also important as the battery will undergo peaks in current during the charging and discharging cycles. Another important factor that needs to be taken into account is the role of chemical kinetics in the functioning of the battery. The insertion and extraction of Lithium-ions shuttling inside the cell should be as rapid as possible. This is linked directly to a given material's electronic and ionic conductivity values. One must also consider the compatibility of the electrode and the electrolyte as there is an interface formed between them; interface dynamics play an important role in cell reactions. It is generally preferred that the formation of the electrode-electrolyte interface should be stable over time. This is particularly important in the case of the negative electrode. Figure 4 – Orbital Picture for Oxidation and Reduction[22] Figure 4 – Orbital Picture for Oxidation and Reduction[22] Let EF+ and EF- be the fermi levels of the positive and negative electrodes as shown in Figure 4. The positive electrode which has the higher potential has the lowest fermi-level energy. As its job is to accept electrons from the negative electrodes during the discharge cycle. A similar assumption can be made for the negative electrode, which would imply that the fermi level energy is the highest for it. Note that in the plot, we have inverted the energy and the potential scales. Here, HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) are marked. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 Table 1- Active Material Properties for Electrodes [21] During the reduction, an electron is added to the electrolyte's LUMO, while removing an electron from its HOMO results in oxidation. It is beneficial for the Fermi level of the positive electrode to be higher than the electrolyte's HOMO. This would imply that there will be no electron transfer between the electrolyte and positive electrode and that it will not oxidize. The electrolyte hence will remain stable in contact with the electrode. This remains true theoretically for almost all cases as the potential of the positive electrode does not go beyond 4.5V vs Li+ /Li. But for the negative electrode, as long as the Fermi level is below the LUMO of the electrolyte, electron transfer is prevented, and the electrolyte remains stable. Unfortunately, it has been seen that once the negative electrode has been lithiated (battery charged), a majority of them have shown a Fermi level that is above the LUMO of the electrolyte. At this point, one would think that this would lead to the reduction of the electrolyte. However, it has been observed that on the surface of the negative electrode, a passivation layer gets formed, which provides protection by slowing down the kinetics of the reduction. This layer allows Li+ ions to diffuse but isolates the electrode's material contact with the electrolyte. Hence, we can say that the layer is electronically insulating while ionically conducting. This passivation layer enables the functioning of a Li-ion battery, and the instability of which is also a primary reason for the major reasons for loss of performance and the quick aging of batteries. The stability and ease of formation of the SEI layer (Solid Electrolyte Interphase) are major considerations for the selection of anode materials. 3. COMMERCIAL LiB ELECTRODES Figure 5 – Market Shares of Different types of LiB Cathodes and Anodes[23] Figure 5 – Market Shares of Different types of LiB Cathodes and Anodes[23] From the Figure 5 it can be seen that although the market share is quite diverse for the cathode chemistries with each cathode type occupying 5-35%, the anode market share is dominated by Graphite (98%)[23]. 3.1 COMMERCIAL CATHODES In the following section commercial LiB cathodes observed from Figure 6 have been extensively discussed. 3.1.1 Lithium Cobalt Oxide (LCO) 3.1.1 Lithium Cobalt Oxide (LCO) https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 Figure 6- Lithium Cobalt Oxide (LCO) Unit Cell[24] Figure 6- Lithium Cobalt Oxide (LCO) Unit Cell[24] The structure of Lithium Cobalt Oxide (LCO) is a form of layered transition metal oxide compound as shown in Figure 6. It has a rock salt type structure. The unit cell consists of cubic close packing of oxygen with the presence of three CoO2 sheets per unit cell. The lithium ions occupy the octahedral interstitial sites. The LCO battery chemistry has been restricted to use in small scale applications like portable electronics and is not suitable for electric vehicle and hybrid electric vehicle application [25]. Primarily because of its high cobalt content which makes it costly. It has poor chemical stability after extraction of 50% of all the lithium (Li0.5CoO2). Hence, the practical reversible capacity is only around 130 mAh/g [26]though the theoretical capacity is about 274 mAh/g. Extraction of lithium greater than 50% leads to oxidation of O2- leading to the evolution of oxygen gas and breakdown of the structure[27]. This happens due to the overlapping of the t 2g band of the cobalt ions with the 2p band of oxygen ions [28]. Another problem associated with the LCO chemistry is that when the cut-off voltage is increased beyond 4.2V, cobalt undergoes dissolution into the electrolyte due to phase transformation effects. This leads to strains and development of micro cracks increasing inter-facial resistance and leading to chemical degradation of the battery electrode. Though LCO gives us a high discharge voltage but it suffers from disadvantages like high cost, poor rate capability and poor thermal stability which restricts its use in high-load applications. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 Figure 7- Spinel Structure of LiMn2O4[29] Figure 7- Spinel Structure of LiMn2O4[29] LMO is characterized by a spinel type structure. The lithium ions occupy the tetrahedral sites while the Mn3+/4+ ions occupy the octahedral sites in the close-packed oxygen structure as shown in Figure 7. It is characterized by alternative layers of cubic close packed oxygen spinel ([Mn2]O4) framework. The Mn3+ containing layer is present in a 3:1 ratio. The cubic close packed oxygen array gets stabilized leading to reversible movement of lithium ions. The charging cycle leads to the oxidation manganese ions from Mn3+ to Mn4+. 3.1.1 Lithium Cobalt Oxide (LCO) The major issues encountered by the LMO cathode are the dissolution of manganese into the electrolyte and irreversible phase transitions due to the John Teller distortion of Mn3+[30]. This leads to poor cyclic stability [31]. The dissolution of manganese into the electrolyte is detrimental to the SEI layer of the anode and further reduces stability of the cell. Another problem with the LMO cathode is that the Mn4+ ion has high oxidation ability and it leads to the decomposition of the solvent in the electrolyte. 3.1.3 Lithium Nickel Manganese Cobalt Oxide (LiNiMnCoO2) (NMC) https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 Figure-8 Layered Structure of NMC Cathode[32] Figure-8 Layered Structure of NMC Cathode[32] The LiMnO2 electrode has various challenges one of which is that it is not defect-free and in turn the spinel structure lose capacity irreversibly upon cycling. Another issue is that, in the LiNiO2 cathode material, Ni2+ ions tend to occupy the Li+ sites. This ion mixing is detrimental as it decreases Li+ ion concentration. The reason for this mixing is due to the similarity of sizes of Li+ and Ni2+ radii [33]. Both the LiNiO2 and LiMnO2 electrode chemistries are prone to John Teller distortion. This can be overcome by using a mixed a transition metal layered oxide. NMC or nickel manganese cobalt oxide was thus conceptualised[34]. It is similar to the other layered oxide structures as shown in Figure-8. In the lithiated state, Ni2+, Mn4+ and Co3+ are present. NMC is generally named in regards to the nickel, manganese and cobalt ratios, for instance NMC 1:1:1 corresponds to Li1–x (Ni0.33 Mn0.33 Co0.33) O2. In NMC-111 to maintain structural integrity only 66% of the lithium in the structure can be used, thus NMC gives us a gravimetric capacity of 160 mAh/g compared to the theoretical capacity of 274 mAh/g[35]. The cobalt in the NMC structure contributes to good electrical conductivity and electrochemical performance. It further prevents nickel lithium site mixing and its associated structural distortion. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 Manganese, which has an oxidation state of +4 is detrimental towards ionic conductivity as it interferes with the charge carriers. However, it stabilizes the structure as a whole and improves cyclic stability. The NMC chemistry can be considered as a balancing act between the advantages and disadvantages of lithium cobalt oxide, lithium manganese oxide and nickel oxide thereby leading to higher capacity and better cyclic stability. The major problems associated with NMC is that there are phase transformations occurring in the material, effect of the mixed population (Ni, Co, Mn) on the lithium positions leading to a high irreversible capacity, possibility of oxygen evolution during charging. Another problem is that it has a lower discharge voltage (3.7V) vs Li/Li+ as compared to 3.9V of LCO. 3.1.4 Lithium Nickel Cobalt Aluminum Oxide (LiNiCoAlO2) (NCA) The difference between the NCA cathode which and nickel rich NMC is that the chemically inactive element Mn is replaced by Al. The presence of Al in the structure adds stability, however, large concentrations of Al3+ results in capacity decrease due to inactiveness of aluminium. One of the major issues of NCA cathode is that it is not stable when operated at high current, cut-off charging voltage and high charging temperature [38]. NCA cathodes have been observed to undergo secondary phase change reactions which affects their performance detrimentally. NCA gets converted to nickel oxide at the surface where a cationic defect is present. The NiO phase increases resistance during cycling and is detrimental to the battery performance. Secondary phases of Li2CO3 and LiOH are sometimes formed at the electrode-electrolyte interface leading to reduced capacity[39]. This is detrimental to cyclability and rate capability. 3.1.3 Lithium Nickel Manganese Cobalt Oxide (LiNiMnCoO2) (NMC) Increasing the nickel content has been studied to improve the usable capacity to 190 mAh/g [36]. NMC 442 and NMC 922 have shown that with slight increase of lithium and manganese concentration, the discharge capacity can be improved marginally while retaining good rate capability. This has led to an effective decrease in cobalt concentration [37]. Batteries with NMC cathode with Si anode have been predicted to be a future gamechanger in the domain of electric mobility[38]. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 3.2 COMMERCIAL ANODES In the following section commercial LiB anodes observed from Figure 6 have been extensively discussed. 3.1.5 Lithium Iron Phosphate (LiFePO4) ( LFP) In the layered lithium oxide spinel structures, a major problem that is encountered is that of oxygen release due to overlapping of 3d band of the transition metal and 2p band of O2-. To overcome this problem, Fe2+/ Fe3+ as a transition metal has been used as its 3d bands are far above the 2p bands of oxygen. This leads to structural stability and has resulted in the birth of phospho-olivine cathode chemistries with LiFePO4 being the most prominent. In the LFP crystal structure, oxygen atoms form hexagonal close packing with lithium atoms and iron atoms being located at octahedral voids. The tetrahedral voids are occupied by phosphorus atoms. The phosphorus atoms form covalent bonds with four neighbouring oxygen atoms to distort the structure as shown in Figure 9. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 Figure-9 LFP Crystal Structure[33] Figure-9 LFP Crystal Structure[33] Figure-9 LFP Crystal Structure[33] In this distorted octahedral structure, there is a presence of a one-dimensional diffusion path (parallel to b axis) for the lithium-ion movement [40]. The P-O covalent bond leads to high polarisation of the oxygen atoms due to inductive effect of phosphorous. This is turn weakens the covalence of Fe-O bond leading to a higher circuit voltage (VOC) [41]. The LFP is characterised by a flat charging and discharging curve due to structural similarity of the two boundary phases. This leads to high cycling stability. The major problems associated with LFP chemistry is that it has poor electrical and lithium-ion conductivity. The problem of oxygen evolution is solved in LFP due to strong covalent bonds with the phosphate molecule [42]. LFP also shows excellent thermal capacity [43]. It is characterized by a stable structure with it being inexpensive and ecologically acceptable, even though it has the lowest electronic and ionic conductivity of any commercial LiB cathode material. 3.2.1 Graphite It consists of a planar 2D structure of hexagonal graphene sheets composed of sp2 hybridized carbon. They are stacked together along the c axis due to week van der Waals forces. Lithium occupies every third layer of the structure as shown in Figure-10. The C-C bonds in between the planes are covalent and it leads to good intercalation for Lithium. The theoretical capacity of graphite anode in its final stage LiC6 is 372 mAh/g. The major advantages of graphite include its low cost due to abundance, non-toxicity and structural stability [45]–[48]. This low theoretical https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 capacity limits its usage to low power applications[49]–[51]. The delithiation and lithiation results in volume changes in graphite which in turn produces stress on the electrodes. This detrimentally affects cycling stability [52]–[54]. Lithium deposition on the graphite electrode is also a problem that is observed and it poses a serious safety hazard[55], [56]. Figure-10 Graphite Crystal Structure[44] Figure-10 Graphite Crystal Structure[44] 3.2.2 Lithium Titanium Oxide (LTO: Li4Ti5O12) 3.2.2 Lithium Titanium Oxide (LTO: Li4Ti5O12) 3.2.3 Silicon Silicon is a prominent anode material for lithium-ion batteries. It gives the highest theoretical capacity 4200 mAh/g among all anode materials [62]. However, the major problem that is associated with the silicon anode is that it undergoes massive volume change during the lithiation process resulting in stresses being developed in the material. This leads to de-lamination [63]. Due to the volume expansion of silicon, the SEI layer also ruptures and further lithium is required to form new SEI layer leading to consumption of lithium to re-form the layer. This leads to low cycling performance [64], [65]. 3.2.2 Lithium Titanium Oxide (LTO: Li4Ti5O12) 3.2.2 Lithium Titanium Oxide (LTO: Li4Ti5O12) Figure-11 LTO Crystal Structure[57] Figure-11 LTO Crystal Structure[57] https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 Lithium titanate (LTO) Li4Ti5O12 is another promising anode material for lithium-ion batteries. It crystallizes as a spinel-structure and reversibly intercalates lithium at 1.55V. In the spinel structure of LTO, lithium occupies the tetrahedral sites while the octahedral voids are filled with titanium as shown in Figure-11. It shows a reversible capacity of around 160 mAh/g. The intercalation and de-intercalation process does not cause any major structural volume change. LTO cathode has the unique property of no SEI formation leading to low cell impedance. The major advantages of this include high safety and cycling stability. It can be summarized that the major advantages of LTO include its high cycle life, thermal stability and capacity. That is why even though it has reduced cell voltage and high cost due to the presence of titanium, it has still been commercialised[58]– [60]. The major problem associated with LTO is that it has low conductivity, which hinders electron transport and high polarisation, especially at a high current density. Another issue with LTO is that it undergoes gassing during cycling[61] . https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 4.2 ADVANCES IN LMO CATHODE Various modification techniques have been studied for increasing the electrochemical performance of the LMO cathode. Doping of various elements like Al, Cr, Fe and Co in LMO with the objective of improving the electrode performance have been studied[71], [72]. These elements have an oxidation state that is less than the average oxidation state of manganese (+3.5). It suppresses John Teller distortion, phase transformation and Mn dissolution increasing cyclic stability. However, it is detrimental to the capacity. Nickel doping has also been considered for increasing rate capability by inducing a disordered spinel structure [73]. Organic coatings like polymers and acetyl acetone in LMO have been considered as a surface engineering mechanism to prevent decomposition of electrolytes. The coatings neutralise the surface-active sites in Mn and preventing dissolution of manganese in the electrolyte. This results in increased electrochemical performance at high temperature[74]. Other surface modification improvements of LMO have been considered as well[75]. 4.1 ADVANCES IN LCO CATHODE For LCO electrode chemistry, doping with elements like lanthanum and aluminium has been observed to increase the capacity to almost the capacity limit[66]. Other elements like Mg and Mn when doped into LCO have been observed to improve the cycling performance[67]. Magnesium diffuses into the LCO crystal structure and stabilises the lattice thus preventing undesired phase transformations at high potentials. Surface coatings such as ZrO2 have been explored as they improve the cycle life and rate capability at high voltages[68]. The surface coating acts as protective layer and prevents LCO to come in direct contact with the electrolyte thus preventing its oxidation and subsequent cobalt ion dissolution. The coatings also suppress resistance caused due to the intercalation and de-intercalation of lithium ions. Coatings have also been studied to decrease the activation energy at the electrode - electrolyte interface, thereby positively affecting the kinetics of lithium-ion transfer. Surface engineering techniques[69] and growth of Al2O3 and Al-W-Fluoride on LCO have been studied to increase cyclic stability[70]. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 5.1 ADVANCES IN GRAPHITE ANODE Two major approaches are employed to improve the electrochemical performance of graphite anodes and they include accelerating lithium-ion diffusion and enhancing interfacial reaction between graphite and the liquid electrolyte to form a thin yet stable SEI layer. Multi-channel graphite [101] has been seen to improve the entry of lithium ions into the graphite structure. This is particularly useful for enabling fast charging of LiBs. They also show enhanced discharge rate capability. Expandable graphite[102] has been developed with the objective of increasing inter- layer spacing and they show very high reversible capacity and rate capability. Surface modifications of the graphite anode by modifying Al2O3 on the surface has been seen to improve fast charging ability[103]. Non-graphitised carbon is another intercalation material that has been studied due to its higher layer spacing [104]. But it suffers from issues like voltage hysteresis and high initial irreversible capacity [105], [106]. Other carbon-based materials like graphene [107], [108], nanofibers [109]–[111] and nanotubes [112]–[114] are also being explored as potential materials for LIB anodes. 4.3 ADVANCES IN NCA & NMC CATHODE Surface modification studies have been carried out to suppress side reactions for both NCA and NMC cathodes [76]. SiO2, TiO2 and Li2ZrO3 have been investigated as coating materials to improve the thermal stability of NMC. Fluorine has been studied as one of the coating materials for NCA. Fluorine coating substitutes oxygen and enhances the stability of the lattice due to its higher electronegativity and strong metal-fluoride bonds [77]. A combination of fluorine and zirconium coating [78] and FeF3 coatings have also been proposed [79]. These coatings improve capacity retention even at high cycle life. MgF2 coated NCA gives good rate capability[80]. Other coatings like SiO2 and TiO2 nanoparticles have also been explored. For NCA, potassium doping has been seen to protect the structure from side reactions with the electrolyte [81]. Dopants like copper and iron decrease the capacity but improve cyclability and rate capability [82]. Chromium doping has been seen to enhance NCA capacity. Zr-doped NCA has been seen to improve capacity retention [83]. For NMC cathodes, performance improvement has been seen by coating materials like SiO2 (ref 167), MoO3 (ref 168), Al2O3 [84]–[86]. Other coatings like Li3PO4 [87], PPy and Li3PO4[88], FePO4 [89], LiAlF4 [90] have been studied. Core shell structures consisting of a nickel enriched core (to maximise capacity) and a nickel deficient surface layer (to address the problems of Ni at the surface) have been used to improve the performance of NMC. This has been used extensively in the case of NMC 811. Doping of NMC has been extensively studied with elements such as magnesium, aluminium, titanium and zirconium. However, it is to be noted that all dopants come with their advantages & drawbacks[91] . 4.4 ADVANCES IN LFP CATHODE The most common ways of improving Li+ and electron conductivity in LFP is to reduce particle size [92] or to employ conductive material coating [93]. Carbon based coatings (ref 42) are majorly used as they increase the electrical conductivity to a large extent. However, if the coating becomes too thick, it leads to reduction in gravimetric capacity. Another approach is to synthesize LFP particles in sub-micron or nanometre range and then using it to prepare composites[94]. LFP core shell composites have also been explored[95], [96]. This leads to improvement in lithium-ion conductivity but does not lead to improvement in electrical conductivity. Various doping elements have been studied for increasing the electrical and ionic conductivity of LFP [93]. LFP/C composites with a core shell structure have been observed to improve electrode performance[97], [98]. Carbon-free LFP materials with narrow size distribution are also being researched as it shortens diffusion lengths. Doping of LFP with C and Zn4+ shows increased electron transport[99], [100]. 5.2 ADVANCES IN LTO ANODE To improve the electrical conductivity of LTO electrode, one approach is to synthesize nano-sized LTO particles and coat them with conductive carbon.[115] .The problem of gassing has been addressed by the application of inorganic coatings like AlF3 that improves cyclic performance https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 [116]. Li5Cr7Ti6O25 is another promising material that is being considered for improved performance [117]. CeO2 coating has been seen to improve the performance of Li5Cr7Ti6O25[118]. Nanostructured LTO electrodes with high power density have been shown to improve performance by decreasing electrode polarisation. Highly porous LTO electrodes are also being considered as they increase the number of sites for lithium-ion insertion, thus reducing polarisation[119].Various modification techniques like morphology control, size control, heteroatom doping, surface modifications have been explored for improving the performance of LTO[120] . 5.3 ADVANCES IN SILICON ANODE Silicon anodes are also being constantly improved. Nano particles of silicon have shown to be ideal materials for anode application as they do not rupture below a size of 150 nm [121]. Nano particles help in reduction of stress within the material thereby preventing enough issues. Porous silicon has also been seen as a promising material for anode application. The pores accommodate the silicon volume expansion. Silicon carbon nanocomposites have been observed to reduce capacity fading caused by volume changes of silicon anode [122]. Other nanostructures of silicon such as 0D [123], 1D [124]–[126], 2D thin films [127] and 3D porous structures [128] have been seen to increase the cycling stability. Si/C composite materials have also shown promise as a potential commercial anode material for LiBs[129]. CONCLUSIONS The conclusion of this comprehensive review paper summarizes the working of a Li-ion battery, the analyses various commercial grade Li-ion battery electrodes. By examining various aspects including the theoretical considerations for electrode design and various commercial electrodes we have gained valuable insights into their current technological development states. Firstly, our review highlights the working of a LiB by giving a general overview of various parts involved in the construction of a LiB. The theoretical considerations for cathode, anode and electrolyte combination were discussed with the help of the orbital picture. Commercial grade Li- ion battery electrodes and the challenges involved with each of them were shed light upon. It was noted that the advantages and disadvantages of electrode materials are often trade-offs. Addressing these tradeoffs will be crucial for further advancements and wider implementation of the commercial LiB electrodes. Furthermore, our analysis has unveiled emerging trends and promising directions for future research in improving the performance of commercial LiB electrodes. Areas such as novel coatings, surface modifications, doping, morphology control along with the designing of porosity enhanced and core shell structuring hold great potential for expanding the knowledge and impact of designing better LiB electrodes. Additionally, exploring interdisciplinary collaborations with https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 nanomaterials could lead to innovative approaches and applications for the development of commercial grade electrodes with improved electrochemical performance. nanomaterials could lead to innovative approaches and applications for the development of commercial grade electrodes with improved electrochemical performance. In conclusion, this comprehensive review underscores the significance of improving commercial grade LiB electrodes. While progress has been made, further research and development are necessary to overcome challenges and fully unlock the potential of designing novel commercial LiBs. Researchers, practitioners, and stakeholders in the domain of improving the preexisting commercial LiBs should prioritize addressing the identified limitations and actively explore emerging trends and collaborations to advance the field. By synthesizing the findings of this review, we hope to inspire and guide future research endeavors, ultimately contributing to the improvement of the preexisting commercial LiB electrodes. https://doi.org/10.26434/chemrxiv-2023-8v37k ORCID: https://orcid.org/0009-0000-1758-6835 Content not peer-reviewed by ChemRxiv. License: CC BY 4.0 REFERENCES [1] S. Pacala and R. Socolow, “Stabilization Wedges: Solving the Climate Problem for the Next 50 Years with Current Technologies,” Science (1979), vol. 305, no. 5686, pp. 968– 972, Aug. 2004, doi: 10.1126/science.1100103. [2] M. S. Whittingham, “Electrical Energy Storage and Intercalation Chemistry,” Science (1979), vol. 192, no. 4244, pp. 1126–1127, Jun. 1976, doi: 10.1126/science.192.4244.1126. [2] M. S. 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https://openalex.org/W4280624499	https://www.frontiersin.org/articles/10.3389/fphar.2022.856747/pdf	English	null	Adenosine in Acute Myocardial Infarction-Associated Reperfusion Injury: Does it Still Have a Role?	Frontiers in pharmacology	2,022	cc-by	5,714	MINI REVIEW MINI REVIEW published: 13 May 2022 doi: 10.3389/fphar.2022.856747 Adenosine in Acute Myocardial Infarction-Associated Reperfusion Injury: Does it Still Have a Role? Corrado De Marco 1,2, Thierry Charron 1,2 and Guy Rousseau 1,3* 1CIUSSS du Nord-de-l’Île-de-Montréal, Hôpital du Sacré-Coeur, Department of Medicine, QC, Montréal, Canada, 2Department of Medicine, Université de Montréal, Montréal, QC, Canada, 3Department of Pharmacology and Physiology, Université de Montréal, Montréal, QC, Canada The mainstay of acute myocardial infarction has long been timely reperfusion of the culprit obstruction. Reperfusion injury resulting from a multitude of pathophysiological processes has been demonstrated to negatively affect myocardial recovery and function post- infarction. Adenosine interacts directly with the sequential pathophysiological processes culminating in reperfusion injury by inhibiting them upstream. The evidence for adenosine’s beneﬁt in acute myocardial infarction has produced mixed results with regards to myocardial salvage and long-term mortality. The heterogenous evidence with regards to beneﬁts on clinical outcomes has resulted in modest uptake of adenosine in the clinical setting. However, it is critical to analyze the variability in study methodologies. The goal of this review is to evaluate how adenosine dose, route of administration, timing of administration, and site of administration play essential roles in the molecule’s efﬁcacy. The beneﬁts of adenosine, as highlighted in the following review, are clear and its role in the treatment of acute myocardial infarction should not be discounted Edited by: Xianwei Wang, Xinxiang Medical University, China Reviewed by: Gerd Heusch, University of Duisburg-Essen, Germany *Correspondence: Guy Rousseau guy.rousseau@umontreal.ca Keywords: myocardial infarction, no-reﬂow, reperfusion injury, adenosine, reperfusion, myocardial ischemia INTRODUCTION Adenosine has been shown to act on all mechanisms of reperfusion injury, distal embolization excluded, by promoting Received: 17 January 2022 Accepted: 26 April 2022 Published: 13 May 2022 INTRODUCTION Reperfusion therapy for acute myocardial infarction (AMI) has been established as the mainstay of therapy for reduction of the extent of myocardial damage and, ultimately, of mortality (Fibrinolytic Therapy Trialists’ (FTT) Collaborative Group, 1994). Moreover, earlier reperfusion is related to improved survival (GUSTO Angiographic Investigators, 1993). That said, reperfusion itself has been associated with certain deleterious effects, including myocyte death, microvascular injury, myocardial stunning, no-reﬂow, and reperfusion arrhyhtmias (Kloner, 1993). Specialty section: This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology Received: 17 January 2022 Accepted: 26 April 2022 Published: 13 May 2022 Citation: De Marco C, Charron T and Rousseau G (2022) Adenosine in Acute Myocardial Infarction- Associated Reperfusion Injury: Does it Still Have a Role? Front. Pharmacol. 13:856747. doi: 10.3389/fphar.2022.856747 Specialty section: This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology Received: 17 January 2022 Accepted: 26 April 2022 Published: 13 May 2022 Nowadays, the ﬁrst-choice therapy for ST-elevation myocardial infarction (STEMI) is percutaneous coronary intervention (PCI) (Keeley et al., 2003). Although PCI is successful in most cases, up to 40% of patients do not achieve complete myocardial reperfusion despite successful angioplasty of the culprit lesion (Gao et al., 2015). Critically, the myocardial outcome of the “ischemia-reperfusion” sequence exhibited in AMI depends not only on the time delay between ischemia and reperfusion, but so too on whether lethal reperfusion injury occurs (Monassier, 2008a). Nowadays, the ﬁrst-choice therapy for ST-elevation myocardial infarction (STEMI) is percutaneous coronary intervention (PCI) (Keeley et al., 2003). Although PCI is successful in most cases, up to 40% of patients do not achieve complete myocardial reperfusion despite successful angioplasty of the culprit lesion (Gao et al., 2015). Critically, the myocardial outcome of the “ischemia-reperfusion” sequence exhibited in AMI depends not only on the time delay between ischemia and reperfusion, but so too on whether lethal reperfusion injury occurs (Monassier, 2008a). A multitude of therapeutic options aimed at minimizing reperfusion injury, and thereby optimising results and outcomes of coronary artery revascularization following AMI, have historically been proposed. Adenosine, an endogenous nucleoside produced by the degradation of adenosine triphosphate (ATP) (Forman et al., 2006), ﬁgures among them. Citation: De Marco C, Charron T and Rousseau G (2022) Adenosine in Acute Myocardial Infarction- Associated Reperfusion Injury: Does it Still Have a Role? Front. Pharmacol. 13:856747. doi: 10.3389/fphar.2022.856747 A multitude of therapeutic options aimed at minimizing reperfusion injury, and thereby optimising results and outcomes of coronary artery revascularization following AMI, have historically been proposed. Adenosine, an endogenous nucleoside produced by the degradation of adenosine triphosphate (ATP) (Forman et al., 2006), ﬁgures among them. Adenosine has been shown to act on all mechanisms of reperfusion injury, distal embolization excluded, by promoting May 2022 \| Volume 13 \| Article 856747 1 Frontiers in Pharmacology \| www.frontiersin.org De Marco et al. Adenosine in Acute Myocardial Infarction FIGURE 1 \| A schematic representation of adenosine’s protective role in myocardial ischemia. FIGURE 1 \| A schematic representation of adenosine’s protective role in myocardial ischemia. preservation of microvascular ﬂow, inhibiting neutrophils and the resultant inﬂammatory cascade, stabilizing cellular membranes and reducing synthesis of radical oxygen species, restoring calcium homeostasis, and mediating post-conditioning (Figure 1) (Forman et al., 2006; Monassier, 2008b). caused by increased capillary permeability and consequent edema in the infarcted/reperfused area, endothelial and vascular smooth muscle damage as well as release of vasoconstrictive substances from atherosclerotic lesions with resulted impaired vasomotion, and neutrophil inﬁltration and aggregation, all of which culminate in capillary destruction and hemorrhage (Heusch and Gersh, 2017; Heusch, 2019). Three large, randomised trials of adenosine completed in the late 1990s and early 2000s showed variable results. While adenosine consistently showed beneﬁt in terms of minimizing infarct size, none of the studies were able to demonstrate a statistically signiﬁcant mortality beneﬁt, be it all-cause or cardiovascular mortality (Mahaffey et al., 1999; Quintana et al., 2003; Ross et al., 2005). Consensus states that a large part of cell death resulting from reperfusion injury occurs within minutes of reperfusion. In addition to the mechanisms of microvascular dysfunction and vasoconstriction contributing to reperfusion injury, there seems to be an as-of-yet obscure but equally critical role mediated by the mitochondrial permeability transition pore (MPTP). Free radical oxygen species generation, which occurs during the ischemic event, and delivery to the infarcted area, which occurs following reperfusion, may contribute to MPTP opening. Citation: Opening of the MPTP appears to induce sarcolemmal rupture within the ﬁrst minutes of reperfusion, potentially through the hypercontracture of neighbouring myocytes induced by high calcium levels in the presence of ATP (Komamura et al., 1994; Hausenloy et al., 2017; Heusch, 2020). A systematic review published in 2015 (Gao et al., 2015) conﬁrmed the same ﬁndings demonstrated in the three randomised control trials discussed above. Adenosine treatment was not found to have any effect on mortality or re- infarction, though did appear to offer beneﬁcial effects on myocardial reperfusion, no reﬂow, and post-infarction left ventricular ejection fraction (Gao et al., 2015). Multiple hypotheses for the lack of consistent beneﬁcial effects of adenosine have been emitted, including the inconsistent route of adenosine administration and the timing of infusion (Gao et al., 2015). Reperfusion of the culprit lesion in AMI equally results in embolization of microthrombi and particles of plaque material downstream, thereby plugging small arteries and arterioles (Heusch and Gersh, 2017; Heusch, 2020; Kleinbongard and Heusch, 2022). These distal microinfarcts result in an inﬂammatory response and, moreover, reduce contractile function in the surviving myocardium around the distal microinfarcts via a mechanism of inﬂammatory signal-mediated myoﬁbrillar oxidation (Kleinbongard and Heusch, 2022). Microvascular dysfunction in both the culprit artery, but so too in adjacent arteries via collateral vessels, may subsequently be induced by such embolic events (Heusch and Gersh, 2017; Heusch, 2020). The conﬂicting ﬁndings in the literature, as well as the lack of statistically signiﬁcant beneﬁts in terms of endpoints pertaining to mortality and re-infarction, have resulted in sparse uptake in use of adenosine in the treatment of acute myocardial infarction. UNDERSTANDING ADENOSINE AND THE RATIONALE FOR ITS USE AS A CARDIOPROTECTIVE AGENT Adenosine is a naturally occurring purine nucleoside comprised of an adenine molecule linked to a ribose sugar moiety via a beta- N9-glycosidic bond (Layland et al., 2014). It is synthesized in the intracellular space by purine synthesis or it may accumulate as a result of the breakdown of adenosine triphosphate (ATP) (Layland et al., 2014). Concentrations of intracellular adenosine have been shown to increase in situations in which there is a mismatch between ATP synthesis and use (e.g., ischemia) (Saito et al., 1999). Ischemic pre-conditioning is a phenomenon wherein a brief period of ischemia renders the myocardium more resistant to injury following a subsequent episode of ischemia (Vinten-Johansen et al., 1999). Seminal work done by Schulz et al (Schulz et al., 1995) has highlighted the integral role that endogenous release of adenosine plays in limitation of infarct size. The group’s work (Schulz et al., 1995), published in 1994, compared four groups: a ﬁrst group subjected to 90 min of ischemia, a second group subjected to an initial 10-minute period of ischemia (i.e., ischemic pre- conditioning) followed by a 15-minute period of reperfusion prior to a 90-minute period of ischemia, a third group receiving intra-coronary infusion of adenosine deaminase maintained throughout the 90-minute ischemic period, and a ﬁnal group undergoing ischemic pre-conditioning with concomitant adenosine deaminase infusion and subsequent 90-minute ischemia. Their results demonstrated a signiﬁcantly reduced infarct size in the ischemic pre- conditioning alone (i.e., without adenosine deaminase infusion) group (Schulz et al., 1995). In so doing, this pioneering article conﬁrmed the central role of endogenous adenosine in ischemic pre-conditioning in swine hearts, since the infarct size reduction in ischemic pre-conditioning was signiﬁcantly attenuated when catabolism of adenosine was increased through administration of adenosine deaminase (Schulz et al., 1995). A similar principle, dubbed ischemic post- conditioning, is a phenomenon whereby repetitive short episodes of ischemia following a longer, more prolonged ischemic episode result in signiﬁcantly decreased myocardial damage (Liu et al., 1991; Zhao et al., 2003). Adenosine, via A1 receptor-induced activation of protein kinase C and subsequent protein function modulation by phosphorylation mediates and potentiates the phenomena of pre- and post-conditioning (Vinten-Johansen et al., 1999). Lending further credence to this theory, research demonstrated that pre-conditioning protection could be blocked by use of adenosine receptor blocking agents (Liu et al., 1991; Schulz et al., 1995), and was conversely potentiated by use of an A1-speciﬁc agonist (Liu et al., 1991). REVISITING THE CONCEPTS OF REPERFUSION INJURY Monassier succinctly summarizes the concept of reperfusion injury by separating it into its two main facets: no-reﬂow, resulting from endothelial microvascular injury, and reperfusion syndrome, involving cardiomyocytes (Monassier, 2008b). The no-reﬂow phenomenon was initially described in 1966 by Krug et al (Krug et al., 1966). This phenomenon results from microvascular dysfunction The processes above coalesce to ultimately contribute to abnormalities of potassium, sodium, and calcium homeostasis in injured myocytes, to an inability to restore myocyte energy May 2022 \| Volume 13 \| Article 856747 Frontiers in Pharmacology \| www.frontiersin.org 2 Adenosine in Acute Myocardial Infarction De Marco et al. balance, and to microvascular injury that are the hallmarks of the no-reﬂow phenomenon (Forman et al., 2006). of new blood vessels following vascular injury (Dubey et al., 2002; Montesinos et al., 2004). These properties of adenosine are attributable to its stimulation of angiogenic factors such as IL- 8, vascular endothelial growth factor, and basic ﬁbroblast growth factor from microvascular endothelial cells (Feoktistov et al., 2003). One may infer from the above properties that adenosine may play an integral role in the repair of injured endothelium after reperfusion and, thereby, prevent ventricular remodeling by favouring collateral blood ﬂow. Frontiers in Pharmacology \| www.frontiersin.org UNDERSTANDING ADENOSINE AND THE RATIONALE FOR ITS USE AS A CARDIOPROTECTIVE AGENT The A2A and A3 receptors may equally play a role in pre- and post-conditioning, though this has been less well established. Adenosine’s transportation occurs rapidly into vascular endothelial cells and erythrocytes, where it is catabolized by adenosine deaminase to inosine (Layland et al., 2014). Notably, adenosine deaminase is found on the plasmatic membranes of erythrocytes and platelets (Franco et al., 1990). Understanding adenosine’s transportation and, most importantly, its short half-life is critical to comprehending its pharmacokinetics. Adenosine, when administered via the intra- coronary route, has a peak effect of < 10 s and a duration of action of roughly 20 s (Layland et al., 2014). The four receptors that bind adenosine are A1, A2A, A2B, and A3 (Shryock and Belardinelli, 1997). Adenosine A2A receptors are found on neutrophils, endothelial cells, vascular smooth muscle, and platelets (Vinten-Johansen et al., 1999). Activation of the A2A and A2B receptors increases adenylate cyclase activity and cAMP levels via coupling to Gs proteins (Heusch, 2010), consequently resulting in potent vasodilation of the coronary circulation and an increase in myocardial blood ﬂow (Shryock and Belardinelli, 1997; Vinten-Johansen et al., 1999; Heusch, 2010). Furthermore, adenylate cyclase activation via the A2A receptor results in inhibition of neutrophil superoxide generation and adherence to the endothelium (Vinten-Johansen et al., 1999). Therein, adenosine and its receptor-ligand induction of adenylate cyclase target neutrophil aggregation and adherence, reducing neutrophilic arterial plugging that has been evoked as one of the central mechanisms of reperfusion injury. Additionally, adenosine’s inhibition of neutrophil activation targets and prevents what is otherwise one of the initiating factors of the inﬂammatory cascade that culminates in reperfusion injury (Vinten-Johansen et al., 1999). Adenosine’s administration initiates a multitude of metabolic events that could be beneﬁcial in the setting of ischemia and reperfusion (Forman et al., 2006). Exogenous adenosine is responsible for the restoration of ATP levels in viable but energy-deﬁcient ischemic cells, as demonstrated in a study wherein the presence of an adenosine deaminase inhibitor resulted increased levels of ATP following reperfusion (Forman and Velasco, 1991). Moreover, the ischemic pre-conditioning triggered by adenosine has been demonstrated to be unique when compared to that triggered by other molecules mediating pre- conditioning, such as acetylcholine, bradykinin, and opioids (Cohen et al., 2001). PROPERLY TIMING ADENOSINE ADMINISTRATION demonstrated that the pre-conditioning induced by adenosine release, unlike that of the other molecules mentioned, seems independent of either free radicals or mitochondrial KATP channels (Cohen et al., 2001). The evidence supports that it is therefore most likely that adenosine results in direct activation of protein kinases (Cohen et al., 2001), more speciﬁcally protein kinase C, whose role in ischemic pre-conditioning is well established (Vinten-Johansen et al., 1999). Recalling that the pathophysiology underpinning reperfusion injury is, as the name implies, coronary reperfusion, it is inherently logical that the optimal timing for adenosine administration be prior to reperfusion, whether achieved with thrombolysis or percutaneous coronary intervention. If administered late, once reperfusion has already occurred, the pathophysiological cascade culminating in reperfusion injury will already have been triggered, and adenosine’s inhibitory effects on this chain of events are more likely to be inefﬁcient. The beneﬁcial effects of adenosine on myocardial blood ﬂow and on limitation of infarct size have been extensively studied and demonstrated in animals (Boucher et al., 2004; Yetgin et al., 2015). To the above point, an animal study on rats published in 2004 (Boucher et al., 2004) compared the effects of early versus late administration of an adenosine A2A agonist (CGS21680) on infarct size, which was calculated as a percentage of the area at risk by occlusion of the target coronary artery—in this case, the left anterior descending artery. In the study, the early administration group received CGS21680 5 min prior to reperfusion, whereas the late administration group received adenosine 5 min after the initiation of reperfusion. Study groups were compared to control subjects. Infarct size was signiﬁcantly reduced in the early compared to the control group (25.7 ± 5.3% versus 46.5 ± 5.3%, p < 0.05). Conversely, there was no statistically signiﬁcant difference when comparing the late group infarct size area (38.2 ± 6.2%) to that of the control group. Indeed, such a study lends credence to the belief that timing adenosine administration before reperfusion is crucial for optimising treatment effects. HIGHLIGHTING THE IMPORTANCE OF THE ADMINISTRATION ROUTE Early studies of adenosine in the setting of AMI in humans were performed with administration of adenosine via the intravenous route. In the AMISTAD (Mahaffey et al., 1999; Ross et al., 2005) trials, low (50 mcg/kg/min) and high (70 mcg/kg/min) infusions of adenosine started within 15 min of thrombolysis or before coronary intervention were compared in a 1:1:1 fashion to placebo. AMISTAD II demonstrated no difference in the primary endpoint of new congestive heart failure > 24 h after randomization or ﬁrst re-hospitalization for congestive heart failure, or death from any cause within 6 months, but that patients treated with adenosine had a tendency towards smaller infarct sizes (17% of the left ventricular size versus 27%, p = 0.074). Furthermore, infarct size was shown to be signiﬁcantly related (p < 0.001) to the occurrence of the primary endpoint. To date, there has been inconsistency in study methodology as it pertains to adenosine administration (Yetgin et al., 2015). Ultimately, results have been mixed, though there is a demonstrable trend towards improved outcomes in terms of infarct size limitation with earlier administration of adenosine. In the ATTAC (Quintana et al., 2003) study, a single dose (10mcg/kg/min) of adenosine was infused at the time of thrombolysis and maintained for 6 h and was compared to placebo. No beneﬁcial effect of adenosine was found regarding echocardiographic indices of left ventricular systolic or diastolic function. Despite the trial being stopped early due to lack of apparent effect after an interim analysis, 12-month follow-up data showed that cardiovascular mortality was 8.9% with adenosine and 12.1% with placebo (OR 0.71, 95% CI 0.4-1.2, p = 0.2) and 8.4 versus 14.6% (OR 0.53, 95% CI 0.23-1.24, p = 0.09) in patients with anterior AMI. UNDERSTANDING ADENOSINE AND THE RATIONALE FOR ITS USE AS A CARDIOPROTECTIVE AGENT More speciﬁcally, it has been Studies suggest that adenosine may play a critical role in promoting vascular repair and in accelerating the development May 2022 \| Volume 13 \| Article 856747 Frontiers in Pharmacology \| www.frontiersin.org 3 Adenosine in Acute Myocardial Infarction De Marco et al. Frontiers in Pharmacology \| www.frontiersin.org THE IMPORTANCE OF LOCATION OF ADENOSINE ADMINISTRATION The logical question stemming from growing interest in intracoronary administration of adenosine pertains to selection of the optimal location for such intracoronary delivery. Studies to date have employed various methodologies, with intracoronary injection sites being quite variable. The three main intracoronary administration sites identiﬁed and examined in the literature thus far are at the catheter site, at the site of percutaneous coronary intervention, or distal to the occlusion site (Yetgin et al., 2015). While use of adenosine in the acute treatment of AMI has yet to garner uptake at large, it remains an avenue of interest worth exploring further. Most critically, the varying study methodologies make it difﬁcult to draw broad conclusions from the existing evidence. The evidence is becoming increasingly clearer that dose, timing of administration, route of administration, and site of administration play critical roles in adenosine’s ultimate efﬁcacy. An understanding of adenosine’s pharmacokinetics and pharmacodynamics is critical when examining studies done to date and when planning and executing future studies. In the case of intra-coronary administration, especially, the presence of blood in the catheter may lead to rapid inactivation of adenosine, recalling that adenosine deaminase is found on the plasmatic membrane of erythrocytes and platelets, prior to its arrival at the target site. Overall, by drawing from the literature, it appears as though a bolus of adenosine delivered intra-coronary distal to the occlusion site at or just prior to reperfusion is most effective in reducing reperfusion injury, though this hypothesis has yet to be tested in human subjects. An animal study (Charron et al., 2013) in male pigs harmonizes many of the elements discussed above by comparing three different doses of adenosine—2, 4, and 8 mg—administered prior to reperfusion as a bolus either intra-coronary, downstream of the occlusion site, or extra- coronary. Results demonstrated a signiﬁcant reduction (p < 0.05) in infarct size expressed as a percentage of area at risk in the 4 mg (33 ± 6%) and 8 mg (30 ± 5%) intra-coronary groups compared to the placebo group (46 ± 3%). Critically, there was no statistically signiﬁcant reduction in the 2 mg intra- coronary group (38 ± 2%) and, most interestingly, in the 8 mg intracoronary group in whom adenosine was injected upstream of the occlusion site (47 ± 6%). DISCUSSION control group, p = 0.02) and no reﬂow (26 ± 6% of the infarct area versus 49 ± 6% in the control group, p = 0.03), with bolus administration of adenosine without subsequent prolonged infusion yielding results that were similar to those of the control group. The mainstay of therapy for patients with AMI remains reperfusion, either via timely percutaneous coronary intervention in capable centers or via thrombolysis. Successful reperfusion has been extensively demonstrated to reduce cardiac mortality and minimize infarct size and left ventricular damage. That said, reperfusion, as discussed extensively in this review, may itself induce injury deleterious to left ventricular function both in the short- and long-term. Therefore, exploration and identiﬁcation of therapy tailored to those at the highest risk of reperfusion injury may provide additional avenues dedicated to improving left ventricular function and clinical outcomes. Given the extremely short half-life of adenosine, it stands to reason that bolus injections, unless perfectly timed and optimally delivered, may be inadequate when treating reperfusion injury in AMI. Conversely, more prolonged intracoronary delivery initiated just prior to reperfusion may increase local drug concentrations multi-fold and may achieve adequate levels in the target microvascular bed, thereby potentially improving therapeutic efﬁcacy. That said, There remains at present a lack of evidence in the literature to support this hypothesis. Adenosine has widely been demonstrated to have multiple favorable effects in mitigating no-reﬂow and reperfusion injury, primarily but not exclusively through inhibition of neutrophil-mediated vascular plugging and vascular damage. In experimental models, both intravenous and intracoronary routes of administration resulted in marked reduction of infarct size. In human trials, intravenous adenosine has similarly shown beneﬁt in infarct size reduction, though this beneﬁt has yet to translate to concrete clinical endpoints, most notably cardiovascular mortality and heart failure hospitalization. ESTABLISHING AN OPTIMAL DURATION OF ADENOSINE INFUSION Duration of infusion represents yet another point of discussion when examining adenosine’s role in AMI. The early intravenous studies (Mahaffey et al., 1999; Quintana et al., 2003; Ross et al., 2005), most notably AMISTAD, AMISTAD II, and ATTACC, used variable infusion durations ranging from one to six h. That said, the limitations of intravenous adenosine administration have been discussed previously and, indeed, dose adjustments to infusion rates were often made in these studies because of adverse effects related to the adenosine infusion. The primary limitation pertaining to intravenous adenosine administration is that maximal doses are difﬁcult to achieve because of the marked systemic hypotension associated with higher adenosine doses (Charron et al., 2013). Whether maximal doses of adenosine were achieved in studies employing the intravenous route is unclear. The cardioprotective properties observed, however, are non- negligible and highlight that even intravenous adenosine, despite potentially sub-optimal dosing, may result in stimulation of remote pathways of cardioprotection. Intracoronary administration of adenosine, unlike intravenous administration, enables delivery of much higher doses without direct systemic effects, most notably hypotension. In contrast, while there may be no human studies evaluating continuous infusions of adenosine via the intracoronary route, an animal study on porcine subjects (Yetgin et al., 2015) did compare bolus with prolonged infusions of intracoronary adenosine. Results of said study (Yetgin et al., 2015) demonstrated that only the prolonged, high-dose infusion of adenosine was able to limit infarct size (46 ± 4% of the area at risk versus 59 ± 3% in the May 2022 \| Volume 13 \| Article 856747 Frontiers in Pharmacology \| www.frontiersin.org 4 Adenosine in Acute Myocardial Infarction De Marco et al. THE IMPORTANCE OF LOCATION OF ADENOSINE ADMINISTRATION Translation of such animal studies to humans, with the beneﬁcial effects of intra-coronary adenosine downstream of the occlusion site prior to the onset of reperfusion, has been demonstrated by Marzilli et al. (Marzilli et al., 2000). Such an administration route was associated with beneﬁcial effects on both coronary blood ﬂow and ventricular function, indicating likely minimization of the mechanisms underpinning reperfusion injury (Marzilli et al., 2000). In fact, a meta- analysis comparing randomized controlled trials studying intracoronary adenosine versus control to trials studying intravenous adenosine versus control found improved clinical outcomes in terns of less heart failure in patients administered intracoronary adenosine (Bulluck et al., 2016). Frontiers in Pharmacology \| www.frontiersin.org REFERENCES Heusch, G., and Gersh, B. J. (2017). The Pathophysiology of Acute Myocardial Infarction and Strategies of Protection beyond Reperfusion: a Continual Challenge. Eur. Heart J. 38, 774–784. doi:10.1093/eurheartj/ehw224 Boucher, M., Pesant, S., Falcao, S., de Montigny, C., Schampaert, E., Cardinal, R., et al. (2004). Post-ischemic Cardioprotection by A2A Adenosine Receptors: Dependent of Phosphatidylinositol 3-kinase Pathway. J. Cardiovasc Pharmacol. 43, 416–422. doi:10.1097/00005344-200403000-00013 Heusch, G. (2020). Myocardial Ischaemia-Reperfusion Injury and Cardioprotection in Perspective. Nat. Rev. Cardiol. 17, 773–789. doi:10. 1038/s41569-020-0403-y Keeley, E. C., Boura, J. A., and Grines, C. L. (2003). Primary Angioplasty versus Intravenous Thrombolytic Therapy for Acute Myocardial Infarction: a Quantitative Review of 23 Randomised Trials. Lancet 361, 13–20. doi:10. 1016/S0140-6736(03)12113-7 Bulluck, H., Sirker, A., Loke, Y. K., Garcia-Dorado, D., and Hausenloy, D. J. (2016). 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(2014). Adenosine: Physiology, Pharmacology, and Clinical Applications. JACC Cardiovasc Interv. 7 (6), 581–591. doi:10.1016/j.jcin.2014.02.009 Feoktistov, I., Ryzhov, S., Goldstein, A. REFERENCES E., and Biaggioni, I. (2003). Mast Cell- mediated Stimulation of Angiogenesis: Cooperative Interaction between A2B and A3 Adenosine Receptors. Circ. Res. 92, 485–492. doi:10.1161/01.RES. 0000061572.10929.2D Liu, G. S., Thornton, J., Van Winkle, D. M., Stanley, A. W., Olsson, R. A., and Downey, J. M. (1991). Protection against Infarction Afforded by Preconditioning Is Mediated by A1 Adenosine Receptors in Rabbit Heart. Circulation 84, 350–356. doi:10.1161/01.cir.84.1.350 Fibrinolytic Therapy Trialists’ (Ftt) Collaborative Group (1994). Indications for Fibrinolytic Therapy in Suspected Acute Myocardial Infarction: Collaborative Overview of Early Mortality and Major Morbidity Results from All Randomised Trials of More Than 1000 Patients. Fibrinolytic Therapy Trialists’ (FTT) Collaborative Group. 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Cardiovasc Dis. 101 (7- 8), 491–500. doi:10.1016/j.acvd.2008.06.014 Gusto Angiographic Investigators (1993). The Effects of Tissue Plasminogen Activator, Streptokinase, or Both on Coronary-Artery Patency, Ventricular Function, and Survival after Acute Myocardial Infarction. N. Engl. J. Med. CONCLUSION It is critical that reperfusion injury be seen not only as a theoretical phenomenon, but rather as an important determinant of ﬁnal infarct size and consequent ventricular dysfunction. Adenosine, despite its perceived lack of beneﬁt relative to clinical outcomes, remains one of the few molecules to consistently reduce infarct size, May 2022 \| Volume 13 \| Article 856747 Frontiers in Pharmacology \| www.frontiersin.org 5 Adenosine in Acute Myocardial Infarction De Marco et al. AUTHOR CONTRIBUTIONS especially in anterior AMI, by directly combatting the pathophysiological underpinnings of reperfusion injury. Its clinical use and continued study in well-designed experiments is not only necessary, but so too vital to the goal of improving patient outcomes in AMI. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. The ﬁgure was made with www.Biorender.com. REFERENCES 329, 1615–1622. doi:10.1056/NEJM199311253292204 Montesinos, M. C., Shaw, J. P., Yee, H., Shamamian, P., and Cronstein, B. N. (2004). Adenosine A(2A) Receptor Activation Promotes Wound Neovascularization by Stimulating Angiogenesis and Vasculogenesis. Am. J. Pathol. 164, 1887–1892. doi:10.1016/S0002-9440(10)63749-2 Hausenloy, D. J., Botker, H. E., Engstrom, T., Erlinge, D., Heusch, G., Ibanez, B., et al. (2017). Targeting Reperfusion Injury in Patients with ST-Segment Elevation Myocardial Infarction: Trials and Tribulations. Eur. 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Cardiol. 114, 45. doi:10.1007/s00395-019-0756-8 May 2022 \| Volume 13 \| Article 856747 Frontiers in Pharmacology \| www.frontiersin.org 6 De Marco et al. Adenosine in Acute Myocardial Infarction during Reperfusion: Comparison with Ischemic Preconditioning. Am. J. Physiol. Heart Circ. Physiol. 285, H579–H588. doi:10.1152/ajpheart.01064. 2002 Myocardial Infarction (AMISTAD-II). J. Am. Coll. Cardiol. 45, 1775–1780. doi:10.1016/j.jacc.2005.02.061 Saito, H., Nishimura, M., Shinano, H., Makita, H., Tsujino, I., Shibuya, E., et al. (1999). Plasma Concentration of Adenosine during Normoxia and Moderate Hypoxia in Humans. Am. J. Respir. Crit. Care Med. 159, 1014–1018. doi:10. 1164/ajrccm.159.3.9803100 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Schulz, R., Rose, J., Post, H., and Heusch, G. (1995). Involvement of Endogenous Adenosine in Ischaemic Preconditioning in Swine. Pﬂugers Arch. 430, 273–282. doi:10.1007/BF00374659 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Frontiers in Pharmacology \| www.frontiersin.org REFERENCES Shryock, J. C., and Belardinelli, L. (1997). Adenosine and Adenosine Receptors in the Cardiovascular System: Biochemistry, Physiology, and Pharmacology. Am. J. Cardiol. 79, 2–10. doi:10.1016/s0002-9149(97)00256-7 Shryock, J. C., and Belardinelli, L. (1997). Adenosine and Adenosine Receptors in the Cardiovascular System: Biochemistry, Physiology, and Pharmacology. Am. J. Cardiol. 79, 2–10. doi:10.1016/s0002-9149(97)00256-7 Vinten-Johansen, J., Thourani, V. H., Ronson, R. S., Jordan, J. E., Zhao, Z. Q., Nakamura, M., et al. (1999). Broad-spectrum Cardioprotection with Adenosine. Ann. Thorac. Surg. 68, 1942–1948. doi:10.1016/s0003-4975(99)01018-8 Vinten-Johansen, J., Thourani, V. H., Ronson, R. S., Jordan, J. E., Zhao, Z. Q., Nakamura, M et al (1999) Broad spectrum Cardioprotection with Adenosine Ann Thorac Vinten-Johansen, J., Thourani, V. H., Ronson, R. S., Jordan, J. E., Zhao, Z. Q., Nakamura, M., et al. (1999). Broad-spectrum Cardioprotection with Adenosine. Ann. Thorac. Surg. 68, 1942–1948. doi:10.1016/s0003-4975(99)01018-8 Copyright © 2022 De Marco, Charron and Rousseau. 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. Yetgin, T., Uitterdijk, A., Te Lintel Hekkert, M., Merkus, D., Krabbendam-Peters, I., van Beusekom, H. M. M., et al. (2015). Limitation of Infarct Size and No- Reﬂow by Intracoronary Adenosine Depends Critically on Dose and Duration. JACC Cardiovasc Interv. 8, 1990–1999. doi:10.1016/j.jcin.2015.08.033 Cardiovasc Interv. 8, 1990–1999. doi:10.1016/j.jcin.2015.08.033 Zhao, Z. Q., Corvera, J. S., Halkos, M. E., Kerendi, F., Wang, N. P., Guyton, R. A., et al. (2003). Inhibition of Myocardial Injury by Ischemic Postconditioning May 2022 \| Volume 13 \| Article 856747 Frontiers in Pharmacology \| www.frontiersin.org 7
https://openalex.org/W2745012473	https://biblio.ugent.be/publication/8535959/file/8535960	English	null	Built environmental correlates of older adults’ total physical activity and walking: a systematic review and meta-analysis	The international journal of behavioural nutrition and physical activity	2,017	cc-by	20,525	Built environmental correlates of older adults’ total physical activity and walking: a systematic review and meta-analysis David W. Barnett1, Anthony Barnett1, Andrea Nathan1, Jelle Van Cauwenberg2,3, Ester Cerin1,4,5* and on behalf of the Council on Environment and Physical Activity (CEPA) – Older Adults working group * Correspondence: ester.cerin@acu.edu.au 1Institute for Health and Ageing, Australian Catholic University, Level 6, 215 Spring Street, Melbourne, VIC 3000, Australia 4School of Public Health, The University of Hong Kong, 7 Sassoon Road, Pokfulam, Hong Kong, Special Administrative Region, China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Identifying attributes of the built environment associated with health-enhancing levels of physical activity (PA) in older adults (≥65 years old) has the potential to inform interventions supporting healthy and active ageing. The aim of this study was to first systematically review and quantify findings on built environmental correlates of older adults’ PA, and second, investigate differences by type of PA and environmental attribute measurement. Methods: One hundred articles from peer-reviewed and grey literature examining built environmental attributes related to total PA met inclusion criteria and relevant information was extracted. Findings were meta-analysed and weighted by article quality and sample size and then stratified by PA and environmental measurement method. Associations (p < .05) were found in relation to 26 individual built environmental attributes across six categories (walkability, residential density/urbanisation, street connectivity, access to/availability of destinations and services, infrastructure and streetscape, and safety) and total PA and walking specifically. Reported individual- and environmental-level moderators were also examined. Results: Positive environmental correlates of PA, ranked by strength of evidence, were: walkability (p < .001), safety from crime (p < .001), overall access to destinations and services (p < .001), recreational facilities (p < .001), parks/ public open space (p = .002) and shops/commercial destinations (p = .006), greenery and aesthetically pleasing scenery (p = .004), walk-friendly infrastructure (p = .009), and access to public transport (p = .016). There were 26 individual differences in the number of significant associations when the type of PA and environmental measurement method was considered. No consistent moderating effects on the association between built environmental attributes and PA were found. Conclusions: Safe, walkable, and aesthetically pleasing neighbourhoods, with access to overall and specific destinations and services positively influenced older adults’ PA participation. However, when considering the environmental attributes that were sufficiently studied (i.e., in ≥5 separate findings), the strength of evidence of associations of specific categories of environment attributes with PA differed across PA and environmental measurement types. Future research should be mindful of these differences in findings and identify the underlying mechanisms. Higher quality research is also needed. Keywords: Older adults, Built environment, Physical activity, Walking, Correlates, Systematic review, Meta-analysis © The Author(s). Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 DOI 10.1186/s12966-017-0558-z Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 DOI 10.1186/s12966-017-0558-z Open Access Background Further reasons for inconsistent findings on the associ- ations between the built environment and PA may relate to the methodologies of the systematic reviews and/or the studies being reviewed. For example, Gebel and col- leagues [26] recommended that systematic reviews should: a) consider article/study quality in the synthesis of findings; and b) include relevant data from grey litera- ture. Also, small sample sizes, a large variety of built environmental exposures and PA outcomes [27, 28], and the inappropriate categorisation of continuous variables [29] in examined studies may have contributed to incon- sistent findings. To date, no systematic review on the built environment and total PA has considered these issues. g Worldwide, the proportion of older adults (65 years or older) is forecast to grow exponentially from 524 million in 2010 to approximately 1.5 billion individuals by 2050 [1]. This will pose a major economic challenge for soci- eties globally, given the healthcare expenditure associ- ated with individuals experiencing age-related chronic diseases [1, 2]. Evidence suggests that regular engage- ment in PA is particularly important for healthy ageing. For example, it reduces the risk of coronary heart dis- ease, some cancers, type 2 diabetes, depression, cognitive impairment and social isolation [3, 4]. Older adults worldwide, however, are often inactive [4–7]. Thus, it is important to identify modifiable factors with a high level of reach that may help increase total PA in this age group. As it is ultimately the overall dose of PA that confers benefit/detriment upon health [8], irrespective of the domain/s in which it was accrued, it is import- ant to focus on factors contributing to total PA. Fur- thermore, walking is the most prevalent and preferred form of PA in older adults [9], low-risk and beneficial to health and can contribute substantially to daily en- ergy expenditure [10]. Hence, this review will focus on total PA and total walking. A synthesis of evidence would also need to distinguish between findings based on objective- and self-report mea- sures of exposures (environmental attributes) and out- comes (PA). Self-report measures are more likely to be influenced by culture [30, 31] and, thus, yield different findings across geographical locations due to measure- ment rather than substantive reasons. Also, perceptions of the local environment may not accurately represent the ‘real’ environment [32, 33]. Indeed, associations of PA with objective and perceived measures of the built environment tend to differ [34, 35]. Background This does not necessarily mean that one type of measurement is better than the other, how- ever, as perceived environmental measures may be more closely associated with PA than objective alternatives [27], a consideration of these differences would help better in- form policies and interventions. Socio-ecological models posit that PA behaviour is shaped by complex and dynamic interrelations between individual, social, and environmental factors [11, 12]. The built environment offers substantial public health potential, due to people being regularly exposed to it across their life span. Understanding the impact of built environmental attributes on older adults’ PA is particu- larly pertinent as their diminished physical capacity makes them more vulnerable to the detrimental effects of physically challenging environments (e.g., inclines, un- even surfaces, absence of walk-friendly infrastructure) on PA [13]. In turn, this may lead to less venturing out- side of the home due to fear of falls [14]. However, a previous review of the built environment and older adults’ PA identified no consistent correlates [15]. With regard to measurement of total PA, objective PA measures are considered to provide more valid assess- ments of intensity, duration, and frequency of PA than subjective alternatives [36]. They are also less likely to be influenced by cultural biases [30]. Additionally, differ- ences in environment-PA associations between self- reported and objectively-measured PA have previously been reported in adults [37]. This indicates that there is a need for a synthesis of findings on built environment correlates of total PA by type of PA assessment (object- ive and self-reported). One postulated reason for the lack of consistent significant associations between environmental corre- lates and PA was the relative ‘western’ bias of the reviewed research – 68% of the 31 articles reviewed by Van Cauwenberg and colleagues were from North America alone [15]. This is an important limitation since homogenously low-density western cities lack environ- mental variability potentially resulting in the underesti- mation of the strength of associations between the built environment and PA. Also, western cities differ from the built environments of Africa, Asia, and South America, limiting the generalizability of findings [16–18]. In consideration of the recent growing evidence from non- western countries (e.g., [19–25]), an update of the litera- ture was necessary. Therefore, this systematic review aimed to provide a timely, robust overview of studies that investigated asso- ciations of built environmental attributes and estimates of total PA, including total walking. Abstract 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 2 of 24 Literature search strategy gy Our systematic literature search followed Preferred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) guidelines [39] and was an extended update of Van Cauwenberg et al.’s 2011 review [15] in- cluding additional electronic databases and search terms to account for grey literature, experimental research, a greater variety of built environmental attributes and older adults’ PA. Systematic searches were performed in the following electronic databases (September – November 2016): Cinahl, PubMed, Scopus, SPORTDiscus, TRIS, and Web of Science. An example of the complete search terms syntax (used in PubMed) is presented in the resulting PRISMA flowchart (Fig. 1). All electronic database searches used the same search terms and syntax. However, filters varied depending on available options in each data- base. Manual searches were then undertaken of previously published systematic reviews, meta-analyses and authors’ personal archives. The websites of Active Living Research, SUSTRANS, the National Institute for Health and Clinical Excellence, Heart Foundation, and Open Grey were also searched for grey literature (e.g., unpublished studies, theses and reports). Three reviewers independently screened articles for eligibility criteria by title and then by abstract. If ab- stracts met the inclusion criteria, the article text was in- dependently read by two reviewers, and a decision for inclusion made accordingly. The reviewers discussed and resolved any disputed inclusions. Post article inclu- sion, first authors’ publication histories and eligible arti- cles’ reference lists were screened for additional eligible articles. One hundred articles were eligible for inclusion in this review (Fig. 1). Methods This systematic review and meta-analysis was registered in PROSPERO (Registration no. CRD42016051227 [38]) in November 2016. Background This included ad- dressing some key methodological limitations of previ- ous reviews by stratifying findings by measurement methods (objective and self-reported) and applying a meta-analytic procedure. The latter incorporated study quality data to more robustly quantify the direction of associations between the built environment and older adults’ PA [27] and assisted the formulation of objective conclusions based on statistical theory rather than on subjective criteria (e.g., defining >50% of significant Page 3 of 24 Page 3 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 positive associations as convincing evidence of a positive association between a specific environment characteris- tic and PA). and 3rd September 2016; (2) quantitative study; (3) study with a cross-sectional, longitudinal or quasi-experimental design; (4) a sample with a mean age ≥65 years; (5) inves- tigated the association between any objective or perceived attribute of the built environment and objectively mea- sured or self-reported PA and/or walking that was not specific to a single PA domain only; (6) did not exclusively focus on clinical populations (e.g., overweight, disabled or institutionalised participants); and (7) did not investigate associations between PA and the built environment with an ill-defined aggregated environmental measure that, for example, combined two weakly correlated attributes such as access to/availability of recreational facilities and traffic/ pedestrian safety. Data extraction Two reviewers independently extracted all relevant in- formation from eligible papers and cross-checked each other’s work upon completion. Any disputes were re- solved in consultation with a third member of the team. A table (Additional file 1) was then constructed contain- ing the following data: study name (if any), first author and publication year; participant information – sample size, study setting (e.g., urban or rural), mean age, per- centage of females, study response rate, community dwellers or not, geographical location; study design – Quantification of buffer effects and/or moderators Findings from studies reporting associations for a given combination of environmental attribute and PA outcome for multiple environmental buffer sizes or values of a moderator were assigned fractional weights totalling 1.0 (per study). Associations from studies reporting on mul- tiple buffer sizes were each assigned a fractional weight equal to 1 divided by the number of buffer sizes. For ex- ample, Nathan and colleagues examined associations for two buffer sizes (400 m and 800 m). They found a nil as- sociation for 400 m buffers around the home between access to social infrastructure and total walking and a positive one for 800 m buffers [49]. In this case, a result- ing score of 0.5 nil (400 m buffer) and 0.5 positive asso- ciations (800 m buffer) was recorded. For studies that examined moderator effects, associations were reported as main effects only if the findings across all examined values of the moderator were consistent in direction and statistical significance. When this did not occur, weights were assigned for each examined value of the moderator dependent upon the (approximate) proportion of the total sample represented by the subgroup of participants. For example, Jefferis and colleagues found a positive as- sociation between access to social and leisure activities for males and a nil association for females [50]. Since males represented 65% of the total sample, a fractional weight of 0.65 was assigned to the positive association and a 0.35 weight for the nil association (females). For studies that used a continuous measure of moderators, associations recorded at the average value of the moder- ator were assigned a weight of 0.60, while those above and below the mean were each assigned a weight of 0.20 (i.e., ±1 standard deviation (SD)). This follows the logic that, if data are normally distributed, ≈20% of values would fall below and 20% above 1SD (accounting for some uncertainty around the value of the moderator at ±1 SD). When an association was moderated by numer- ous factors, weights were assigned according to the aforementioned procedure and, again, with all fractional weights totalling 1.0. In this review, the variable “Total PA” represents all (combined) PA outcomes relevant to this review and ex- tracted from the selected articles. These include the PA outcomes reported in the selected articles as “Total PA”, “Total MVPA” and “Total walking”. “Total PA” was then stratified into objective and self-report PA, congruent with study aims. Selection criteria Article eligibility for inclusion was based on the following criteria: (1) published in English between 1st January 2000 Fig. 1 PRISMA flowchart Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 4 of 24 Page 4 of 24 dataset (e.g., Senior Neighborhood Quality of Life Study (SNQLS) [43–48]) to avoid duplication of extracted data. sampling method for clusters (areas or neighbourhoods) and individuals, stratification of recruitment site by en- vironmental attribute/s (if any), neighbourhood defin- ition (if any); list of covariates; PA outcome information – type of measurement (objective or self-reported), in- strument or device used and whether or not it was vali- dated, how outcome measures were operationalised (e.g., continuously or categorically); environmental exposure information – type of measurement (objective or per- ceived), name of measure, environmental attributes as named in study and category of environmental attribute according to the classification used in this review (see below); moderator information (if any); analytical ap- proach information; findings; and additional comments important for the assessment or interpretation of the study (if any). Coding and quantification of associations g q Following the protocol used by Van Cauwenberg and colleagues in their earlier systematic review [15], associa- tions between built environmental attributes and PA outcomes were categorised as statistically significantly positive (P), significantly negative (N), or not statistically significant (Ø). When available, preference was given to findings within articles that adjusted for socio- demographic covariates and self-selection (i.e., factors that may affect neighbourhood choice and subsequent PA level, for example, preference for PA resources [42]). Often, one article reported multiple environmental attri- butes that were coded under the same environmental category (e.g., ‘absence of litter’ and ‘presence of trees’ would both fall under the common category of ‘greenery and aesthetically pleasing scenery’). Similarly, one article may have reported multiple PA outcomes related to the same environmental attribute (e.g., ‘total walking’ and ‘total PA’ with ‘crime/personal safety’). Four distinct find- ings would be extracted from these two examples. We then cross-examined other articles from the same Quantification of buffer effects and/or moderators Total walking was also considered sep- arately for reasons aforementioned. Built environmental attributes were categorised according to an expanded list of factors included in the Neighbourhood Environment Walkability Scale (NEWS), which is the most frequently used neighbourhood environment questionnaire inter- nationally [40, 41] (Table 1). The detailed list of mea- sures included under each category of environmental attributes can be found in Supplementary Table S1 (Additional file 1). Article quality and sample size assessment A checklist for article quality assessment was developed based on ten set criteria [with a maximum total quality score of 9] (Additional file 2): (1) study design [score: cross- sectional = 0, longitudinal = 1, quasi-experimental = 2]; (2) study areas or participant recruitment stratified by key environmental attributes [yes = 1, no = 0]; (3) adequate par- ticipant response rate (≥60%) [51] or evidence of a repre- sentative sample [yes = 1, no = 0]; (4) outcome measures valid and reliable based on published metric properties of rition and Physical Activity (2017) 14:103 Page 5 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 5 of 24 Barnett et al. Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 5 of 24 Table 1 Characteristics of selected articles (N = 100) Characteristic Number of articles % Article reference Study designa Cross-sectional 95 94 [19–25, 32, 43–50, 53, 54, 59, 60, 65, 67–80, 87, 92, 93, 100, 118–173] Longitudinal 5 5 [61–65] Quasi-experimental 1 1 [66] Geographical area: continent Africa 1 1 [25] Asia 16 16 [19–22, 60, 73, 75, 80, 124, 139, 142, 146, 160, 164, 169, 173] Europe 22 22 [50, 66, 74, 79, 87, 119, 120, 123, 129, 136, 137, 147, 148, 152, 154, 156, 157, 162, 163, 165, 166, 170] North America 46 46 [32, 43–48, 53, 54, 61–65, 67–70, 76, 78, 92, 93, 125–127, 130–135, 138, 140, 141, 143, 144, 149–151, 155, 158, 159, 161, 167, 168, 172] Oceania 13 13 [49, 59, 71, 72, 77, 100, 118, 121, 122, 128, 145, 153, 171] South America 2 2 [23, 24] Geographical area: country Australia 13 13 [49, 59, 71, 72, 77, 100, 118, 121, 122, 128, 145, 153, 171] Belgium 2 2 [74, 170] Brazil 1 1 [23] Canada 10 10 [54, 61, 62, 78, 125–127, 135, 138, 172] China 1 1 [80] Colombia 1 1 [24] Czech Republic, Poland, & Slovakia (pooled analysis) 1 1 [154] Hong Kong, China 2 2 [73, 124] Iceland 1 1 [119] Iran 1 1 [146] Ireland 2 2 [147, 152] Japan 7 7 [19–22, 160, 164, 169] Lithuania 1 1 [120] Malaysia 1 1 [139] Netherlands 4 4 [123, 136, 137, 166] Norway 1 1 [157] Singapore 1 1 [142] South Africa 1 1 [25] South Korea 2 2 [75, 173] Thailand 1 1 [60] United Kingdom 10 10 [50, 66, 79, 87, 129, 148, 156, 162, 163, 165] United States of America 36 36 [32, 43–48, 53, 63–65, 67–70, 76, 92, 93, 130–134, 140, 141, 143, 144, 149–151, 155, 158, 159, 161, 167, 168] Geographical setting Urban 56 56 [23–25, 32, 43–48, 50, 53, 59, 61–64, 66, 71, 72, 74, 80, 87, 92, 93, 100, 120 122 124 127 129 131 133 135 138 142 144 146 149 153 trition and Physical Activity (2017) 14:103 Page 6 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 6 of 24 Barnett et al. Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Barnett et al. Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 6 of 24 Sample sizeb ≤100 9 9 [19, 25, 53, 61, 66, 118, 127, 135, 161] 101–300 27 27 [32, 43, 53, 59, 60, 67, 69, 76, 87, 119, 120, 122, 123, 129, 132, 133, 140–142, 146, 150, 156, 162–166] 301–500 23 23 [23, 47, 63, 64, 68, 71–75, 93, 100, 121, 124, 126, 131, 134, 153, 154, 160, 167, 169, 170] 501–1000 16 16 [44–46, 48, 62, 78, 79, 92, 125, 128, 138, 144, 147, 148, 158, 159] 1001–2500 11 11 [20, 22, 24, 50, 54, 77, 130, 151, 155, 172, 173] > 2500 16 16 [21, 49, 54, 65, 70, 80, 136, 137, 139, 143, 145, 149, 152, 157, 168, 171] Study with multiple articles Active Living Study 3 3 [71, 72, 153] BEPAS Seniors 2 2 [74, 170] Great Britain older adults 1 2 2 [162, 163] Health and Wellbeing Surveillance System 2 2 [49, 171] LL-FDI study (Late-Life Function and Disability Instrument) 2 2 [132, 150] Melbourne older adults study 1 2 2 [121, 122] Netherlands Housing Survey (WoON) 2 2 [136, 137] Nurses’ Health Study 2 2 [70, 168] PACS (Physical Activity Cohort Scotland) 2 2 [79, 148] Project OPAL (Older People Active Living) 3 3 [87, 129, 165] SHAPE (Senior Health and Physical Exercise) 4 4 [32, 63, 92, 93] SNQLS (Senior Neighborhood Quality of Life Study) 6 6 [43–48] TILDA (The Irish Longitudinal Study on Ageing) 2 2 [147, 152] VoisiNuAge 2 2 [62, 138] Single publication (named study) 33 33 [21, 50, 54, 59, 61, 64–66, 73, 77, 78, 80, 100, 124, 128, 130, 133–135, 139, 140, 143–145, 149, 155, 157–159, 166, 169, 172, 173] Single publication (unnamed study) 31 31 [19, 20, 22–25, 53, 60, 67–69, 75, 76, 118–120, 123, 125–127, 131, 141, 142, 146, 151, 154, 156, 160, 161, 164, 167] Neighbourhood recruitment stratificationa Environmental attributes 57 57 [19, 21–25, 32, 44–50, 54, 60, 63, 66, 68, 71–76, 78–80, 87, 92, 118, 119, 123, 124, 126, 129, 130, 134, 135, 139, 146–148, 151, 153, 155, 159, 160, 162–166, 168, 170–172] Urbanisation 22 22 [19, 21, 22, 49, 54, 60, 68, 75, 78, 80, 118, 119, 123, 134, 139, 155, 159, 160, 166, 168, 171, 172] Area-level socio-economic status 6 6 [23, 25, 87, 129, 151, 165] Area-level socio-economic status & walkability 12 12 [44–48, 73, 74, 124, 126, 135, 146, 170] Area-level socio-economic status & urbanisation 10 10 [50, 63, 76, 79, 92, 130, 147, 148, 162, 163] Walkability 4 4 [32, 71, 72, 153] Streetscape 2 2 [66, 164] Demographics 31 31 [20–22, 50, 62, 64, 69, 70, 79, 119, 121, 122, 132–134, 136–138, 141–144, 148, 150, 151, 155, 156, 161–163, 173] None 21 21 [43, 53, 59, 61, 65, 67, 77, 93, 100, 120, 125, 128, 131, 140, 145, 149, 154, 157, 158, 167, 169] Neighbourhood definitiona Objective Ad i i t ti / / t l d 25 25 [19 54 65 68 70 74 80 118 134 136 139 142 145 146 148 149 25 25 [19, 54, 65, 68, 70, 74, 80, 118, 134, 136–139, 142, 145, 146, 148, 149, 157, 158, 160, 166, 170, 172, 173] 25 Barnett et al. Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 8 of 24 Barnett et al. Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 7 of 24 Page 7 of 24 Table 1 Characteristics of selected articles (N = 100) (Continued) Buffer (crow-fly or road-network) ≤250 m 3 3 [21, 64, 171] 300 m 1 1 [123] 400–500 m 16 16 [21, 24, 44, 46–49, 59, 64, 72, 73, 93, 135, 153, 159, 171] 800–1000 m 15 15 [21, 49, 59, 63, 64, 73, 92, 93, 130, 132, 135, 155, 161, 168, 171] > 1000 m 3 3 [161, 168, 171] Variable/not fixed 4 4 [32, 135, 151, 164] Retirement village 3 3 [43, 72, 153] Unknown (not reported) 3 3 [53, 62, 128] Perceived 10–20 min walk from home 24 24 [20, 22, 23, 44–46, 48, 60, 71, 72, 75, 79, 100, 121, 122, 124, 126, 131, 132, 141, 143, 150, 159, 169] Other participant delineation 22 22 [24, 25, 32, 50, 61, 62, 67, 69, 76, 78, 87, 120, 125, 129, 133, 140, 147, 151, 152, 154, 165, 167] Retirement village 2 2 [71, 153] Unknown (not reported) 6 6 [63, 77, 92, 134, 144, 156] Physical activity outcome by type of measurement and its operationalisationa Total PA (all PA outcomes from all articles) Continuous outcome 59 55.1 [19, 21, 43–48, 54, 59, 60, 62, 63, 65–67, 69, 71, 73, 74, 79, 80, 87, 92, 93, 118, 119, 121, 123–125, 127, 129, 131, 132, 134–138, 140–144, 147–151, 155, 160, 161, 164–166, 170, 173] Categorical outcome 48 44.9 [20, 22–25, 32, 49, 50, 53, 54, 61, 64, 65, 68, 70, 72, 75–78, 80, 93, 100, 120, 122, 124, 126, 128, 130, 133, 135, 139, 144, 146, 152–154, 156–159, 162, 163, 167–169, 171, 172] Objective PA 28 27.2 Continuous outcome 23 [19, 43–48, 59, 73, 74, 79, 118, 127, 129, 132, 135, 141, 148, 150, 164–166, 170] Categorical outcome 5 [25, 50, 61, 72, 135, 153] Self-reported PA 75 73.8 Continuous outcome 37 [19, 21, 54, 60, 62, 63, 65–67, 69, 71, 73, 80, 87, 92, 93, 119, 121, 123–125, 131, 134, 136–138, 140–144, 147, 149, 151, 155, 160, 161, 173] Categorical outcome 45 [20, 22–25, 32, 49, 53, 54, 64, 65, 68, 70, 72, 75–78, 80, 93, 100, 120, 122, 124, 126, 128, 130, 133, 139, 144, 146, 152–154, 156–159, 162, 163, 167–169, 171, 172] Total PA (as detailed in article) 31 27.2 Objective PA 8 8 Continuous outcome 8 8 [48, 79, 129, 135, 148, 150, 164, 166] Categorical outcome 0 0 Self-reported PA 23 23 Continuous outcome 19 19 [19, 60, 65–67, 69, 73, 87, 119, 121, 125, 136, 137, 141, 143, 147, 151, 160, 161] Categorical outcome 4 4 [65, 70, 126, 157] Total walking (as detailed in article) 55 48.2 Objective PA 9 9 Continuous outcome 8 8 [19, 118, 127, 129, 132, 135, 141, 165] Categorical outcome 2 2 [61, 135] Self-reported PA 47 47 Continuous outcome 19 19 [21, 62, 63, 65, 67, 71, 80, 92, 93, 123, 124, 131, 134, 138, 142, 144, 149, 155, 173] C t i l t 32 32 [20 22 24 32 49 53 64 65 70 72 75 76 80 93 100 122 124 Table 1 Characteristics of selected articles (N = 100) (Continued) Total PA (all PA outcomes from all articles) Continuous outcome 59 55.1 [19, 21, 43–48, 54, 59, 60, 62, 63, 65–67, 69, 71, 73, 74, 79, 80, 87, 92, 93, 118, 119, 121, 123–125, 127, 129, 131, 132, 134–138, 140–144, 147–151, 155, 160, 161, 164–166, 170, 173] Categorical outcome 48 44.9 [20, 22–25, 32, 49, 50, 53, 54, 61, 64, 65, 68, 70, 72, 75–78, 80, 93, 100, 120, 122, 124, 126, 128, 130, 133, 135, 139, 144, 146, 152–154, 156–159, 162, 163, 167–169, 171, 172] Objective PA 28 27.2 Continuous outcome 23 [19, 43–48, 59, 73, 74, 79, 118, 127, 129, 132, 135, 141, 148, 150, 164–166, 170] Categorical outcome 5 [25, 50, 61, 72, 135, 153] Self-reported PA 75 73.8 Continuous outcome 37 [19, 21, 54, 60, 62, 63, 65–67, 69, 71, 73, 80, 87, 92, 93, 119, 121, 123–125, 131, 134, 136–138, 140–144, 147, 149, 151, 155, 160, 161, 173] Categorical outcome 45 [20, 22–25, 32, 49, 53, 54, 64, 65, 68, 70, 72, 75–78, 80, 93, 100, 120, 122, 124, 126, 128, 130, 133, 139, 144, 146, 152–154, 156–159, 162, 163, 167–169, 171, 172] Total PA (as detailed in article) 31 27.2 Objective PA 8 8 Continuous outcome 8 8 [48, 79, 129, 135, 148, 150, 164, 166] Categorical outcome 0 0 Self-reported PA 23 23 Continuous outcome 19 19 [19, 60, 65–67, 69, 73, 87, 119, 121, 125, 136, 137, 141, 143, 147, 151, 160, 161] Categorical outcome 4 4 [65, 70, 126, 157] Total walking (as detailed in article) 55 48.2 Objective PA 9 9 Continuous outcome 8 8 [19, 118, 127, 129, 132, 135, 141, 165] Categorical outcome 2 2 [61, 135] Self-reported PA 47 47 Continuous outcome 19 19 [21, 62, 63, 65, 67, 71, 80, 92, 93, 123, 124, 131, 134, 138, 142, 144, 149, 155, 173] Categorical outcome 32 32 [20, 22–24, 32, 49, 53, 64, 65, 70, 72, 75, 76, 80, 93, 100, 122, 124, 130, 144, 146, 153, 154, 158, 159, 162, 163, 167–169, 171, 172] Total PA (as detailed in article) Objective PA Continuous outcome Categorical outcome Self-reported PA Continuous outcome Categorical outcome Total walking (as detailed in article) Objective PA Continuous outcome Categorical outcome Self-reported PA Continuous outcome Categorical outcome Total PA (as detailed in article) Objective PA Continuous outcome Categorical outcome Self-reported PA Continuous outcome Categorical outcome Total walking (as detailed in article) Objective PA Continuous outcome Categorical outcome Self-reported PA Continuous outcome Categorical outcome Barnett et al. Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 8 of 24 Table 1 Characteristics of selected articles (N = 100) (Continued) Total MVPAc (as detailed in article) 28 24.6 Objective PA 15 15 Continuous outcome 11 11 [43–48, 73, 74, 129, 166, 170] Categorical outcome 4 4 [25, 50, 72, 153] Self-reported PA 14 14 Continuous outcome 3 3 [54, 140, 173] Categorical outcome 12 12 [25, 54, 68, 77, 78, 120, 128, 130, 133, 139, 152, 156] Environmental attribute by type of measurementa Objective environment 49 48 Perceived environment 53 52 Walkability 13 13 Objective 11 11 [46, 48, 72–74, 146, 153, 155, 167, 171, 172] Perceived 2 2 [126, 141] Residential density/urbanisationd 35 35 Objective 21 21 [19, 21, 47, 54, 65, 70, 73, 80, 92, 118, 119, 123, 128, 134, 139, 148, 158–160, 168, 173] Perceived 15 15 [20, 22, 25, 48, 76, 132, 140, 145, 147, 150, 152, 154, 159, 162, 169] Street connectivityd 24 24 Objective 10 10 [21, 24, 43, 53, 73, 93, 159, 161, 166, 168] Perceived 16 16 [20, 23, 25, 48, 53, 71, 76, 79, 127, 132, 140, 142, 150, 154, 159, 170] Access to/availability of destinations & servicesa 65 65 Objective 29 29 Perceived 45 45 Overall access to destinations & servicesd 24 24 Objective 6 6 [21, 53, 62, 93, 135, 168] Perceived 21 21 [20, 23, 25, 48, 50, 53, 62, 67, 71, 76, 87, 100, 126, 127, 131, 132, 140, 141, 150, 154, 159] Land-use mix—destination diversityd 16 16 Objective 8 8 [49, 72, 80, 123, 135, 153, 159, 161] Perceived 9 9 [25, 48, 67, 76, 125, 132, 150, 159, 170] Shops/commercial destinationsd 26 26 Objective 17 17 [32, 43, 47, 49, 53, 72, 73, 80, 93, 135, 148, 158, 159, 161, 166, 168] Perceived 10 10 [22, 23, 32, 75, 129, 141, 142, 156, 165, 169] Food outlets 11 11 Objective 8 8 [43, 47, 49, 53, 73, 135, 166, 168] Perceived 3 3 [23, 141, 142] Government/finance services 8 8 Objective 7 7 [43, 47, 49, 53, 73, 135, 158] Perceived 1 1 [23] Education 7 7 Objective 6 6 [21, 132, 135, 158, 161, 168] Perceived 1 1 [141] Health & aged care 10 10 Objective 8 8 [43, 49, 53, 72, 135, 153, 158, 166] Perceived 2 2 [23 156] Table 1 Characteristics of selected articles (N = 100) (Continued) Barnett et al. 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Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 10 of 24 Table 1 Characteristics of selected articles (N = 100) (Continued) Pollution (air) 3 3 Objective 1 1 [124] Perceived 2 2 [23, 156] Safety 46 46 Objective 7 7 Perceived 40 40 Traffic/pedestrian safety 28 28 Objective 4 4 [47, 72, 93, 124] Perceived 24 24 [22–25, 45, 48, 53, 61, 69, 71, 75, 76, 79, 92, 100, 125, 132, 136, 142, 150, 156, 159, 169, 170] Crime/personal safetyd 41 41 Objective 5 5 [43, 93, 124, 149, 157] Perceived 37 37 [22, 23, 25, 45, 48, 50, 53, 61, 63, 66, 68, 69, 71, 75, 76, 79, 100, 121, 122, 125, 132–134, 137, 140, 142–145, 150, 156, 157, 159, 163, 167, 169, 170] Moderator of environmental attribute-PA associationa 39 39 Individual factors 24 24 [20–23, 44, 45, 48–50, 59, 69, 70, 73, 77, 79, 100, 122, 125, 131, 144, 152, 155, 157, 159] Socio-demographics 16 16 [20–23, 45, 49, 50, 69, 70, 73, 122, 125, 144, 152, 155, 157] Health status/functionality 7 7 [59, 73, 77, 100, 131, 155, 159] Psychosocial factors 3 3 [44, 79, 100] Driving status/car ownership 2 2 [48, 73] Duration of residency 1 1 [21] Environmental factors 18 18 [19, 21, 25, 45, 46, 64, 66, 74–76, 92, 100, 141, 146, 155, 160, 162, 168] Socioeconomic status/area-level income 5 5 [25, 45, 64, 74, 146] Residential density/urbanisation 6 6 [19, 75, 76, 160, 162, 168] Infrastructure and streetscape aspects (e.g., walkability) 3 3 [46, 92, 100] Intervention 2 2 [66, 100] Geographical scale (e.g., 400 m buffer) 6 6 [21, 64, 73, 93, 135, 161] Tested, but unknown 1 1 [24] None 61 61 [32, 43, 47, 53, 54, 60–63, 65, 67, 68, 71, 72, 78, 80, 87, 93, 118–124, 126–130, 132–143, 145, 147–151, 153, 154, 156, 158, 163–167, 169–173] Notes: aMultiple options allowed in single articles b l h d h l b d h d ff l ([ ]) h l b f l dd d Table 1 Characteristics of selected articles (N = 100) (Continued) the instrument used [52], or outcome measures well- established in the field [yes = 1, no = 0]; (5) adjustment for key socio-demographic covariates (at least age, sex, and education considered) [yes = 1, no = 0]; (6) adjust- ment for self-selection [yes = 1, no = 0]; (7) appropriate analytical approach – adjustment for clustering (if needed) [yes = 1/3, no = 0]; (8) appropriate analytical approach – accounting for distributional assumptions [yes = 1/3, no = 0]; (9) appropriate analytical approach – analyses conducted and presented correctly [yes = 1/ 3, no = 0]; and (10) did not inappropriately categorise continuous environmental exposures [yes = 1, no = 0]. Article quality and sample size assessment International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 9 of 24 Table 1 Characteristics of selected articles (N = 100) (Continued) Religious 5 5 Objective 3 3 [43, 135, 161] Perceived 2 2 [23, 141] Public transport 18 18 Objective 8 8 [24, 47, 72, 73, 80, 93, 158, 166] Perceived 10 10 [22, 23, 48, 50, 62, 68, 100, 141, 156, 169] Parks/public open spaced 30 30 Objective 17 17 [21, 24, 32, 43, 44, 48, 53, 64, 73, 92, 93, 124, 132, 135, 151, 158, 161] Perceived 15 15 [23, 32, 46, 66, 68, 69, 75, 100, 125, 141, 142, 144, 151, 156, 163] Recreational facilities 25 25 Objective 10 10 [43, 47, 64, 72, 73, 124, 132, 135, 153, 168] Perceived 15 15 [22, 23, 46, 60, 63, 68, 77, 78, 92, 120, 125, 142, 156, 169, 170] Social recreational facilities 13 13 Objective 6 6 [43, 49, 73, 135, 138, 166] Perceived 7 7 [23, 50, 71, 141, 142, 153, 156] Other destinations 1 1 Objective 1 1 [43] Perceived 0 0 Infrastructure & streetscapea 43 43 Objective 12 12 Perceived 34 34 Overall cycle/walk-friendly infrastructure 8 8 Objective 0 0 Perceived 9 9 [25, 48, 66, 76, 127, 132, 150, 154, 159] Walk-friendly infrastructure 21 21 Objective 7 7 [43, 47, 80, 93, 124, 132, 161] Perceived 14 14 [22, 44, 53, 61, 62, 68, 69, 71, 100, 121, 125, 142, 169, 170] Cycle-friendly infrastructure 4 4 Objective 1 1 [47] Perceived 3 3 [22, 125, 169] No physical environmental barriers (e.g., hills) 16 16 Objective 8 8 [21, 24, 32, 43, 47, 72, 124, 164] Perceived 9 9 [23, 32, 66, 71, 100, 125, 142, 169, 170] Pavement/footpath quality 8 8 Objective 2 2 [47, 124] Perceived 6 6 [23, 24, 66, 121, 142, 163] Street lighting 6 6 Objective 1 1 [124] Perceived 5 5 [23, 53, 69, 75, 122] Greenery & aesthetically pleasing sceneryd 33 33 Objective 6 6 [32, 43, 47, 80, 124, 161] Perceived 28 28 [20, 22, 23, 25, 32, 44, 48, 53, 61, 71, 75, 76, 79, 100, 122, 125, 127, 132, 137, 142, 147, 150, 154, 156, 159, 163, 169, 170] Table 1 Characteristics of selected articles (N = 100) (Continued) Table 1 Characteristics of selected articles (N Religious Objective Perceived Public transport Objective Perceived Parks/public open spaced Objective Perceived Recreational facilities Objective Perceived Social recreational facilities Objective Perceived Other destinations Objective Perceived Infrastructure & streetscapea Objective Perceived Overall cycle/walk-friendly infrastructure Objective Perceived Walk-friendly infrastructure Objective Perceived Cycle-friendly infrastructure Objective Perceived No physical environmental barriers (e.g., hills) Objective Perceived Pavement/footpath quality Objective Perceived Street lighting Objective Perceived Greenery & aesthetically pleasing sceneryd Objective Perceived Barnett et al. Data synthesis h Each separate positive, negative, and nil association be- tween a built environmental attribute and PA outcome (either total PA, total MVPA, or total walking) was tallied [27] and, where appropriate, multiplied by a buffer-size or moderator-related fractional weight (see section above on ‘Quantification of buffer effects and/or moderators’). All of these findings were then included into a “Total PA” outcome, which was then stratified by PA measurement type (objective or self-report). “Total walking” was considered separately and was based on findings from self-report walking only, as the majority of associations between built environmental attributes and objectively assessed walking was insufficiently examined (i.e., <5 findings) (Additional file 3). The findings related to both “Total PA” and “Total walking” were then strati- fied by environmental measurement type (objective or perceived). All examined moderators of environment-PA associa- tions were counted and summarised by the category of moderator tested. This included multiple factor (i.e., higher-order) moderating effects, tested formally (e.g., [59]) or not (e.g., [20]). Reporting higher-order moderat- ing effects by moderator category resulted in findings being counted multiple times, i.e., once for each moder- ator category. For example, Chen and colleagues re- ported sex by employment status interactions, so the findings were reported twice, once under sex and once under employment status as moderators [20]. For this reason, the total number of moderating effects reported in this review is greater than the number of moderating effects reported in the articles. Due to the large range of different environmental and PA measures reported in the selected articles, it was not possible to conduct a ‘traditional’ meta-analysis involving the estimation of pooled effect sizes and their 95% confi- dence intervals. Hence, to quantitatively synthesise the findings, a meta-analytic approach was applied to define conservative estimates of p-values for each examined combination of environmental attribute and PA outcome [27]. These p-values represented the probability of ob- serving a certain distribution of findings (e.g., 4 positive, 2 nil, and 0 negative associations) under a null hypoth- esis of no association. These computations were under- taken accounting for: (1) sample size and quality scores of included articles (see previous section); (2) sample size score only; (3) article quality score only; and (4) nei- ther – with 2–4 providing a sensitivity analysis on how each of these influenced meta-analytical findings. Data synthesis h Asso- ciations of specific built environmental attributes with PA outcomes were examined by type of PA and environ- mental measurement (objective and perceived) only when ≥3 articles provided data on each type of measure, consistent with recommendations for meta-analyses from Cochrane’s database of systematic reviews [55]. Environment-PA outcome associations totalling <5 Article quality and sample size assessment Higher scores reflect higher quality: 0–3.5 (low quality), 3.6–5.9 (moderate quality), and ≥6 (high quality). Apart from providing descriptive information on article qual- ity, these scores were also used as weights in the meta- analyses described below so that higher quality articles had a greater contribution to the synthesis of findings. Page 11 of 24 Page 11 of 24 Page 11 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 separate findings were deemed as insufficiently studied to reach a robust conclusion [56]. To account for sample size in the meta-analyses, the following weights were assigned to findings: 0.25 (≤100 participants), 0.50 (101–300 participants), 1.00 (301–500 participants), 1.25 (501–1000 participants), 1.50 (1001– 2500 participants), and 1.75 (>2500 participants). In two instances, different sample weights were applied to find- ings derived from the same study because of differences in sample size between objectively assessed and per- ceived environmental exposures [53] and between data collection periods [54]. A thorough rationale for these weights can be found elsewhere [27]. To perform the meta-analyses, we first assigned a z- value to each separate built environmental attribute and PA finding, specifically: 1.96 for a positive; −1.96 for a negative; and 0 for a nil finding [27]. We then calculated a summary two-tailed p-value using Rosenthal’s ap- proach [57], reporting a summary weighted z-value and its associated two-tailed probability value as detailed in Cerin et al.’s recent systematic review and meta-analysis of built environmental correlates of active transport [27]. For the sensitivity analysis aforementioned, we also calculated non-weighted z values and their associated two-tailed p-values. All computations were performed in a Microsoft Excel spreadsheet using algorithms devel- oped by the authors. P-values were interpreted as follows: .05 to .01 – evidence of an association; <.01 – strong evidence of an association; and <.001 – very strong evidence of an association [58]. Results Of 19,005 potential articles, we fully read 530 and in- cluded 100 in our analysis – two of which were PhD the- ses [60, 61] (Fig. 1). From the 100 articles, 1553 individual associations were extracted. Characteristics of included articles Details of article characteristics can be found in Table 1. Cross-sectional studies accounted for 94% of articles, with five papers reporting longitudinal [61–65] and one quasi-experimental [66] findings. Almost half of all stud- ies (46%) were based in North America, followed by Europe (22%), Asia (16%), Oceania (13%), two studies from South America [23, 24], and one pilot study from South Africa [25]. Regarding countries, the USA (36%), Australia (13%), the UK (10%), and Canada (10%) con- ducted the most research. Only 3% of articles specifically studied rural participants [67–69], about half researched those from urban settings (56%), and 32% from a mix- ture of both. Page 12 of 24 Page 12 of 24 Page 12 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Sample sizes ranged from 44 [25] to 69,253 [70], with over a third of samples (36%) regarded as small (i.e., ≤300 participants). Recruitment of neighbour- hoods was stratified by key built environmental attributes in 57% of articles; urbanisation being the most prevalent (22%), followed by area-level socio- economic status (SES) and walkability combined (12%), and area-level SES and urbanisation combined (10%). Neighbourhoods were most frequently defined objectively by administrative and census areas. When buffers were applied to define neighbourhoods, a 400- 500 m radius was the most frequently used buffer size. A 10–20 min’ walk from home was the most com- monly reported perceived neighbourhood definition. availability of parks/public open space and recreational facilities (both 15%). The most commonly evaluated objectively assessed environmental attributes were urbanisation/residential density (21%), and access to/ availability of shops/commercial destinations and of parks/public open space (17% each). Thirty-nine percent of articles investigated moderating effects on associations between built environmental attributes and total PA. At the individual level, socio- demographics were the most frequently examined moderator, and at the environmental level, residential density/urbanisation was the most frequently reported moderator. (Note: Full details of article characteristics can be found in Additional file 1). Overall, 66% of articles used a validated or established PA measure. Almost three quarters of articles reported findings based on older adults’ self-reported PA (74%). The most commonly reported PA outcome was total walking (55%). Approximately 55% of PA outcomes were operatio- nalised as continuous measures (e.g., minutes/day) and 56% of self-report PA outcomes were measures of total walking. Article quality Only 9% of articles were deemed of high quality, 55% of moderate quality, and the remaining 36% of low quality. Key socio-demographic covariates (i.e., at least age, sex, and education) were adjusted for in 66% of articles; much fewer analyses adjusted for self-selection (12%) (Table 2) [62, 69, 71–80]. Approximately three quarters of the included articles conducted appropriate analyses (76%). (Note: Full details of article quality can be found in Additional file 3). Overall, the most researched attributes were crime- related personal safety (41%), residential density/urban- isation (35%), greenery and aesthetically pleasing scenery (33%), access to/availability of parks/public open space (30%), pedestrian-related traffic safety (28%), and access to/availability of shops/commercial destinations (26%). Similarly, the most investigated per- ceived attributes were crime-related personal safety (38%), greenery and aesthetically pleasing scenery (28%), pedestrian-related traffic safety (25%), access to/ Characteristics of included articles The most commonly reported objective PA out- come was total MVPA (47% of objective PA articles). Built environmental correlates of older adults’ PA Total PA There was very strong evidence that neighbourhood walkability (p < .001), overall access to destinations and services (p < .001) and recreational facilities (p < .001), and crime-related personal safety (p < .001) were posi- tively associated with older adults’ total PA (Table 3). Table 2 Summary of article quality assessment (N = 100) Quality-assessment item [score] % 1. Study design [cross-sectional: 0; longitudinal: 1; quasi-experimental: 2] cross-sectional 94 longitudinal 5 quasi-experimental 1 2. Study areas or participant recruitment stratified by key environmental attributes [1] 56 3. Response rate ≥60% or sample representative of the population [1] 32 4. Physical activity measures (outcomes) valid, or well-established in the field [1] 66 5. Analyses adjusted for key socio-demographic covariates (at least age, sex, and education considered) [1] 66 6. Analysis adjusted for self-selection [1] 12 7. Analytical approach – adjustment for clustering (if needed) [1/3] 58 8. Analytical approach – accounting for distributional assumptions [1/3] 84 9. Analytical approach – analyses conducted and presented correctly [1/3] 76 10. Did not, inappropriately, categorise continuous environmental exposure/s [1] 74 Total quality score [theoretical range: 0–9]; mean ± SD 3.9 ± 1.3 Notes: SD Standard deviation Table 2 Summary of article quality assessment (N = 100) Quality-assessment item [score] % 1. Study design [cross-sectional: 0; longitudinal: 1; quasi-experimental: 2] cross-sectional 94 longitudinal 5 quasi-experimental 1 2. Study areas or participant recruitment stratified by key environmental attributes [1] 56 3. Response rate ≥60% or sample representative of the population [1] 32 4. Physical activity measures (outcomes) valid, or well-established in the field [1] 66 5. Analyses adjusted for key socio-demographic covariates (at least age, sex, and education considered) [1] 66 6. Analysis adjusted for self-selection [1] 12 7. Analytical approach – adjustment for clustering (if needed) [1/3] 58 8. Analytical approach – accounting for distributional assumptions [1/3] 84 9. Analytical approach – analyses conducted and presented correctly [1/3] 76 10. Did not, inappropriately, categorise continuous environmental exposure/s [1] 74 Total quality score [theoretical range: 0–9]; mean ± SD 3.9 ± 1.3 Notes: SD Standard deviation Table 2 Summary of article quality assessment (N = 100) Quality-assessment item [score] Table 2 Summary of article quality assessment (N = 100) Quality-assessment item [score] Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 13 of 2 Barnett et al. Table 2 Summary of article quality assessment (N = 100) Quality-assessment item [score] International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 13 of 24 Table 3 Associations of environmental attributes/correlates with older adults’ physical activity by physical activity outcomes Environmental attribute Total PA1 Total walking only P Ø N pa Da P Ø N pa Da Walkability 12.33 6.67 0 <.001 P 4.37 3.63 0 .001 P Residential density/urbanisation 11.53 33.50 12.97 .394 Ø 8 14.50 3.50 .036 P Street connectivity 8.80 26.06 2.14 .094 Ø 5.80 13.20 2 .185 Ø Access to/availability of destinations & services Overall access to destinations & services 12.55 38.15 0.50 <.001 P 6.93 25.57 0.50 .009 P Land-use mix—destination diversity 5.68 19.32 2 .148 Ø 1 8 2 .439 Ø Shops/commercial 9.96 57.04 0 .006 P 8.58 23.42 0 .001 P Food outlets 0.72 21.28 1 .932 Ø 0.72 6.28 1 .873 Ø Government/finance services 0.33 11.67 0 .834 Ø 0 6 0 1.00 Ø Education 0.31 11.69 0 .765 Ø 0.14 2.85 0 .826 Ø Health & aged care 4.61 26.39 1 .275 Ø 3.61 7.39 1 .191 Ø Religious 0 8 0 1.00 Ø 0 1 0 1.00 Ø Public transport 7.50 25.50 1 .016 P 5.50 11.50 1 .011 P Parks/public open space 11.29 47.54 0.17 .002 P 6.05 23.78 0.17 .014 P Recreational facilities 13.34 39.66 0 <.001 P 3.07 15.93 0 .135 Ø Social recreational facilities 4.15 25.95 0 .105 Ø 1.50 10.50 0 .413 Ø Other destinations 0 3 0 1.00 Ø - - - - - Infrastructure & streetscape Overall access to cycle/walk-friendly infrastructure 1 9 0 .612 Ø 0 3 0 1.00 Ø Walk-friendly infrastructure 9 21.49 1.51 .009 P 5 15 0 .042 P Cycle-friendly infrastructure 0 5 0 1.00 Ø 0 3 0 1.00 Ø No physical barriers (e.g., hills) 5 20.40 2.60 .208 Ø 2 14.40 0.60 .384 Ø Pavement/footpath quality 3 6 1 .155 Ø 2 5 0 .169 Ø Street lighting 3 6 0 .059 Ø 3 4 0 .042 P Greenery & aesthetically pleasing scenery 13.01 45.49 0.5 .004 P 10.51 19.49 0 .002 P Pollution (air) 0 5 0 1.00 Ø 0 4 0 1.00 Ø Safety Traffic/pedestrian safety 7 47 3 .463 Ø 5 25 3 .705 Ø Crime/personal safety 20.52 50.48 4 <.001 P 10.49 28.01 2.50 .027 P Notes: 1Objective and self-report total PA (including total walking) combined. Table 2 Summary of article quality assessment (N = 100) Quality-assessment item [score] P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). In bold font: statistically significant evidence of a directional association that has been sufficiently studied (i.e., ≥5 findings reported on specific combinations of environmental exposure and PA variables) Notes: 1Objective and self-report total PA (including total walking) combined. P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). In bold font: statistically significant evidence of a directional association that has been sufficiently studied (i.e., ≥5 findings reported on specific combinations of environmental exposure and PA variables) Notes: 1Objective and self-report total PA (including total walking) combined. P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). In bold font: statistically significant evidence of a directional association that has been sufficiently studied (i.e., ≥5 findings reported on specific combinations of environmental exposure and PA variables) Moreover, there was strong evidence of positive associations between total PA and access to both parks/public open space (p = .002) and shops/commercial destinations (p = .006), the presence of greenery and aesthetically pleas- ing scenery (p = .004), and walk-friendly infrastructure (p = .009). In addition, there was evidence that access to public transport was positively associated with total PA (p = .016). No other significant associations were found in relation to built environmental attributes and total PA. walkability (p = .001), access to/availability of shops/ commercial destinations (p = .001) and overall desti- nations and services (p = .009) and more greenery and aesthetically pleasing scenery (p = .002) (Table 3). We also found evidence that access to/availability of neighbourhood public transport (p = .011) and parks/public open space (p = .014), crime/personal safety (p = .027), residential density (p = .036), walk-friendly infrastructure (p = .042), and street lighting (p = .042) were positively associated with total walking. No significant associations were found for the remaining 15 built environmental attributes (Table 3). Total PA For nine out of 18 environmental exposures, associations with total PA differed by environmental measurement type (Table 5). For five environmental attributes, positive associations with total PA were found with perceived but not objectively assessed measures. Perceptions of crime-related personal safety (p < .001), access to/avail- ability of recreational facilities (p < .001), access to/avail- ability of parks/public open space (p = .003), greenery and aesthetically pleasing scenery (p = .003), and destin- ation diversity (land-use mix) (p = .002) were all posi- tively associated with total PA. Built environmental correlates of older adults’ PA by measurement method friendly infrastructure (p = .031) and destination diversity (land use mix) (p = .019). Built environmental correlates of older adults’ PA by measurement method friendly infrastructure (p = .031) and destination diversity (land use mix) (p = .019). friendly infrastructure (p = .031) and destination diversity (land use mix) (p = .019). PA measurement type (objective and self-report) Irrespective of the PA measurement type used, neigh- bourhood walkability (both p < .001) and overall ac- cess to destinations and services (both p < .01) were positively associated with older adults’ total PA (Table 4). Seven other positive associations between attri- butes of the built environment and PA were PA- measurement dependent. Five positive associations were specific to self-reported total PA, namely: green- ery and aesthetically pleasing scenery (p = .001), ac- cess to shops/commercial destinations (p = .002), parks/public open space (p = .002), recreational facil- ities (p = .002) and public transport (p = .006). Two remaining positive associations were in relation to objectively assessed total PA only, specifically: walk- Total walking We found strong evidence for positive associations be- tween older adults’ total walking and neighbourhood Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 14 of 24 Built environmental correlates of older adults’ PA by measurement method PA measurement type (objective and self-report) Irrespective of the PA measurement type used, neigh- bourhood walkability (both p < .001) and overall ac- cess to destinations and services (both p < .01) were positively associated with older adults’ total PA (Table 4). Seven other positive associations between attri- butes of the built environment and PA were PA- measurement dependent. Five positive associations were specific to self-reported total PA, namely: green- ery and aesthetically pleasing scenery (p = .001), ac- cess to shops/commercial destinations (p = .002), parks/public open space (p = .002), recreational facil- ities (p = .002) and public transport (p = .006). Two remaining positive associations were in relation to objectively assessed total PA only, specifically: walk- frie dive Env and Tota For with typ asso but crim abil abil and atio tive Table 4 Associations of environmental attributes/correlates with older a method (objective and self-report) Environmental attribute Total PA Objective P Ø N Walkability 5.96 2.04 0 Residential density/urbanisation 1 7 0 Street connectivity 3 7.86 0.14 Access to/availability of destinations & services Overall access to destinations & services 3.89 8.31 0 Land-use mix—destination diversity 3.17 8.83 0 Shops/commercial 1.38 26.62 0 Food outlets 0 13 0 Government/finance services 0.34 5.66 0 Education 0.17 6.83 0 Health & aged care 1 18 0 Religious 0 5 0 Public transport 1 12 0 Parks/public open space 1.75 14.25 0 Recreational facilities 4.29 16.71 0 Social recreational facilities 2.65 12.35 0 Infrastructure & streetscape Overall access to cycle/walk-friendly infrastructure 1 5 0 Walk-friendly infrastructure 3 3 0 Total PA Objectively assessed Table 4 Associations of environmental attributes/correlates with older adults’ physical activity by physical activity measurement method (objective and self-report) Environmental attribute Total PA Objective Self-report P Ø N pa Da P Ø N pa Da Walkability 5.96 2.04 0 <.001 P 6.37 4.63 0 <.001 P Residential density/urbanisation 1 7 0 .377 Ø 10.53 26.50 12.97 .240 Ø Street connectivity 3 7.86 0.14 .262 Ø 5.71 18.20 2 .215 Ø Access to/availability of destinations & services Overall access to destinations & services 3.89 8.31 0 .005 P 8.66 29.84 0.50 .004 P Land-use mix—destination diversity 3.17 8.83 0 .019 P 2.51 12.49 2 .884 Ø Shops/commercial 1.38 26.62 0 .507 Ø 8.58 29.42 0 .002 P Food outlets 0 13 0 1.00 Ø 0.72 8.28 1 .884 Ø Government/finance services 0.34 5.66 0 .377 Ø 0 6 0 1.00 Ø Education 0.17 6.83 0 .818 Ø 0.14 4.86 0 .845 Ø Health & aged care 1 18 0 .612 Ø 3.61 8.39 1 .206 Ø Religious 0 5 0 1.00 Ø 0 3 0 1.00 Ø Public transport 1 12 0 .520 Ø 6.50 13.50 1 .006 P Parks/public open space 1.75 14.25 0 .296 Ø 9.54 33.29 0.17 .002 P Recreational facilities 4.29 16.71 0 .056 Ø 9.05 22.95 0 .002 P Social recreational facilities 2.65 12.35 0 .118 Ø 1.50 13.50 0 .432 Ø Infrastructure & streetscape Overall access to cycle/walk-friendly infrastructure 1 5 0 .529 Ø 0 4 0 1.00 Ø Walk-friendly infrastructure 3 3 0 .031 P 6 18.49 1.51 .059 Ø No physical barriers (e.g., hills) 3 5 1 .135 Ø 2 15.40 1.60 .631 Ø Greenery & aesthetically pleasing scenery 1.50 15 0.50 .741 Ø 11.51 30.49 0 .001 P Safety Traffic/pedestrian safety 2 14 0 .408 Ø 5 33 3 .737 Ø Crime/personal safety 3 8 0 .063 Ø 17.52 42.48 4 .001 P Notes: P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). Total PA In bold font: statistically significant evidence of a directional association that has been sufficiently studied (i.e., ≥5 findings reported on specific combinations of environmental exposure and PA variables) of environmental attributes/correlates with older adults’ physical activity by physical activity measurement d self-report) Notes: P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). In bold font: statistically significant evidence of a directional association that has been sufficiently studied (i.e., ≥5 findings reported on specific combinations of environmental exposure and PA variables) Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 15 of 24 Barnett et al. Total PA In bold font: evidence of a difference between environmental measures of an association between a sufficiently studied exposure and PA variable (i.e., ≥3 articles’ reported findings on specific combinations of environmental exposure and physical activity variables) Notes: 1Objective and self-report total PA (including total walking) combined. P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). In bold font: evidence of a difference between environmental measures of an association between a sufficiently studied exposure and PA variable (i.e., ≥3 articles’ reported findings on specific combinations of environmental exposure and physical activity variables) (fully-adjusted: p = .036; unadjusted: p = .055) and walk- friendly infrastructure (fully-adjusted: p = .042; article quality-adjusted: p = .057). In addition, some built envir- onmental attributes were significant when unadjusted, but not when taking into account sample size and/or article quality. These were street lighting (total PA and self- report total PA), street connectivity (total walking), and crime/personal safety (objective total PA). access to/availability of shops/commercial destinations (p = .006) and public transport (p = .004), presence of walk-friendly infrastructure (p = .028), and absence of physical environmental barriers (e.g., hills) (p = .048) were all positively associated with total PA, whereas as- sociations with these attributes were non-significant when using perceived measures. Discussion In the last decade, world bodies have been advocating the importance of healthy ageing and the enabling role played by PA and built environments (e.g., [81]). As a re- sult, the number of published articles on the associations between built environmental attributes and older adults’ PA increased over three times since the last systematic review in 2011 [15]. Moreover, there was a greater per- centage of articles from outside of North America, with Moderators of environment-PA associations There were five positive PA associations with objectively measured environment variables only and four others with measures based on perceptions only. Evidence of a positive association with total walking was found for perceived mea- sures of neighbourhood greenery and aesthetically pleasing scenery (p < .001), crime/personal safety (p = .012), traffic-safety (p = .043), and access to/availability of recreational facilities (p = .050). Regarding objectively measured environmental attributes, access to/availabil- ity of shops/commercial destinations (p < .001), public transport (p < .001), overall destinations and services (p = .004), parks/public open space (p = .035), and residential density/urbanisation (p = .032) were all positively related to total walking. Sixteen moderators of built environmental attribute-PA as- sociations were examined in 39 articles (Additional file 5). The most frequently examined moderators by number of articles were sex (7 articles), health status/functionality (7 articles), residential density/urbanisation (6 articles), and SES/area-level income (5 articles). Buffer size (121 findings) and sex (83 findings) were the most frequently examined by estimating regression interaction terms. The direction of effects for all significant interaction terms was inconsist- ent (Additional file 5). Total PA International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 16 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 16 of 24 Table 5 Associations of environmental attributes/correlates with older adults’ physical activity by physical activity and environmental measures (objective and perceived) (Continued) Objective 5 9 0 .028 P 3 7 0 .103 Ø Perceived 4 12.49 1.51 .137 Ø 2 8 0 .222 Ø No physical barriers (e.g., hills) 5 20.40 2.61 .208 Ø 2 14.40 0.61 .384 Ø Objective 5 8.40 1.60 .048 P 2 4.40 0.60 .227 Ø Perceived 0 12 1 .629 Ø 0 10 0 1.00 Ø Greenery & aesthetically pleasing scenery 13.01 45.49 0.5 .004 P 10.51 19.49 0 .002 P Objective 3 18 0 .252 Ø 3 9 0 .199 Ø Perceived 10.01 27.49 0.50 .003 P 7.51 10.49 0 <.001 P Safety Traffic/pedestrian safety 7 47 3 .463 Ø 5 25 3 .705 Ø Objective 1 13 3 .407 Ø 0 11 3 .150 Ø Perceived 6 34 0 .126 Ø 5 14 0 .043 P Crime/personal safety 20.63 50.58 3.99 <.001 P 10.49 28.01 2.5 .027 P Objective 4 5.50 2.50 .510 Ø 3 5 2 .627 Ø Perceived 16.52 44.98 1.50 <.001 P 7.49 23.01 0.50 .012 P Notes: 1Objective and self-report total PA (including total walking) combined. P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). In bold font: evidence of a difference between environmental measures of an association between a sufficiently studied exposure and PA variable (i.e., ≥3 articles’ reported findings on specific combinations of environmental exposure and physical activity variables) Table 5 Associations of environmental attributes/correlates with older adults’ physical activity by physical activity and environmental measures (objective and perceived) (Continued) Notes: 1Objective and self-report total PA (including total walking) combined. P = positive association; Ø = nil association; N = negative association; p = p-value; D = direction of association supported by the data; subscript “a” = fully adjusted (for sample size and article quality). Total PA International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 15 of 24 Table 5 Associations of environmental attributes/correlates with older adults’ physical activity by physical activity and environmental measures (objective and perceived) environmental attributes/correlates with older adults’ physical activity by physical activity and environmental i d) Table 5 Associations of environmental attributes/correlates with older adults’ physical activity by physical activity and environmental measures (objective and perceived) Environmental attribute Total PA1 Total walking only P Ø N pa Da P Ø N pa Da Walkability 12.33 6.67 0 <.001 P 4.37 3.63 0 .001 P Objective 9.05 6.95 0 <.001 P - - - - - Perceived 3 0 0 .003 P - - - - - Residential density/urbanisation 11.53 33.5 12.97 .394 Ø 8 14.5 3.5 .036 P Objective 10 18.50 11.50 .388 Ø 7 6.50 3.50 .032 P Perceived 1.53 15 1.47 .855 Ø 1 8 0 .652 Ø Street connectivity 8.71 26.06 2.14 .094 Ø 5.71 13.20 2 .185 Ø Objective 2.80 14.20 1 .366 Ø 1.80 9.20 1 .543 Ø Perceived 6 11.86 1.14 .076 Ø 4 4 1 .210 Ø Access to/availability of destinations & services Overall access to destinations & services 12.55 38.15 0.5 <.001 P 6.93 25.57 0.5 .009 P Objective 3.76 12.24 0 .003 P 3.43 9.57 0 .004 P Perceived 8.79 25.91 0.50 .008 P 3.50 16 0.50 .277 Ø Land-use mix—destination diversity 5.68 19.32 2 .148 Ø 1 8 2 .439 Ø Objective 1.17 10.83 2 .504 Ø - - - - - Perceived 4.51 8.49 0 .002 P - - - - - Shops/commercial 9.96 57.21 0 .006 P 8.58 23.42 0 .001 P Objective 8.25 34.75 0 .006 P 7.08 12.92 0 <.001 P Perceived 1.71 21.29 0 .475 Ø 1.50 10.50 0 .422 Ø Food outlets 0.72 21.28 1 .932 Ø 0.72 6.28 1 .873 Ø Objective 0.72 14.28 0 .685 Ø - - - - - Perceived 0 7 1 .521 Ø - - - - - Education 0.31 11.69 0 .765 Ø 0.14 2.85 0 .826 Ø Objective 0.31 8.69 0 .727 Ø - - - - - Perceived 0 3 0 1.00 Ø - - - - - Health & aged care 4.61 26.39 1 .275 Ø 3.61 7.39 1 .191 Ø Objective 4 24 1 .382 Ø - - - - - Perceived 0.61 2.39 0 .290 Ø - - - - - Public transport 7.5 25.6 1 .013 P 5.5 11.5 1 .011 P Objective 6.50 12.50 0 .004 P 5.50 5.50 0 <.001 P Perceived 1 13 1 .918 Ø 0 6 1 .501 Ø Parks/public open space 11.29 47.54 0.17 .002 P 6.05 23.78 0.17 .014 P Objective 4.42 28.58 0 .083 Ø 4.42 13.58 0 .035 P Perceived 6.87 18.96 0.17 .003 P 1.63 10.20 0.17 .201 Ø Recreational facilities 13.34 39.66 0 <.001 P 3.07 15.93 0 .135 Ø Objective 4.58 21.42 0 .092 Ø 0.29 6.71 0 .848 Ø Perceived 8.76 18.24 0 <.001 P 2.78 9.22 0 .050 P Social recreational facilities 4.15 25.95 0 .105 Ø 1.5 10.5 0 .413 Ø Objective 3.50 14.50 0 .094 Ø 1.50 4.50 0 .291 Ø Perceived 0.65 11.45 0 .687 Ø 0 6 0 1.00 Ø Infrastructure & streetscape Barnett et al. Sensitivity analyses None of the significant correlates of total PA, objective total PA or self-report total PA differed based on any ad- justment (partial or none) (Additional file 4). Regarding total walking, only two significant correlates differed based on adjustment, namely: residential density/urbanisation Page 17 of 24 Page 17 of 24 Page 17 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 notable increases in research conducted in Asia and Eur- ope, which expanded the range of examined geograph- ical settings and cultures. We undertook a systematic review and applied a meta-analytic procedure to statisti- cally identify built environmental attributes related to total PA and total walking, stratifying by measurement method. and Germany, where cycling levels remain high even among older people [85]. Future research examining the differential influence of residential density on different types of PA as well as identifying the optimal threshold of density for supporting all types of PA will be import- ant for informing planning policy and practice [86]. In terms of access to specific types of destinations in the neighbourhood, we found evidence supporting shops and commercial destinations, public transport, parks and public open space, and recreational facilities as pos- sible facilitators of PA. No evidence was found for the seven other destination types examined. Overall, this is in line with the work of others who highlight that certain types of destinations may be more conducive to higher PA levels than other destination types [15, 27]. In general, while the findings from Van Cauwenberg and colleagues’ systematic review published in 2011 were inconclusive [15], we found strong evidence of positive associations between walkability, access to destinations and services, personal safety from crime and PA. Also, while the relatively small number of articles included in Van Cauwenberg and colleagues’ review [15] precluded the examination of differences in findings by measure- ment method, this review and meta-analysis revealed important differences in associations when using object- ive versus perceived measures of environmental attri- butes and when using self-report versus objective measures of PA. These new findings and their implica- tions are discussed in detail below. Shops/commercial destinations and public transport, particularly for objectively assessed measurement types, were positively associated with total walking and total PA, specifically self-reported measures of total PA. Built environmental correlates of older adults’ PA Walkability and access to destinations/services We found strong to very strong evidence supporting the benefits of neighbourhood walkability on total PA and walking, regardless of measurement method. Two of the three components comprising walkability were found to individually relate to PA: strong evidence supported the association between access to destinations and services and total PA, for both objective and perceived environ- mental and PA measurement types. Evidence was also found supporting the impact of access to destinations and services and residential density on total walking, particularly when these attributes were measured ob- jectively. Our findings highlight the importance of having local neighbourhood destinations for older adults to not only walk to and walk around, but to also engage in other types of PA. Furthermore, local destinations may provide a location for social activity and engagement, potentially reducing risk of social isolation and loneliness [82]. g We found strong to very strong evidence for parks and public open space and recreational facilities as cor- relates of total PA, particularly for self-reported types of measurement. This is consistent with findings in adults [91]. When comparing environmental measurement methods, evidence was found for positive associations between total PA and perceived, but not objectively assessed, access to parks, public open space and recre- ational facilities. Access to parks and public open space was also found to be positively associated with total walking. However, when comparing the environmental measurement type, it was the objectively assessed mea- sures showing a positive association with total walking, not perceptions. Here, it should be noted that most of the objectively assessed positive findings were from stud- ies based in Portland, USA [64, 92, 93], a city renowned for its walk-friendliness and management of parks in the presence of urban growth [94, 95]. Having accessible parks and public open space and recreational facilities in local areas may be beneficial beyond PA, as green spaces and visual cues of nature in parks may impart further Although easier access to destinations and services tend to be highly correlated with greater residential density [83], our findings suggest that walking may be the only type of PA positively related to residential dens- ity. High levels of residential density may not be condu- cive to other forms of active transport, such as cycling. Sensitivity analyses The im- portance of shops and commercial destinations for PA is consistent with findings highlighting that shopping is the most prevalent reason for older adults leaving their homes [87], and thus an important part of daily life. Therefore, ensuring neighbourhoods have ease of access to shops means that health-enhancing levels of PA can be incorpo- rated into daily living. Availability and access to public transport not only facilitates PA levels but has the poten- tial to also reduce car dependence [88] with co-benefits of environmental sustainability [89]. For older adults espe- cially, access to public transport enables those who are not confident with driving or no longer able to drive to still travel outside of home, thus maintaining their mobility and reducing risk of loneliness [90]. Safety Safety from crime, especially when perceived measures were used, was found to be positively associated with total PA (primarily self-reported measures) and total walking. This adds to the evidence base as previous re- search in older adults has mostly shown inconsistent findings [31, 108]. Our findings are in line with the no- tion that perceptions of crime have more influence on behaviour (e.g., leaving the home) than objective crime rates [109]. This speaks in favour of interventions aimed at positively changing perceptions of safety (when appro- priate) and encouraging older people to get out of home. This may be particularly important as the frequency of daily out of home trips is predictive of PA participation in this demographic [87]. We found evidence supporting a positive association be- tween street lighting and total walking only. This high- lights the importance of providing street lighting along pedestrian infrastructure so that its use is not dependent on the time of day. For older adults especially, ensuring neighbourhoods are well-lit at night may also contribute to a heightened sense of safety from crime [98]. This is be- cause street lighting helps contribute to natural surveil- lance by allowing pedestrians to be seen. Overall, we found no evidence to support the relation- ship between traffic-related safety and total PA and total walking. However, when only perceived measures of traffic safety were considered, there was evidence of a positive association with total walking only. It is possible that older adults may have no choice other than partici- pating in walking near heavy neighbourhood traffic be- cause they do not own a car and/or have limited access to public transport [73]. It may be that a substantial amount of walking and PA in older adults comes from actively travelling to and from destination-rich areas where traffic is typically heavy [27]. We found no evidence of an association between pavement/footpath quality and PA. This is in contrast to qualitative research findings indicating quality of infrastructure to be particularly pertinent in facilitat- ing PA among older adults [98, 99, 101]. Our findings may be explained by the diversity of measures used and/or lack of clear definition of pavement/footpath quality. For example, what constituted ‘footpath qual- ity’ ranged from “curb quality” (objectively assessed) [47] to “quality and maintenance of sidewalks” (as perceived by study participants) [24]. Infrastructure and streetscape Pedestrian-friendly infrastructure, particularly when measured objectively, was found to be positively associ- ated with both total PA and total walking. This reflects qualitative [98] and experimental research [99] findings highlighting the importance of pavements/footpaths and other infrastructure, such as benches for resting, for older adults’ PA. Ensuring the provision of walk- friendly infrastructure, especially along routes to des- tinations within the neighbourhood, may be particu- larly pertinent. It is plausible that the relationship between walk-friendly infrastructure and PA may dif- fer based on physical functionality. However, we iden- tified only one study that had examined this, finding no difference in the associations [100]. Built environmental correlates of older adults’ PA Walkability and access to destinations/services One study has found that Flemish older adults living in urban areas were less likely to cycle everyday than those living in semi-urban (i.e., less dense) areas [84]. While walking is the most popular type of PA that older adults participate in [9], cycling is also a popular PA mode in European countries such as the Netherlands, Denmark, Page 18 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 18 of 24 Page 18 of 24 psychological benefits on individuals [96, 97]. Moreover, both destinations provide an opportunity for fostering social connectedness/activities (e.g., a walk with friends in a park and a game of squash at a recreational facility). ‘tactical urbanism’, which are low cost interventions to make areas more attractive and pedestrian-friendly [107], environmental modifications such as planting trees and flora are micro-scale interventions that can be more easily implemented than macro-level interventions to street design and layout. Differences in built environmental correlates by type of PA measurement method We found more significant environmental correlates for self-reported PA than for objectively measured PA. One reason for this may relate to common method bias associated with self-reported PA and environmental attributes – systematic error variance introduced by measurement methods that do not accurately assess the constructs they represent and may be due to factors such as social desirability [110]. Another reason may be that the environmental attributes measured in these studies primarily influence walking behaviours that may be more easily measured by self-reports than by accel- erometry. An additional reason may relate to issues with the accelerometry-based operationalisation of older adults’ MVPA. Sixteen of the 28 reviewed articles report- ing objective PA findings used accelerometer cut-points and half of those applied an MVPA cut-point of 1952 ac- celerometer counts per min derived for adults [111]. As Strong evidence supported the role of greenery and aesthetically pleasing scenery on levels of total PA (espe- cially self-reported measurement types) and total walk- ing. These findings are in line with recent research highlighting the importance of green, clean, and attract- ive neighbourhoods and streetscapes in facilitating PA [98, 102]. When stratifying by environmental measure- ment type, only perceived measures were found to be significant. Beyond facilitating PA, it is plausible that there are synergistic benefits of streetscape trees and vegetation, for example, in reducing urban heat island effect [103] and air pollution [104] – environmental fac- tors linked with premature mortality [105] and global disease burden [106]. Following design principles of Page 19 of 24 Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 19 of 24 differently to different behaviours [115]. Instead, a public health perspective examining built environmen- tal attributes associated with total PA focuses on the identifications of environmental attributes enabling health-enhancing levels of PA, which is accrued across all domains. Notably, nearly all built environmental correlates of older adults’ total PA were also identified as being environmental correlates of either active trans- port [27] and/or leisure-time PA [116], thus explaining the behavioural pathways through which the built form impacts on total levels of PA. There was one exception, however, with crime/personal safety being positively associated with total PA, but no evidence found for a relationship with either active transport [27] or leisure- time PA [116]. Other behavioural or psychosocial factors may explain the associations between crime/ personal safety and total PA. Research design issues Longitudinal and quasi-experimental studies are needed to establish causal relationships between the built envir- onment and PA. Insofar as possible, future research designs would also benefit from assessing and adjusting for residential self-selection to account for biases at the individual level (e.g., an older adult who enjoys PA or chooses to live near a park) and thus enabling, to a cer- tain extent, the controlling of reverse causation. The findings of this review may help inform researchers in- volved with natural experiments on what environmental attributes to measure, given the environmental manipu- lation itself will be out of their control. Better quality research may also come from conceptually- driven choices of built environmental attributes and validated PA measures. Where accelerometer cut-points and the classification of older adults’ PA intensities are concerned, it is important that the thresholds for moderate intensity activity are appropriate (e.g., 1013 counts per min [112]). Applying suggestions such as these also allows for the possibility of pooling data across countries. For ex- ample, there has been evidence of curvilinearity related to perceived access to destinations and services and objectively assessed MVPA in a multi-country study of adults [31]. This finding was only possible because of the use of Differences in built environmental correlates by type of PA measurement method Given the medium to strong evidence of these associations, it is especially im- portant for future research to unpack the mechanisms through which crime/personal safety relates to total health-enhancing PA in order to better inform the im- plementation of suitable interventions. For example, a better understanding of fear of crime and assessment of the emotional rather than cognitive response to crime may be warranted [108]. Moderators of the relationship between safety and PA that warrant further consider- ation may include self-efficacy and physical function- ing/capacity [117]. older adults have a lower MVPA cut-point due to lower resting metabolic rates [112], using the adult accelerom- eter cut-point likely resulted in lower estimates of MVPA, potentially masking associations. To accurately classify different intensities of older adults’ PA, future re- search using objectively assessed PA should be under- pinned by appropriate cut-points. Differences in built environmental correlates by type of environmental measurement method Overall, there were numerous differences in the associa- tions between built environmental attributes and total PA and walking, based on type of environmental meas- ure. Attributes that can be classed within the functional (e.g., pedestrian infrastructure) and destination domains in Pikora’s framework tended to be significantly related to PA when objectively assessed [113]. In contrast, those attributes that fall within the safety and aesthetics do- mains were associated with PA when perceived measures were used. This may be explained by attributes within safety and aesthetics domains being more subjective in their interpretation and thus depend on perceptions that may vary greatly between individuals. Attributes related to function and destinations are more objective and, hence, are associated with lower levels of interpersonal differences in perceptions (e.g., a pavement is either present or it is not). Effects were generally stronger for associations between the perceived environment and PA, which is consistent with previous research [114]. Unlike the objective environment, perceptions of the same neighbourhood environment can greatly differ across individuals due to differences in socio-demographics (e.g., socioeconomic status), preference, experience, culture and/or amount of walking in the neighbourhood [30]. Regular walkers may have more accurate perceptions of their local environ- ments. Moreover, perceived measures often define neigh- bourhood in terms of time to reach a destination (e.g., 10–20 min’ walk from home) [40, 41], rather than set dis- tances (e.g., objective 400 m home-centred buffers), and therefore may be more closely aligned with the individual and their own definition of ‘neighbourhood’. Competing interests h h d l h Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Implications for future research and planning policy/ practice Socio-ecological models of health behaviour underpin the majority of research undertaken in the built environment and PA field. One of the key tenets of this approach is its emphasis on the importance of behaviour specificity [12], and for PA this means considering the domain in which PA was accrued. While taking a behavioural perspective allows for the pathways or mechanisms through which the built environment influences PA to be understood, it is possible that built environmental attributes may relate Barnett et al. International Journal of Behavioral Nutrition and Physical Activity (2017) 14:103 Page 20 of 24 Abbreviations MVPA: Moderate- to vigorous-intensity PA; PA: Physical activity; PhD: Doctor of Philosophy; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; SD: Standard deviation; UK: United Kingdom of Great Britain and Northern Ireland; USA: United States of America Strengths and limitations of this review and meta-analysis This systematic review and meta-analysis has several strengths. It addressed publication bias by including both peer-reviewed scientific articles and grey literature. It provided a quantitative synthesis of associations based on non-standardised environmental and PA measure- ment instruments and stratified findings by measure- ment types. It incorporated an extensive article quality assessment into the meta-analytical procedure and, therefore, adjusted the synthesis of evidence for study methodology quality. Limitations include: (1) not ac- counting for potentially correlated findings from the same article; (2) an inability to account for potential mod- erating effects of neighbourhood size and definition; (3) using a meta-analytic method that relied on statistical sig- nificance testing rather than effect size estimates and, thus, likely underestimating the evidence of environment- PA associations; and (4) including only articles published in English. Funding David William Barnett is supported by an international postgraduate research scholarship. Ester Cerin is supported by an Australian Research Council Future Fellowship (FT3 140100085). Jelle Van Cauwenberg is supported by a postdoctoral fellowship of the Research Foundation Flanders (FWO, 12I1117N). The funding bodies had no role in the design of the review and meta-analysis, interpretation of data, nor writing of the manuscript. Availability of data and materials All information generated and analysed in this systematic review and meta- analysis are included in this published article and its supplementary information files (Additional files 1, 2, 3, 4 and 5). Authors’ contributions All authors conceptualised the systematic review. EC developed the review protocol and the meta-analytical approach. DWB conducted the meta-analyses and AN verified its validity. DWB extracted the data from the included articles, drafted the manuscript, and supplementary materials. AB, AN, and JVC verified the validity of the extracted data from the included articles. JVC and AN searched the grey literature. All authors conducted searches, contributed to the screening of article eligibility, revised the manuscript for intellectual content, and approved the final manuscript. Additional files comparable environmental exposure and PA outcome mea- sures across a large range of diverse geographical locations combined with a high variability in exposures across coun- tries (another issue that future research may care to ad- dress). Thus, the multi-country pooling of data based on valid, comparable measures are needed to address issues surrounding limited variability in environmental exposures and non-linear associations between exposures and PA out- comes. Other statistical analysis decisions such as adjusting for key socio-demographic covariates (i.e., age, sex, and education), and not categorising continuous environmental measures would contribute to improving the quality of future research designs. Additional file 1: Table S1. Reviewed total physical activity articles (N = 100) – Information. (DOCX 286 kb) Additional file 1: Table S1. Reviewed total physical activity articles (N = 100) – Information. (DOCX 286 kb) Additional file 2: Table S3. Reviewed total physical activity articles (N = 100) – Quality assessment. (DOCX 106 kb) Additional file 3: Table S2. Associations between built environmental attributes and older adults’ objectively measured walking. (DOCX 16 kb) Additional file 4: Table S4. Meta-analytic results of significance of associations between built environmental correlates of older adults’ PA by outcome and type of adjustment for article characteristics. (DOCX 19 kb) Additional file 5: Table S5. Overview of moderating effects examined in the association between environmental attributes and older adults’ total PA. (DOCX 214 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. Consent for publication Not applicable. Consent for publication Not applicable. Acknowledgements This work has been undertaken as a collaborative effort under the umbrella of the Council on Environment and Physical Activity–Older Adults working group, chaired by Ester Cerin. Conclusions Safe, walkable, and aesthetically pleasing neighbourhoods, with access to destinations and services, specifically, recre- ational facilities, parks/public open space, shops/commer- cial destinations and public transport facilitated older adults’ participation in PA, beyond domain-specificity. However, PA correlates were not consistent across differ- ent PA and environmental measurement types. Future re- search should consider these differences in findings and identify the mechanisms underlying them. 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https://openalex.org/W2790398961	https://www.ajol.info/index.php/njtd/article/download/167910/157325	English	null	Mechanistic evaluation of the effect of calcium carbide waste on properties of asphalt mixes	Nigerian Journal of Technological Development	2,018	cc-by-sa	4,298	I. INTRODUCTION mix, their findings shows that waste HDPE modified bituminous binders provide better resistance against permanent deformation due to their high stability and high Marshall Quotient. Asphalt is a mixture of bitumen, coarse aggregate, fine aggregate and mineral filler in different proportions, depending on the type of asphalt mixture (Garber and Hoel, 2009). One of the major problems associated with the use of asphalt in road works is the high cost of the constituent material which in turn necessitates the development of innovative paving materials. While developing these new paving materials, current attention has been directed at the use of industrial waste to address environmental and economic concerns (Aisien et al., 2006). The vast amount of waste materials produced daily is one of the major worldwide problems in waste management (Ratnasamy et al., 2009). Developing countries like Nigeria produce considerable amounts of wastes from ceramic, steel industry and coal-fired power plants every year. These huge amounts of waste create a significant problem with respect to handling and storage. The use of these materials can actually add value to what was once a costly disposal problem. Using industrial wastes in asphalt concrete pavement can not only decrease environmental problems but also improve some properties of the pavement (Ratnasamy et al., 2009). Fillers as one of the components in asphalt mixture, consist of finely divided mineral matter such as rock dust, slag dust, hydrated lime, hydraulic cement, fly ash, loess or other suitable mineral matter (Ritter and Paquette, 1951). Conventional fillers as listed above are often used as anti- stripping agents as a result of their respective hydrophobic nature. This plays a major role in determining the properties and the behavior of the mixture, especially the binding and aggregate interlocking effects (Ritter and Paquette, 1951). The filler has the ability to increase the resistance of particle to move within the mix matrix and/or work as an active material when it interacts with the bitumen to change the properties of the mastic. (Ratnasamy et al., 2011) Calcium Carbide waste is a waste material generated from oxy-acetylene gas used in welding works (Chukwudebelu et al., 2013). Calcium Carbide Waste is abundant in our environment and researches have been conducted to determine its use as a civil engineering construction material. Corresponding author’s e-mail address: nuhamz@gmail.com Mechanistic Evaluation of the Effect of Calcium Carbide Waste on Properties of Asphalt Mixes N. Isa1, A. Olowosulu1, M. Joel2 1Department of Civil Engineering, Ahmadu Bello University, Zaria, Nigeria. 2Department of Civil Engineering, University of Agriculture, Makurdi, Nigeria. ABSTRACT: Calcium Carbide Waste (CCW) was used as an alternative to traditional Portland cement mineral filler in hot mix asphalt concrete to rid its disposal problem. Its effect on mechanical properties of hot mix asphalt was assessed using the Marshall method of mix design. Using the optimum bitumen content determined from Marshall Test, Portland cement used as mineral filler was partially replaced with 0, 10, 20, 30, 40 and 50% CCW by dry weight. Results of tests indicated an increase in stability, voids in mineral aggregates, Marshall Stiffness and reduction in flow, unit weight, voids filled with binder when the percentage of CCW increases. Based on results of tests, partial replacement of Portland cement with 40% CCW could be used in asphalt production. This will ensure economy in asphalt production and promote disposal of CCW which constitute environmental hazards. KEYWORDS: Calcium Carbide Waste, Asphalt, Portland cement, Mineral Filler, Mechanical Pr [Received July 26 2017; Revised November 19 2017; Accepted December 20 2017] 20 20 NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 15, NO.1, MARCH 2018 doi: http://dx.doi.org/10.4314/njtd.v15i1.4 C. Methods Laboratory investigations were conducted to determine optimum bitumen content for the control mix using different bitumen content, according to the Marshall method of mix design (ASTM D1559). Although Marshall method is essentially empirical, it is useful in comparing mixtures under specific conditions. Therefore it was used in this research work. Using an optimum bitumen content of 5.8% obtained from the conventional mix, asphalt concrete specimens were prepared with Portland cement partially replaced with 0, 10, 20, 30, 40, 50%, calcium carbide waste and the results were compared with the Nigerian General Specification for Roads and Bridges (NGSRB, 1997) standards presented in Table 2. Table 1: Aggregate Gradation. Table 1: Aggregate Gradation. Table 1: Aggregate Gradation. Particle Size (mm) Particle Size (in) % Passing Mean Grading 19.1 3/4 100 100 12.7 1/2 80 - 100 90 9.52 3/8 70 - 90 80 4.76 No. 4 50 - 70 60 2.38 No. 8 35 - 50 42 0.59 No. 30 18 - 29 23 0.297 No. 50 13 - 23 18 0.119 No. 100 8 - 16 12 0.074 No. 200 4 - 10 7 (Source: FMWHM, 2013) 0 10 20 30 40 50 60 70 80 90 100 0.05 0.5 5 50 PERCENTAGE PASSING (%) SIEVE SIZE (mm) Upper Limit Middle Lower Limit Figure 1: Aggregate gradation used in this study. B. Mineral Filler and Bitumen treat over-wetted soils being used as highway embankment filling materials. The fillers used in this investigation include Portland cement which was obtained from cement retailer and calcium carbide waste from automobile welders shop in Panteka Kaduna. Calcium carbide was used as a percentage of 0%, 10%, 20%, 30%, 40% and 50% Portland cement. 60/70 penetration grade bitumen used in this study was obtained from NNPC Kaduna refinery. The bitumen was subjected to penetration, ductility and specific gravity test in accordance with ASTM D5, D113, D70 respectively to determine its physical properties. Different researchers have investigated the use of CCW in concrete works (Nattapong et al., 2010, Heni et al., 2014, Hongfang et al., 2015) and soil stabilization (Du et al., 2011, Songsuda et al., 2013, Joel et al., 2014), little has been done in the use of the waste in Asphalt production. The high cost of conventional materials used in asphalt production coupled with the hazardous effect of CCW on the environment necessitated this study which is aimed at investigating the effect of partial replacement of mineral filler with calcium carbide waste in hot mix asphalt concrete. Outcome of the research will be beneficial to engineers in the road construction industry. I. INTRODUCTION Nattapong et al., (2010) investigated the effects of calcium carbide residue-fly ash Binder on mechanical properties of concrete and observed that the hardened concrete produced from calcium carbide residue – fly ash mixtures had mechanical properties similar to those from normal Portland cement concrete. Joel and Edeh (2014) investigated the stabilization of Ikpayongo lateritic soil with cement and calcium carbide waste and observed that calcium carbide waste can partially be used in the stabilization of Ikpayongo laterite. Du et al., (2011) investigated the strength and California bearing ratio properties of natural soils treated with calcium carbide residue and reported that calcium carbide residue can be adopted as an alternative binder to Researchers have been investigating the use of waste materials in the construction industry, to enable better management of these wastes and improve the properties of construction materials. Burak and Ali, (2004) investigated the use of asphalt roofing shingles in hot mix asphalt, and observed that waste shingles as an additive can improve the Marshall stability and rutting resistance of the asphalt mixtures. Aisien et al., (2006) investigated the application of ground scrap tyre rubbers in asphalt concrete pavement, and observed that the rubberized asphalt concrete mix has much better mechanical properties than the conventional mix. Sinan and Emine, (2004) investigated the use of waste high density polyethylene (HDPE) as bitumen modifier in asphalt concrete 21 ISA et al: MECHANISTIC EVALUATION OF THE EFFECT OF CALCIUM CARBIDE WASTE A. Aggregates Coarse and fine aggregates used in the preparation of asphalt concrete mixtures were sourced from PW quarry site along Angwa-Maijero road, 0331897E, 1161485N Kaduna with specific gravity values presented in Table 3. In this study, two sizes of coarse aggregate were used. The selected gradation of aggregate incorporated in all asphalt concrete specimens confirms to the mid - point of the standard aggregate gradation specified in the Federal Ministry of Works Highway Design Manual (2013). Table 1 presents the selected mix gradation. Table 2: Properties of compacted asphaltic concrete. Property Base-Course Wearing Course Optimum Bitumen content 4.5% - 6.5% 5.0% - 8% Stability not less than 3.5 kN 3.5 kN Flow 2 mm - 6 mm 2 mm - 4 mm Voids in total mixture 3% - 8% 3% - 5% Voids filled with bitumen (VFB) 65% - 72% 75% - 82% (Source: NGSRB, 1997) NIGERIAN JOURNAL OF TECHNOLOGICAL DEVELOPMENT, VOL. 15, NO.1, MARCH 2018 calcium carbide waste are non-plastic (NP), with specific gravity of Portland cement being 3.15 and that of calcium carbide waste 2.42. The mineral filler properties reported in Table 4 are within the ranges reported by other researchers (Talal et al., 2013; Hassan et al., 2006) and ASTM D242 which makes the result satisfactory. Table 4: Average physical properties of mineral fillers. Although there was inconsistency in these results with respect to CCW content, the difference between the Marshall stability values were not notably high from 0 - 50% replacement. This might be attributed to particles from CCW filling the void between the coarse and fine aggregate component of asphalt. A positive stability results were obtained with the replacement of Portland cement with CCW because they were above the Nigerian General Specification of roads and Bridges (1997) standard of not less than 3.5 kN. The result of the physical properties of bitumen used presented in Table 5 shows that the bitumen is a 60/70 pen grade bitumen with a penetration of 70, a ductility of 100+ and a specific gravity of 1.027. All these values conform to the range specified in the Nigerian General Specifications for Road and Bridges (1997). 6 6.5 7 7.5 8 0.0 10.0 20.0 30.0 40.0 50.0 Marshall Stability (kN) Calcium Carbide Waste Content (%) Figure 2: Variation of Marshall Stability with CCW content. Table 5: Physical properties of bitumen. Test Unit ASTM Result (60/70) Penetration, 100g, 25oC 0.1 mm D5 70 Ductility, 5cm/min., 25oC cm D113 >100 Specific Gravity at 25oC ---- D70 1.027 Table 5: Physical properties of bitumen. Table 5: Physical properties of bitumen. The results of all Marshall Stability tests using the designed asphalt ratio of 5.8% are summarized in Table 6 for mixtures with different CCW content. All results shown for each specimen are the average value of three tests. Figure 2: Variation of Marshall Stability with CCW content. author’s e-mail address: nuhamz@gmail com doi: http://dx doi org/10 Marshall Properties Stability (kN) Flow (mm) Bulk Density (gm/cm3) Air Voids % Voids in Mineral Aggregate VMA % Voids Filled with Binder % Mar-shall Stiff- ness kN/mm NGSRB 1997 Require- ments Not less than 3.5 2-4 - 3-5% Not less than 15% 75-82 - Calcium carbide waste, % (by wt. of filler material) Asphalt content, % (by wt. of total mix) = 5.8 % 0.0 6.93 5.64 2.379 3.65 21.87 83.31 1.23 10.0 7.15 4.90 2.375 3.65 21.76 83.23 1.46 20.0 6.95 4.66 2.369 3.74 22.18 83.14 1.49 30.0 7.15 5.39 2.344 4.59 23.01 80.05 1.33 40.0 7.38 4.17 2.337 4.57 23.26 80.35 1.77 50.0 6.04 4.66 2.312 5.44 23.92 77.26 1.30 Table 6: Marshall Stability test results for mixtures with different carbide waste content at 5.8% bitumen content. III. RESULTS AND DISCUSSION The specific gravities of coarse and fine aggregates obtained are as presented in Table 3. The specific gravity of fine aggregate is higher than those for the coarse aggregates. For a particular aggregate type or source, fine aggregate specific gravities can be slightly higher than coarse aggregate specific gravities because as the aggregate particles get smaller, the fraction of pores exposed to the aggregate surface increases (Pavement Interactive, 2011). The specific gravity values are within the range reported by other researchers (Mahesh et al., 2009; Hassan et al., 2011; Talal et al., 2013; Ratnasamy and Eltaher, 2011). 0 10 20 30 40 50 60 70 80 90 100 0.05 0.5 5 50 PERCENTAGE PASSING (%) SIEVE SIZE (mm) Upper Limit Middle Lower Limit Table 3: Specific gravity of coarse and fine aggregates. Aggregate Type Average Specific Gravity Table 3: Specific gravity of coarse and fine aggregates. Aggregate Type Average Specific Gravity Coarse 12.7mm 2.631 Coarse 9.52mm 2.638 Table 3: Specific gravity of coarse and fine aggregates. Aggregate Type Average Specific Gravity Coarse 12.7mm 2.631 Coarse 9.52mm 2.638 Fine Aggregate 2.667 50 Figure 1: Aggregate gradation used in this study. All the percentage passing sieve 30, 50 and 200 of both Portland cement and calcium carbide waste conformed with the range specified by ASTM D242; Portland cement and 22 A. Effect of CCW on Marshall Stability The relationship between Marshall Stability value and CCW content in Figure 2 clearly shows that the stability value increases with CCW content up to 40%, then decreased. This is due to the aggregates which tend to be finer as CCW content increases; therefore there exists a reduction in the stability value at 50% CCW. The reduction in stability at 50% CCW content can also be attributed to the high voids in mineral aggregates within the mix which requires a high compactive effort to achieve high stability value. Samples with 40% calcium carbide waste after 30minutes immersion in water bath have the highest Marshall stability value. Table 4: Average physical properties of mineral fillers. Sieve (mm) % Passing ASTM D242 Portland Cement Carbide Waste No. 30 (0.59) 100 100 100 No. 50 (0.297) 100 100 95-100 No. 200 (0.074) 94 92 70-100 Plasticity Index NP NP < 4 Specific Gravity 3.15 2.42 ---- Table 4: Average physical properties of mineral fillers. Table 4: Average physical properties of mineral fillers. Table 6: Marshall Stability test results for mixtures with different carbide waste content at 5.8% bitumen content. E. Effect of CCW on Voids in Mineral Aggregate The effect of CCW on voids in mineral aggregates is shown in Figure 6. It is a common trend that, as filler content in the mixes increase, the void in the mineral aggregates decrease up to a minimum value then increases at higher content as could be seen from Figure 6. The voids in mineral aggregates increases with CCW content, this could be attributed to the fact that CCW is coarser than Portland cement. This effect further explains the reduction in the bulk density of the mix. The voids in mineral aggregates values all lie within the permissible limits specified in the Federal Ministry of Works Highway Design manual (2013). Figure 3: Variation of marshall flow with CCW content. ISA et al: MECHANISTIC EVALUATION OF THE EFFECT OF CALCIUM CARBIDE WASTE 23 B. Effect of CCW on Marshall Flow D. Effect of CCW on Voids in Total Mix Variation of flow with CCW content is shown in Figure 3. Decline in Marshall flow was observed at 10%, 20%, 40% CCW replacement. The reduction in flow values with the introduction of CCW can be attributed to CCW particles being finer than OPC particles and stiffens the mixture more than OPC. The increment in flow at 30% and 50% CCW content can be attributed to the sliding action of particles within the mix. The effect of CCW content on voids in total mixtures of compacted mixes is shown in Figure 5. The air void content for mixes with 0% - 40% CCW were all within the range of 3% - 5% specified by the Nigerian General Specification for Roads and Bridges (1997). Values of 5.44% obtained when cement was replaced with 50% CCW were above the value recommended by the Nigerian code. High air voids are associated with high permeability, which will affect the performance of the surfacing made with such mix. This behavior was equally observed by (Hassan et al., 2011) when glass powder was used to replace ordinary Portland cement and limestone powder. The Nigerian general specification for Road and Bridges (1997) flow value of 4mm was satisfied when cement was replaced with 40% CCW. 3.00 3.50 4.00 4.50 5.00 5.50 0.0 10.0 20.0 30.0 40.0 50.0 Voids in Total Mix (%) Calcium Carbide Waste Content (%) Figure 5: Variation of Voids in Total Mix with CCW content Figure 3: Variation of marshall flow with CCW content. 4 4.5 5 5.5 6 0.0 10.0 20.0 30.0 40.0 50.0 Marshall Flow (mm) Calcium Carbide Waste Content (%) Figure 5: Variation of Voids in Total Mix with CCW content E. Effect of CCW on Voids in Mineral Aggregate E. Effect of CCW on Voids in Mineral Aggregate C. Effect of CCW on Bulk Density The effect of CCW content on bulk density of compacted mixes is shown in Figure 4. Mixes showed a trend of decrease in bulk density as CCW content increases. This is because while CCW content increases in the mix, it fills the voids within the aggregates and at higher content of CCW, the stiffness of the mix increased which in turn needs a greater compaction effort then consequently less dense mixtures obtained. The decrease in bulk density can also be attributed to the low specific gravity of CCW. This trend is similar to that observed by Hassan et al., (2011) when glass powder was used to replace limestone powder and ordinary Portland cement. 21 21.5 22 22.5 23 23.5 24 0.0 10.0 20.0 30.0 40.0 50.0 Voids in Mineral Aggregate (%) Calcium Carbide Waste Content (%) Figure 6: Variation of Voids in Mineral Aggregate with CCW content. 21 21.5 22 22.5 23 23.5 24 0.0 10.0 20.0 30.0 40.0 50.0 Voids in Mineral Aggregate (%) Calcium Carbide Waste Content (%) Corresponding author’s e mail address: nuhamz@gmail com doi: http://dx doi org/10 4314/njtd v15i1 4 2.300 2.320 2.340 2.360 2.380 2.400 0.0 10.0 20.0 30.0 40.0 50.0 Bulk Density (g/cm3) Calcium Carbide Waste Content (%) Figure 4: Variation of bulk density with CCW content. 21 21.5 22 22.5 23 23.5 0.0 10.0 20.0 30.0 40.0 50.0 Voids in Mineral Aggreg (%) Calcium Carbide Waste Content (%) Portland cement. Figure 6: Variation of Voids in Mineral Aggregate with CCW content. C di h ’ il dd h @ il 2.300 2.320 2.340 2.360 2.380 2.400 0.0 10.0 20.0 30.0 40.0 50.0 Bulk Density (g/cm3) Calcium Carbide Waste Content (%) Figure 4: Variation of bulk density with CCW content. 2.300 2.320 2.340 2.360 2.380 2.400 0.0 10.0 20.0 30.0 40.0 50.0 Bulk Density (g/cm3) Calcium Carbide Waste Content (%) Figure 6: Variation of Voids in Mineral Aggregate with CCW content. Figure 4: Variation of bulk density with CCW content. 24 G. Effect of CCW on Marshall Stiffness v. Voids filled with Binder decreased from 85.02% at 0% CCW to a minimum value of 76.50% at 50% CCW content. Marshall Stiffness values increased with CCW content and peaked at 40% CCW replacement (1.77 kN/mm). Figure 8 shows the relationship between Marshall Stiffness and CCW content. The Marshall Stiffness (MS), that is, stability divided by its flow, is an empirical stiffness value and is used by some engineers, especially in Europe, to evaluate the strength of asphalt mixture (Talal et al., 2013). A higher value of MS indicates a stiffer mixture and hence, it indicates that the mixture is likely more resistant to permanent deformation (Talal et al., 2013). Marshall Stiffness of mixture as shown in Figure 8 were found to be satisfactory when compared to the value obtained with the control mix of 0% CCW. The plot indicates that Marshall Stiffness increased with CCW content, this is also reflected in Figure 3. IV. CONCLUSION In this study, Calcium Carbide Waste (CCW) was used as an alternative to traditional Portland cement mineral filler in hot mix asphalt concrete to rid its disposal problem. The results are summarized below: F. Effect of CCW on Voids Filled with Binder F. Effect of CCW on Voids Filled with Binder Effect of CCW on voids filled with binder is shown in Figure 7. Voids filled with binder value increase with CCW content, and all are above 75%, where the Marshall criteria for voids filled with binder (VFB) is 75% - 82%. This criterion is important because it affects the durability of mixes and is related to the optimum bitumen content in the mix. If the percentage of voids filled with binder is lower than the limit indicated, there will be less bitumen film around the aggregate particles. On the other hand at CCW contents of 0%, 20%, and 30%, the limit is exceeded and more voids are filled with bitumen than required for durability, this may result in bleeding. i. Results of the tests conducted on CCW to determine its suitability for use as mineral filler showed a specific gravity value of 2.42, plasticity index value of 0 (non- plastic) with 92% passing sieve No.200. These values confirmed its suitability for use as mineral filler in accordance with ASTM D242. ii. Optimum bitumen content value of 5.8 % was achieved after conducting Marshall Stability tests on specimens containing Portland cement as conventional mineral filler. Stability values increased from 6.93 kN at 0% CCW to a maximum value of 7.38 kN at 40% CCW content. The Flow value of 4.17 mm at 40% CCW satisfies the Nigerian General Specification for Roads and Bridges (1997) standards. 76.50 78.50 80.50 82.50 84.50 0.0 10.0 20.0 30.0 40.0 50.0 Void Filled with Binder (%) CalciumCarbide Waste Content (%) Figure 7: Variation of Void Filled with Binder with CCW content. iii. Flow values reduced from 5.64 mm at 0% CCW to a minimum value of 4.17 mm when cement was replaced with 40% CCW which subsequently resulted in a stiffer mix. iv. The Unit Weight of asphalt generally reduced from 2.379 g/cm3 at 0% CCW to a minimum value of 2.312g/cm3 at 50% CCW content. Air voids increased from 3.21% at 0% CCW to a maximum value of 5.44% at 50% CCW content. Voids in Mineral Aggregate values also increased from 21.44% at 0% CCW to a maximum value of 23.19% when cement was partially replaced with 50% CCW. Figure 7: Variation of Void Filled with Binder with CCW content. G. Effect of CCW on Marshall Stiffness REFERENCES Aisien, F.A,; F. K. Hymore and R. O. Ebewele. (2006). Application of Ground Scrap Tyre Rubbers in Asphalt Concrete Pavements. Indian Journal of Engineering and Material Sciences, 13: 333-338. ASTM (2004). International Standard Testing for Road and Paving Materials; Vehicle-Pavement Systems. Burak, S. and Ali, T. (2004). Use of Asphalt Roofing Shingle Waste in HMA. Construction and Building Materials. 19: 337-346. Corresponding author’s e-mail address: nuhamz@gmail.com Figure 8: Variation of marshall stiffness with CCW content. 1.20 1.30 1.40 1.50 1.60 1.70 1.80 0.0 10.0 20.0 30.0 40.0 50.0 Marshall Stiffness (kN/mm) Calcium Carbide Waste Content (%) 1.20 1.30 1.40 1.50 1.60 1.70 1.80 0.0 10.0 20.0 30.0 40.0 50.0 Marshall Stiffness (kN/mm) Calcium Carbide Waste Content (%) Chukwudebelu, J. A.; C. C. Igwe, O. E. Taiwo and O. B. Tojola. (2013). Recovery of pure slaked lime from carbide sludge: Case study of Lagos State, Nigeria. African Journal of Environmental Science and Technology, 7(6): 490-495. Du, Y. J.; Y. Y. Zhang and S. Y. Liu. (2011). Investigation of Strength and California Ratio Properties of Natural Soils Treated by Calcium Carbide Residue. Geo- Frontiers: Advances in Geotechnical Engineering, 1237-1244. Federal Ministry of Works (2013). Highway manual Part 2: Maintenance works", Abuja, Nigeria. Garber, J. and Hoel, A. (2009). Traffic and Highway Engineering (4th ed.). Toronto, Canada: Cengage Learning,. C di h ’ il dd h @ il Figure 8: Variation of marshall stiffness with CCW content. 25 ISA et al: MECHANISTIC EVALUATION OF THE EFFECT OF CALCIUM CARBIDE WASTE of Type and Particle Size of Industrial Wastes Filler on Indirect Tensile Stiffness and Fatigue Performance of Stone Mastic Asphalt Mixtures. Australian Journal of Basic and Applied Sciences, 5(11): 297-308. Hassan, H. J.; F. Mays and Y. J. Israa. (2011). 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https://openalex.org/W3134959386	https://pubs.rsc.org/en/content/articlepdf/2021/ra/d1ra01547h	English	null	Versatile small molecule kinase assay through real-time, ratiometric fluorescence changes based on a pyrene-DPA-Zn2+ complex	RSC advances	2,021	cc-by	5,625	Introduction crucial kinases.15–19 Thus, it remains necessary to develop a small molecule kinase assay method that can be applied in real time and is based on a property shared by most small molecule kinases, regardless of their substrate diversity. Kinases mediate the transfer of a phosphate group to an acceptor for phosphorylation or dephosphorylation of various substrates. This enzymatic reaction is related to a number of biological processes, including metabolism, transcription, cell cycle regulation, and apoptosis.1–4 Because the dysregulation of kinase enzymes can cause various human diseases, the devel- opment of kinase assay methods and inhibitors for each kinase plays a critical role in the diagnosis and therapy of these diseases.5–10 Although many kinase assay methods have been reported for the rapid detection of enzyme malfunctions and inhibitor screening, most of them have focused on protein kinases, despite the importance of small molecule kinases, which utilize small molecules as their substrates. Many protein kinase assay methods have been established by using the characteristic properties of the protein substrates, and the dynamic range of the substrates was also suitable for proteins.11–14 For specic small molecule kinase assays, exam- ples have been developed by using nanoparticles, and coupled enzymes. Unfortunately, the enzyme assays showed obvious drawbacks in their end-point measurement and complex cascade enzyme reaction. Most of them also attempted to target unique structures of substrates that were unsuited for other In many small molecule kinases, a decrease in ATP, accom- panied by an increase in ADP, commonly occurs, and this aspect has received attention as a target for monitoring kinase activity.20–25 However, some small molecule kinases have dis- played an increase in ATP and a decrease in ADP for dephosphor- ylation of the substrate, unlike those kinases using ATP as a phosphate group donor. For example, creatine kinase (CK), which plays an important role in cellular energy transformation, is an example of a kinase that can perform reversible reactions. Mito- chondrial CK catalyzes the transformation of creatine and ATP to phosphocreatine and ADP, whereas the production of creatine and ATP in the cytosol is caused by cytosolic CK to maintain energy homeostasis.26,27 Therefore, to understand small molecule kinases more precisely, it is necessary to monitor the reversible changes, rather than the mono-directional transformation. RSC Advances Cite this: RSC Adv., 2021, 11, 10375 Received 26th February 2021 Accepted 4th March 2021 DOI: 10.1039/d1ra01547h rsc.li/rsc-advances PAPER nder a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue Open Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Department of Chemistry, Gwangju Institute of Science and Technology (GIST), 123 Cheomdangwagi-ro, Buk-gu, Gwangju 61005, Republic of Korea. E-mail: happyhan@ gist.ac.kr † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ra01547h ‡ Both authors contributed equally. Open Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6 This article is licensed under a Creative Commons Attribution 3.0 U A real-time kinase assay method based on a ratiometric ﬂuorescence probe that can be applied to various small-molecule kinases is described herein. The probe can trace the reversible interchange of ATP and ADP, which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric ﬂuorescence change. This property facilitates the monitoring of phosphorylation and dephosphorylation in small- molecule kinases, whereas most of the existing methods focus on one of these reactions. To prove the applicability of this method for small-molecule kinase assays, hexokinase and creatine kinase, which phosphorylate and dephosphorylate substrates, respectively, were analyzed. The ratiometric ﬂuorescence change was correlated with the enzyme activity, and the inhibition eﬃciencies of the well- known inhibitors, N-benzoyl-D-glucosamine and iodoacetamide, were also monitored. Notably, the change in ﬂuorescence can be observed with a simple light source by the naked eye. Received 26th February 2021 Accepted 4th March 2021 DOI: 10.1039/d1ra01547h rsc.li/rsc-advances Jihoon Kim,‡ Jinyoung Oh‡ and Min Su Han * Jihoon Kim,‡ Jinyoung Oh‡ and Min Su Han * Versatile small molecule kinase assay through real- time, ratiometric ﬂuorescence changes based on a pyrene-DPA-Zn2+ complex† Versatile small molecule kinase assay through real- time, ratiometric ﬂuorescence changes based on a pyrene-DPA-Zn2+ complex† Cite this: RSC Adv., 2021, 11, 10375 Experimental Synthesis of [(2,20-dipicolylamino)methyl]pyrene (3). To 1-(4- bromobutyl)pyrene (0.100 g, 0.297 mmol) solution in ACN, 2,20- dipicolylamine (0.071 g, 0.36 mmol), and K2CO3 (0.147 g, 1.07 mmol) was added and reuxed for 28 h. Aer evaporating solvent under reduced pressure, the residue was diluted with water and extracted with DCM. The collected organic layer was concentrated and puried by silica gel column chromatography (EA) to give a pale yellow oil (0.093 g, 69%). 1H-NMR (400 MHz, CDCl3) d 8.49 (d, J ¼ 4.6 Hz, 2H), 8.22–8.14 (m, 3H), 8.08–7.95 (m, 5H), 7.78 (d, J ¼ 7.6 Hz, 1H), 7.55 (td, J ¼ 7.6, 1.8 Hz, 2H), 7.46 (d, J ¼ 7.6 Hz, 2H), 7.08 (ddd, J ¼ 7.2, 5.0, 1.1 Hz, 2H), 3.79 (s, 4H), 3.26 (t, J ¼ 7.6 Hz, 2H), 2.63 (t, J ¼ 7.2 Hz, 2H), 1.88–1.80 (m, 2H), 1.74–1.67 (m, 2H). Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In this study, a general method for a real-time small mole- cule kinase uorescence assay was proposed. To develop the assay, a pyrene-dipicolylamine (DPA)-Zn2+ complex was prepared because this probe allowed discrimination between ATP and ADP with distinct ratiometric uorescence emissions.33,34 It was speculated that a ratiometric uorescence change in the pyrene-DPA-Zn2+ complex would occur in conjunction with the interchange of ATP and ADP, representing the real-time progression of the small molecule kinase reaction (Scheme 1). In addition, the reversible uorescence change of the pyrene-DPA-Zn2+ complex would facil- itate monitoring of the dephosphorylation as well as phosphory- lation of the substrate by various small molecule kinases. As proof of this concept, the activity of two small molecule kinases, namely hexokinase (HK) to phosphorylate glucose and CK to dephos- phorylate phosphocreatine, were analyzed, and inhibitor analyses for the enzymes were also demonstrated. Synthesis of 1-(4-bromobutyl)pyrene (2). To 4-pyrenyl-1- butanol (0.500 mg, 1.82 mmol) solution in anhydrous DCM (30 mL) was added CBr4 (1.212 g, 3.64 mmol), and K2CO3 (0.504 g, 2.73 mmol) at 0 C. PPh3 (0.716 mg, 2.73 mmol) in anhydrous DCM (20 mL) was added dropwise to the solution and stirred at room temperature for 4 h. Aer the reaction mixture was concentrated under reduced pressure, the residue was dissolved with EA and washed with water, brine, and dried on anhydrous Na2SO4, and concentrated under reduced pres- sure. The residue was puried by silica gel column chroma- tography (Hex : DCM ¼ 7 : 1) to give a white solid (0.506 g, 82%). 1H-NMR (400 MHz, CDCl3) d 8.27 (d, J ¼ 9.2 Hz, 1H), 8.18–8.11 (m, 4H), 8.05–7.97 (m, 3H), 7.86 (d, J ¼ 7.9 Hz, 1H), 3.49–3.46 (m, 2H), 3.40–3.36 (m, 2H), 2.05–2.02 (m, 4H). Materials and instruments Chemical reagents were purchased from commercial sources and, unless otherwise stated, were used without further puri- cation. For enzyme assay, hexokinase (HK, EC 2.7.1.1) and creatine kinase (CK, EC 2.7.3.2) were purchased from Sigma Aldrich. Florescence spectra were recorded on an Agilent Cary Eclipse uorescence spectrometer using a 1 cm path length quartz cell. 1H NMR spectrums were recorded on a JEOL 400 MHz NMR spectrometer. Introduction For this reason, it is proposed that real-time assay to trace the reversibly changed ATP and ADP ratio, which is a common feature of small molecule kinases, can be adopted as a general assay method that will be applicable to various small molecule kinases. Ratiometric uorescence probes have received attention because they allow for ratio change between emission bands at distinct wavelength and built-in correction to reduce environ- mental interference and improve sensitivity and accuracy, permitting the development of detection methods for various targets.28–30 In particular, certain ratiometric uorescence probes have been introduced as a method for tracing reversibly changed analytes based on the signicant property that the change RSC Adv., 2021, 11, 10375–10380 \| 10375 © 2021 The Author(s). Published by the Royal Society of Chemistry Paper View Article Online View Article Online Paper RSC Advances Paper of analytes can be monitored directly by the change of uorescence emissions.31,32 For instance, the real-time interchange of bisulfate and hydrogen peroxide in a biological redox cycle was monitored by a ratiometric change of uorescence emission.32 Considering the properties of ratiometric uorescence probes, they could be eﬀective for detecting bidirectional changes between ATP and ADP in a kinase reaction, and thus, they could become an appropriate real-time assay platform for various small molecule kinases asso- ciated with phosphorylation or dephosphorylation. Synthesis of 1-pyrenebutanol (1). 1-Pyrenebutyric acid (1.00 g, 3.45 mmol) in anhydrous THF (22 mL) was stirred at 0 C. To the solution lithium aluminum hydride (1.25 g, 32.5 mmol) dissolved in anhydrous THF (32.5 mL) was added dropwise slowly and stirred for 30 min. Aer then, the solution was reuxed for 23 h and quenched through Fieser method. The residue was evaporated and puried via silica gel column chromatography (MeOH : CHCl3 ¼ 1 : 99) to give a pale yellow solid (0.64 g, 68%). 1H-NMR (400 MHz, CDCl3) d 8.27 (d, J ¼ 9.2 Hz, 1H), 8.16 (d, J ¼ 2.1 Hz, 1H), 8.14 (d, J ¼ 2.4 Hz, 1H), 8.11–8.08 (m, 2H), 8.02–7.96 (m, 3H), 7.86 (d, J ¼ 7.9 Hz, 1H), 3.72–3.68 (m, 2H), 3.37 (t, J ¼ 7.8 Hz, 2H), 1.97–1.90 (m, 2H), 1.77–1.70 (m, 2H) 1.25 (s, 1H). Open Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:1 This article is licensed under a Creative Commons Attribution 3.0 Unpor added concentration, whereas the excimer emission at 476 nm increased gradually as the concentration of ATP increased without a signicant change in the monomer emission (Fig. S1†). As a result, ATP and ADP clearly had diﬀerent eﬀects on the pyrene- DPA-Zn2+ complex and induced two distinct pyrene complexes, as represented by the diverse emission spectra. In fact, it is postulated that the kinase reaction induces an interchange of ATP and ADP, not the uctuation of each molecule, which may be the result of an increase in the ADP/ATP ratio for phosphorylation or decrease in the ratio for dephosphorylation. Therefore, the uorescence change following a change in the ADP/ATP ratio was evaluated as a model study for monitoring kinase reactions. As shown in Fig. 1, a sequential change of the ADP/ATP ratio transformed the overall spectra, with an increase or decrease in the excimer emission accompanied by the opposite change in the monomer emission. This means that the interchange of ADP and ATP as a progression of the kinase reaction can be monitored in real time from the ratiometric uorescence change. Kinetic study of hexokinase for glucose. Various concentra- tions of glucose (from 0 to 1.4 mM) was added to the buﬀer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-Zn2+ complex (20 mM), ATP (10 mM), MgCl2 (80 mM). Aer addition of HK (1 unit), the uorescence intensity was measured at 1 min interval for 10 min with excitation at 341 nm. The Michaelis– Menten constant (Km), maximum initial velocity (Vmax) as the enzymatic parameters were estimated from Lineweaver–Burk plots and the values were compared with reported results. Monitoring dephosphorylation in a kinase reaction. To buﬀer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA- Zn2+ complex (20 mM), ADP (10 mM), MgCl2 (80 mM), and phosphocreatine (0.75 mM), various concentrations (0 to 1 unit) of CK were added. The uorescence intensity was measured at 1 min interval for 15 min with excitation at 341 nm. The observed rate (kobs) for HK was estimated using initial change of uorescence ratio of F476 and F376 from 0 to 4 min. Monitoring phosphorylation in a kinase reaction and its kinetic study Inhibitor assay for the kinases. To buﬀer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-Zn2+ complex (20 mM), ATP (10 mM), MgCl2 (80 mM), and glucose (1 mM), a various concentration of N-benzoyl-D-glucosamine (0 to 4 mM), and without incubation HK (1 unit) was added. Inhibition eﬃciency was obtained by the change of excimer and monomer emission ratio at 20 min aer the measurement. To apply the system for assaying a biological kinase that phos- phorylates its substrates, HK was analyzed as a model enzyme. HK, which plays an important role in the rst step of glycolysis and has been well studied because of its relationship with many diseases, transfers a phosphate group from ATP to the substrate Fig. 2 (a) Enzymatic reaction formula of HK associated with phos- phorylation; (b) ﬂuorescence intensity ratios of monomer (F376) and excimer (F476) emissions over time for the pyrene-DPA-Zn2+ complex with various concentrations of HK (0 to 1 unit) in buﬀer solution (HEPES, 20 mM, pH 7.4); (c) plot of the logarithm of kobs against concentration of HK. [pyrene-DPA-Zn2+] ¼ 20 mM, [ATP] ¼ 10 mM, [Mg2+] ¼ 80 mM, [glucose] ¼ 1 mM, lex ¼ 341 nm. To buﬀer solution(HEPES, 20 mM, pH7.4) containing pyrene-DPA- Zn2+ complex (20 mM), ADP (10 mM), MgCl2 (80 mM), and phospho- creatine (0.75 mM), mixture with various concentrations of iodoace- tamide (0 to 100 mM) and CK (1 unit per mL) incubated for 10 min was added. Inhibition eﬃciency was obtained by the change of excimer and monomer emission ratio at 15 min aer the measurement. Synthesis In situ preparation of pyrene-DPA-Zn2+ complex. Pyrene- DPA-Zn2+ complex was generated in situ by addition of Zn(ClO4)2 to the buﬀer solution (HEPES, 20 mM, pH 7.4) containing compound 3. Without further incubation of the solution, other components were also added to the solution, The pyrene-DPA-Zn2+ complex was prepared following as Scheme 2. Scheme 1 Schematic illustration of the real-time assay method for various small molecule kinases based on a ratiometric ﬂuorescence probe. Scheme 2 Synthetic route for preparation of pyrene-DPA-Zn2+ complex. Scheme 1 Schematic illustration of the real-time assay method for various small molecule kinases based on a ratiometric ﬂuorescence probe. Scheme 2 Synthetic route for preparation of pyrene-DPA-Zn2+ complex. © 2021 The Author(s). Published by the Royal Society of Chemistry 10376 \| RSC Adv., 2021, 11, 10375–10380 © 2021 The Author(s). Published by the Royal Society of Chemistry Fig. 1 (a) Fluorescence spectra of pyrene-DPA-Zn2+ complex with various ratios of ADP and ATP in buﬀer solution (HEPES, 20 mM, pH 7.4); (b) ﬂuorescence intensity ratio of monomer (F376) and excimer (F476) emission against the ratio of ADP and ATP. [pyrene-DPA-Zn2+ complex] ¼ 20 mM, [ADP] + [ATP] ¼ 10 mM, lex ¼ 341 nm. RSC Advances View Article Online Paper and uorescence spectra were recorded with excitation at 341 nm. Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 (a) Fluorescence spectra of pyrene-DPA-Zn2+ complex with various ratios of ADP and ATP in buﬀer solution (HEPES, 20 mM, pH 7.4); (b) ﬂuorescence intensity ratio of monomer (F376) and excimer (F476) emission against the ratio of ADP and ATP. [pyrene-DPA-Zn2+ complex] ¼ 20 mM, [ADP] + [ATP] ¼ 10 mM, lex ¼ 341 nm. Monitoring phosphorylation in a kinase reaction. To buﬀer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA-Zn2+ complex (20 mM), ATP (10 mM), MgCl2 (80 mM), and glucose (1 mM), various concentrations (0 to 1 unit) of HK were added. The uorescence intensity was measured at 1 min interval for 30 min with excitation at 341 nm. The observed rate (kobs) for HK was estimated using initial change of uorescence ratio of F376 and F476 from 0 to 5 min. Fluorescence measurement Model study for real-time uorescence kinase assay. To buﬀer solution (HEPES, 20 mM, pH 7.4) containing pyrene-DPA- Zn2+ complex (20 mM), diﬀerent ratio of ATP and ADP (total 10 mM) were in 1 mL of volume. Emission spectra (lex ¼ 341 nm) were recorded using uorescence spectrometer. Monitoring dephosphorylation in a kinase reaction Although most kinases are involved in phosphorylation of substrates, some are used for dephosphorylation, with transfer of the phosphate group from the substrate to ADP. To establish a more versatile kinase assay method, it is also crucial to analyze the kinases associated with dephosphorylation. Herein, CK, which transfers a phosphate group from phosphocreatine to ADP, was exploited as an example enzyme to show the method's application for the dephosphorylation process (Fig. 3a).26 Progression of the CK reaction caused a decrease in the ADP/ATP ratio, the opposite result to that with HK, which could be inferred from the decreased monomer emission at 376 nm and increased excimer emission at 476 nm (Fig. S4†). The change in ratiometric uorescence was proportional to the concentration of CK from 0 to 1 unit, and the rate constant, which was estimated from the uorescence change, displayed a linear correlation with the concentration of CK (Fig. 3b and c). Therefore, the developed kinase assay system can be applied to analyze dephosphorylation as well as phosphorylation in real time without additional assistance, such as a modied substrate or cascade enzyme reaction. In addition, rapid and simple screening methods for multiple inhibitor candidates are required in the pharmaceu- tical eld. To achieve this purpose, the microwell plates were photographed upon irradiation with a handheld UV lamp to record the uorescence changes for the enzyme activity and inhibitor eﬃciency. As shown in Fig. 4c, during Fig. 4 Inhibition eﬃciency for (a) HK and (b) CK with various concentrations of NBG and IAA, respectively, in buﬀer solution (HEPES, 20 mM, pH 7.4) with each substrate; (c) photographs of the pyrene- DPA-Zn2+ complex in the presence of kinases and inhibitors. [pyrene- DPA-Zn2+ complex] ¼ 20 mM, [ATP] or [ADP] ¼ 10 mM, [Mg2+] ¼ 80 mM, [glucose] ¼ 1 mM, [phosphocreatine] ¼ 0.75 mM, [HK] ¼ 1 unit, [NBG] ¼ 5 mM, [CK] ¼ 1 unit per mL, [IAA] ¼ 100 mM, lex ¼ 341 nm. Fig. 3 (a) Enzymatic reaction formula of CK associated with dephosphorylation; (b) ﬂuorescence intensity ratios of excimer (F476) and monomer (F376) emissions over time for the pyrene-DPA-Zn2+ complex with various concentrations of CK (0 to 1 unit per mL) in buﬀer solution (HEPES, 20 mM, pH 7.4); (c) plot of kobs against concentration of CK. Inhibitor assay for the kinases The fact that most kinases play an essential role in biological processes makes the development of inhibitors crucial. To demonstrate that the suggested kinase assay method can be used for inhibitor assays, the eﬃciency of inhibitors for both HK and CK was measured by using the change in ratiometric uorescence. The inhibition eﬃciencies of well-known inhibitors of HK and CK, N-benzoyl-D-glucosamine (NBG) and iodoacetamide (IAA), respec- tively, were conrmed against an increased concentration of inhibitor (Fig. 4). The half maximal inhibitory concentration (IC50) of the inhibitors estimated with the developed assay system was compared with the reported values (Table S2†). The results corre- lated without any large diﬀerences, indicating that the system was applicable as an inhibitor assay.38,39 Model study for real-time uorescence kinase assay Fig. 2 (a) Enzymatic reaction formula of HK associated with phos- phorylation; (b) ﬂuorescence intensity ratios of monomer (F376) and excimer (F476) emissions over time for the pyrene-DPA-Zn2+ complex with various concentrations of HK (0 to 1 unit) in buﬀer solution (HEPES, 20 mM, pH 7.4); (c) plot of the logarithm of kobs against concentration of HK. [pyrene-DPA-Zn2+] ¼ 20 mM, [ATP] ¼ 10 mM, [Mg2+] ¼ 80 mM, [glucose] ¼ 1 mM, lex ¼ 341 nm. To evaluate the inuence on the pyrene-DPA-Zn2+ complex of ATP and ADP, which are common components for general kinase reactions, the uorescence changes with ATP and ADP were measured. Addition of ADP to the pyrene-DPA-Zn2+ complex increased the monomer emission at 376 nm in proportion to the © 2021 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2021, 11, 10375–10380 \| 10377 Paper View Article Online View Article Online RSC Advances glucose, thereby increasing the ADP/ATP ratio (Fig. 2a).35–37 As the HK reaction proceeded and the ADP/ATP ratio increased gradually, the excimer emission at 476 nm gradually decreased with a concomitant increase in monomer emission at 376 nm, in conjunction with an increase in the concentration of HK from 0 to 1 unit (Fig. S2†); the monomer/excimer emission ratio changed in proportion to the concentration of HK during the reaction time (Fig. 2b). In addition, the logarithm of observed rate constant (kobs) for the enzyme reaction measured by using the ratiometric uorescence change showed a linear correlation with the concentration of HK (Fig. 2c) and enzymatic parame- ters (Km, Vmax) obtained from the method showed little diﬀer- ence with reported value (Fig. S3, Table S1†), allowing quantication and kinetic study of the enzyme from the uo- rescence change. As a result, it was conrmed that the HK activity involved in phosphorylation can be estimated from the developed assay system in real time by tracking the ADP/ATP ratio change, rather than the enzyme substrate, glucose. glucose, thereby increasing the ADP/ATP ratio (Fig. 2a).35–37 As the HK reaction proceeded and the ADP/ATP ratio increased gradually, the excimer emission at 476 nm gradually decreased with a concomitant increase in monomer emission at 376 nm, in conjunction with an increase in the concentration of HK from 0 to 1 unit (Fig. S2†); the monomer/excimer emission ratio changed in proportion to the concentration of HK during the reaction time (Fig. 2b). Model study for real-time uorescence kinase assay In addition, the logarithm of observed rate constant (kobs) for the enzyme reaction measured by using the ratiometric uorescence change showed a linear correlation with the concentration of HK (Fig. 2c) and enzymatic parame- ters (Km, Vmax) obtained from the method showed little diﬀer- ence with reported value (Fig. S3, Table S1†), allowing quantication and kinetic study of the enzyme from the uo- rescence change. As a result, it was conrmed that the HK activity involved in phosphorylation can be estimated from the developed assay system in real time by tracking the ADP/ATP ratio change, rather than the enzyme substrate, glucose. Most typical kinase assays except for recently reported research, which assayed both hexokinase and pyruvate kinase as a sequential monitoring,31 have focused on the analysis of only mono-directional changes, either phosphorylation or dephosphorylation, although the methods were based on changes in ATP and ADP and then extended for several kinases.17,20 However, considering the reversibility of reactions exhibited by kinases, it is necessary for a general kinase assay to analyze the bidirectional changes between ATP and ADP that indicate both phosphorylation and dephosphorylation. The developed assay method in this study provided evidence for the eﬀective utilization of bidirectional changes in kinase reactions, and consequently, the results are suggested as a general method for versatile small molecule kinase assays. Open Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Acknowledgements 23 E. A. Weitz, J. Y. Chang, A. H. Roseneld, E. A. Morrow and V. C. Pierre, Chem. Sci., 2013, 4, 4052–4060. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1A2B5B01002392). 24 S. H. Hewitt, J. Parris, R. Mailhot and S. J. Butler, Chem. Commun., 2017, 53, 12626–12629. 25 F. Huang, G. Hao, F. Wu and G. Feng, Analyst, 2015, 140, 5873–5876. There are no conicts to declare. 22 Y. Kurishita, T. Kohira, A. Ojida and I. Hamachi, J. Am. Chem. Soc., 2010, 132, 13290–13299. ss Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. M. Kraus, H. Keilhack, K. Nagashima, S. Krauss, C. Paweletz, R. C. Hendrickson, A. S. Feldman, C.-L. Wu, J. Rush, J. Vill´en and S. P. Gygi, Nat. Biotechnol., 2009, 27, M. Kraus, H. Keilhack, K. Nagashima, S. Krauss, C. Paweletz, R. C. Hendrickson, A. S. Feldman, C.-L. Wu, Monitoring dephosphorylation in a kinase reaction [pyrene-DPA-Zn2+ complex] ¼ 20 mM, [ADP] ¼ 10 mM, [Mg2+] ¼ 80 mM, [phosphocreatine] ¼ 0.75 mM, lex ¼ 341 nm. Fig. 3 (a) Enzymatic reaction formula of CK associated with dephosphorylation; (b) ﬂuorescence intensity ratios of excimer (F476) and monomer (F376) emissions over time for the pyrene-DPA-Zn2+ complex with various concentrations of CK (0 to 1 unit per mL) in buﬀer solution (HEPES, 20 mM, pH 7.4); (c) plot of kobs against concentration of CK. [pyrene-DPA-Zn2+ complex] ¼ 20 mM, [ADP] ¼ 10 mM, [Mg2+] ¼ 80 mM, [phosphocreatine] ¼ 0.75 mM, lex ¼ 341 nm. Fig. 3 (a) Enzymatic reaction formula of CK associated with dephosphorylation; (b) ﬂuorescence intensity ratios of excimer (F476) and monomer (F376) emissions over time for the pyrene-DPA-Zn2+ complex with various concentrations of CK (0 to 1 unit per mL) in buﬀer solution (HEPES, 20 mM, pH 7.4); (c) plot of kobs against concentration of CK. [pyrene-DPA-Zn2+ complex] ¼ 20 mM, [ADP] ¼ 10 mM, [Mg2+] ¼ 80 mM, [phosphocreatine] ¼ 0.75 mM, lex ¼ 341 nm. Fig. 4 Inhibition eﬃciency for (a) HK and (b) CK with various concentrations of NBG and IAA, respectively, in buﬀer solution (HEPES, 20 mM, pH 7.4) with each substrate; (c) photographs of the pyrene- DPA-Zn2+ complex in the presence of kinases and inhibitors. [pyrene- DPA-Zn2+ complex] ¼ 20 mM, [ATP] or [ADP] ¼ 10 mM, [Mg2+] ¼ 80 mM, [glucose] ¼ 1 mM, [phosphocreatine] ¼ 0.75 mM, [HK] ¼ 1 unit, [NBG] ¼ 5 mM, [CK] ¼ 1 unit per mL, [IAA] ¼ 100 mM, lex ¼ 341 nm. 10378 \| RSC Adv., 2021, 11, 10375–10380 © 2021 The Author(s). Published by the Royal Society of Chemistry View Article Online Paper Paper RSC Advances phosphorylation, the excimer emission observed in the pres- ence of ATP was changed to monomer emission by HK activity, and the inhibition of enzyme activity by NBG allowed the probe to maintain the excimer emission. By contrast, CK changed the monomer emission to excimer emission, but monomer emission was maintained in the presence of IAA. It is believed that these results, in which enzyme activity and inhibitor eﬃciency can be analyzed by the naked eye, will permit the application of the developed system to high-throughput screening of kinase inhibitors. 9 A.-S. Bretonnet, A. Jochum, O. Walker, I. Krimm, P. Goekjian, O. Marcillat and J.-M. Lancelin, J. Med. Chem., 2007, 50, 1865–1875. 10 M. Open Access Article. Published on 10 March 2021. Downloaded on 10/24/2024 6:18:12 A This article is licensed under a Creative Commons Attribution 3.0 Unported In summary, a real-time and versatile small molecule kinase assay system using a ratiometric uorescence probe was proposed. The developed system, based on monitoring of the interchange of ATP and ADP, can be used for various kinases, regardless of their specic substrate. In addition, the reversible uorescence change of the ratiometric uorescence probe is applicable for both phosphorylation and dephosphorylation per- formed by various small molecule kinases, unlike other methods, which have only focused on mono-directional analysis of kinase reactions. As a proof of concept, the activities of HK and CK were analyzed as examples of phosphorylation and dephosphorylation, respectively, and the assay system was also used for inhibitor assays. In addition, the uorescence change was observable with the naked eye, demonstrating the applicability of this method to high-throughput screening. Overall, although a limitation remains that is diﬃcult to apply in cellular environment containing ATP and ADP, the proposed system provides a general method that can be extended to various kinase and inhibitor assays. 12 R. Seethala and R. Menzel, Anal. Biochem., 1998, 255, 257– 262 12 R. Seethala and R. Menzel, Anal. Biochem., 1998, 255, 257– 262. 13 M. D. Shults, K. A. Janes, D. A. Lauﬀenburger and B. Imperiali, Nat. Methods, 2005, 2, 277–283. 14 E. Lukovic, J.-A. Gonzalez-Vera and B. Imperiali, J. Am. Chem. Soc., 2008, 130, 12821–12827. 15 J. Ali, A. Jackson, A. Howells and A. Maxwell, Biochemistry, 1993, 32, 2717–2724. 16 A. Platt, H. C. Ross, S. Hankin and R. J. Reece, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 3154–3159. 17 S. Ren, B. Li and L. Zhang, Analyst, 2013, 138, 3142–3145. 18 J. Bai, L. Liu, Y. Han, C. Jiac and C. 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https://openalex.org/W4235709841	https://www.researchsquare.com/article/rs-24076/latest.pdf	English	null	Stigmasterol Sensitizes Endometrial Cancer Cells to Chemotherapy By repressing Nrf2 Signal Pathway	Research Square (Research Square)	2,020	cc-by	6,809	Primary research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on October 6th, 2020. See the published version at https://doi.org/10.1186/s12935-020-01470-x. Page 1/19 Abstract Background: Chemoresistance reduces the 5-year survival rate of endometrial cancer patient, which is the current major obstacle for cancer therapy. Increasing evidence state that Nrf2 contributes to chemoresistance in several kinds of cancer. However, its role in endometrial cancer cells remains unclarified. Methods: Immunohistochemistry staining was used to detect the expression of Nrf2 in normal patient and endometrial cancer patient. Stable transfection Ishikawa cell line with high level of Nrf2 was established to evaluate its role in chemoresistance. Dot blot assays were used to assess global hydroxymethylation level after stigmasterol treatment. Cellular growth profile was detected by CCK8 assay. Western blot was used to evaluate the changes of the target molecules after various treatments. Results: Nrf2 is overexpressed in endometrial cancer tissues compared with the normal endometrium. Overexpression of Nrf2 resulted in decrease sensitivity to cisplatin. In addition, stigmasterol has been identified as a novel Nrf2 inhibitor. It enhanced the sensitivity of endometrial cancer cells to cisplatin, and the underlying mechanism is that stigmasterol declines the Nrf2 protein level. Conclusions: Our findings identified stigmasterol as a new potential inhibitor of Nrf2 and highlight a critical role of stigmasterol in overcoming chemoresistance in endometrial cancer therapy. Background Endometrial cancer is the main gynecological malignancy and most of cases occur in post-menopausal women [1-4]. In recent decade, it shows an increasing incidence rate, especially in developing country [3, 5]. Usually, surgery is the optimal therapy strategy for the patients with early-stage endometrial cancer, whereas chemotherapy is chosen to administrate the advanced and recurrent patients. As the first-line chemotherapeutics for endometrial cancer therapy, cisplatin and paclitaxel exhibit well-inhibition effect on cancer cell growth, however, chemoresistance is still a major obstacle in endometrial cancer therapy [6]. Therefore, it is urgent to find a biomarker of chemoresistance and develop a new and effective anticancer therapeutic regimen or strategy. As a critical molecule of antioxidant response, Nrf2 has multiple roles in cancer development including chemoresistance, proliferation and anti-apoptosis [7-10]. In previous study, the bad effects of Nrf2 has been addressed, wang et al. demonstrated that high level of Nrf2 enhances chemoresistance, whereas knocking down Nrf2 sensitizes cancer cells to chemotherapy [11]. Consistent with these findings, it was showed that suppression of Nrf2 sensitizes ovarian cancer cells to doxorubicin and cisplatin [8]. Other study also found that xenografts derived from Nrf2-silenced lung cancer cells were more sensitive to carboplatin treatment [12, 13]. These data suggest that Nrf2 play an essential role in chemoresistance. However, how it involves in chemoresistance is not clear in endometrial cancer. Page 2/19 Page 2/19 It has been found that many compounds are able to inhibit the expression of Nrf2 protein and its downstream target genes containing an enhancer sequence known as the antioxidant response element (ARE) [14-17]. Some of the target genes have the function to detoxification and removal of pharmacologic agents, which may contribute to chemoresistance [18-20]. Therefore, targeting Nrf2 and Nrf2-regulated downstream gene is a good strategy for cancer therapy. Donna Zhang et. al first found that brusatol could enhance the chemotherapy sensitivity by facilitating the degradation of Nrf2 protein [21]. Consistent with this study, we also found that brusatol could enhance the metformin-induced inhibition effect on endometrial cancer [22]. In addition, other study also pointed out that metformin can decline the mRNA level and protein level in HepG2 cells, and the decline of Nrf2 is mediated by keap1- independent and AMPK-independent pathway [23]. Some of studies reported a number of Nrf2 inhibitors through high-throughput screening, such as dexamethasone, clobetasol propionate, wogonin, camptothecin [14, 15, 17, 24, 25]. Background Although these compounds are potential candidate inhibitors, the inhibition effect on Nrf2 expression is not very strong. It is far from the inhibition effect of brusatol on Nrf2 expression. Therefore, it is necessary to search more effective compound to enhance chemotherapy sensitivity by downregulation of Nrf2. Previous study mentioned that the extract from mangrove has the effect of antioxidant response and anti-inflammation, this prompts us to investigate whether some of the compound from this plant may have the inhibition effect on Nrf2 expression and Nrf2-induced biological function, such as chemoresistance. Therefore, the objective of this study is to clarify the molecular mechanisms how Nrf2 involves in chemoresistance in endometrial cancer and find some of the candidate inhibitors to overcome drug resistance, which would be relevant to development of new therapeutic strategies that would improve patient care. Cell culture The endometrial cancer cell lines, Ishikawa and SPEC2, were used in this study. MDA-MB-231 is a breast cancer cell line. Ishikawa and MDA-MB-231 cell lines were purchased from the American Type Tissue Type Tissue Collection (Manassas, VA, USA), SPEC2 is kindly provided by Dr. Zheng W (University of Texas Southwestern Medical Center, Dallas, TX, USA) and maintained in our lab. These cell lines were cultured in Medium DMEM: F12 (1:1, GIBCO) with 100µg/ml streptomycin (Life Technologies, Inc., Rockville, MD), 100 U/ml penicillin G and 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA). RNA isolation and quantitative real time PCR Total RNA was harvested with Trizol reagent. 1µg of total RNA was reverse transcribed into cDNA using the reverse transcriptase kit (Qiagen company, Frankfurt, Federal Republic of Germany). The primers used to amplification of Nrf2 and GAPDH were listed table 1. The water was served as negative control. Plasmid transfection Tet1 plasmid was transfected into Ishikawa cells using Lipofectamine 3000 TM (Invitrogen) according to the manufacturer’s protocol. The transfection efficiency was confirmed by western blot. Establishment of stable cell lines To identify the role of Nrf2 in chemotherapy resistance, the stable cell lines with high levels of Nrf2 or Keap1 were established. Ishikawa- and Spece2 derived stable cell lines, with incorporation of Nrf2 or Keap1, were established using lentivirus system as described previously. Stable Ishikawa and spece2 cells were continuously cultured in medium containing 1.5μg/ml puromycin (sigma). The efficiency of transfection was determined with western blot. IHC Total 120 endometrial tissue samples, including 10 cases of normal endometrium, 20 cases of simple hyperplasia and 90 cases of endometrial cancer, were enrolled in this study. These samples were collected from the Department of Obstetrics and Gynecology of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, China. Samples collected in compliance with institutional review board regulations from the Medical College, Tongji University, China. Total 120 endometrial tissue samples, including 10 cases of normal endometrium, 20 cases of simple hyperplasia and 90 cases of endometrial cancer, were enrolled in this study. These samples were collected from the Department of Obstetrics and Gynecology of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, China. Samples collected in compliance with institutional review board regulations from the Medical College, Tongji University, China. Page 3/19 Page 3/19 IHC analysis of Nrf2 expression pattern was performed as previously described. Briefly, firstly samples were deparaffinized in xylene and rehydrated in a graded series of ethanol, subsequently, 3.0% hydrogen peroxide was used to block the endogenous peroxidase activity. Following antigen retrieval, incubation overnight with rabbit anti-human Nrf2 primary antibodies at 4°C in a humid chamber, followed by 60- minute incubation with biotinylated secondary antibody (Dako, Carpinteria, CA, USA). Omitted primary antibodies were used as negative controls. Expression of Nrf2 was assessed as previous described. Western blot The changes of the proteins were detected with western blot. The cells underwent indicated treatment were lysed and loaded to SDS-polyacrylamide gel, electrophoresed and transferred to PVDF membranes, then incubated primary antibodies (Nrf2, #12721, CST company; #ab62352, Abcam company) and second antibodies. After final wash with PBS, the bands was detected with chemiluminescence detection system (ECL detection kit; Pierce, Rockford, IL). Each experiment was performed at least three times. Drug treatment, Cell proliferation and Clonogenic assay Endometrial cancer cells were treated with cisplatin, stigmasterol, or combine to both drugs with indicated doses. Cell proliferation pattern was monitored with CCK8 assay. A clonogenic assay was carried out referring previously study[26]. ARE luciferase activity assay The MDA-MB-231 cell line constructed with NQO1-ARE luciferase reporter gene maintain in our lab. In order to investigate if the stigmasterol plays a role in regulating Nrf2-ARE signal pathway, the different dose of stigmasterol was used to treat the MDA-MB-231 cells. 24 hours later, the cells were harvested and lysed, the luciferase activity was determined by Dual-Luciferase Assay Kit (Promega). Protein and ligand structure preparation The structure of stigmasterol was exported from the data bank of Pubchem with SDF file, following translated to Mol2 format with Open Babel, then imported into SYBYL 2.0 package for Tripos optimization. The 3D structures of Nrf2 and PDB ID (PDB ID: 2FLU, 5F72, 3WN7 and 4ZY3) of the protein were selected from Protein Data Bank (PDB) based on the antagonistic effect of the study. Migration and invasion assay Ishikawa cells were seeded in the upper chamber of 24 transwell inserts with 8-um pores coated with (for invasion assay) or without Matrigel (for migration assay). The lower wells were also cultured with serum- free medium. After another 48h culture with indicated treatment, the cells still stayed in upper champers were wiped off using a cotton swab and the invading cells on the underside of the filter were fixed and stained with crystal violet, five randomly selected fields were counted. Dot blot for detection of 5hmC Dot blot analysis was carried out as previously described. Briefly, the total DNA was extracted and loaded on nitrocellulose membranes, following blocking with 10% skimmed milk for 1 hour after bake at 80°C for Page 4/19 10 min, then incubation with 5hmC primary antibody (1:500 dilutions, Active Motif) overnight at 4°C. After washing the HRP-conjugated secondary antibody, the membrane was treated with ECL and scanned by a scanner. Methylene blue (MB) staining served as a loading control. Apoptosis assay The cells underwent various treatment were harvested and fixed overnight with cold 70% ethanol. After spinning down, the cell pellet was re-suspended in PBS and incubated with Annexin V. The early apoptosis of the cells was analyzed by using flow cytometry. Protein-ligand docking calculations The feasibility of stigmasterol to be ligands for Nrf2 structures was evaluated using molecular docking. This was performed using Surflex-Dock module of SYBYL to evaluate the performance of the protein- ligand docking. If the total score was greater than 5 that indicates the binding between stigmasterol and Nrf2 is good. In addition, combined with a C score, a score close to 5 is considered to have better activity Nrf2 drives chemotherapy resistance Chemotherapy resistance is a pivotal obstacle for endometrial cancer therapy. In order to investigate the role of Nrf2 in chemotherapy resistance, we established Nrf2 overexpressed stable cell line. As showed in figure 2A, the stable cell line showed an approximately 8-fold increasing Nrf2 expression in mRNA level. Similarly, the increase Nrf2 protein expression also has been observed (Fig 2B). Subsequently, we estimated the proliferative activity after cisplatin treatment in Ishikawa cells and Ishikawa-Nrf2 stable cells. It was found that cisplatin could suppress the cellular growth with a dose-dependent manner in both Ishikawa and Ishikawa-Nrf2 cells, however, the parental Ishikawa is more sensitive to cisplatin treatment (Fig 2C). Moreover, we further investigated the expression of Nrf2 in both cell lines. We found that Nrf2 potently declined in Ishikawa cells when it exposure to increase dose of cisplatin, while, the decline is slowly in Ishikawa-Nrf2 cells (Fig. 2D). Therefore, it implies that Nrf2 plays an essential role in endometrial cancer chemotherapy resistance. Nrf2 overexpressed in endometrial carcinoma tissue To identify the role of Nrf2 in endometrial cancer development, we first investigate the expression pattern of Nrf2 in healthy controls and patients with various endometrial disorders, including simple hyperplasia and cancer. It was found that the weak staining signal was present in normal endometrium, whereas, the moderate Nrf2 staining signal was shown in simple hyperplasia tissue samples, the strong staining signal appeared in endometrial cancer samples (Fig1A& B). Meanwhile, we collected fresh endometrial tissue with various lesions and determined the expression of Nrf2 with western blot. An increasing trend of Nrf2 expression was observed accompanying with the lesion progress from normal endometrial tissue, to hyperplasia and carcinoma tissue (Fig1 C). These results suggest that Nrf2 play an important role in endometrial cancer development. Identifications of residues interacting with stigmasterol on Nrf2 binding site LigandScout 3.0 was used to identify protein residues that interact with stigmasterol with greatest affinities. Page 5/19 Page 5/19 Statistical analysis SPSS 19.0 was used for analyzing the cellular growth, changes of western blot bands and apoptosis rate. P < 0.05 was considered significant difference when compared with control group. Stigmasterol enhances the inhibition effect of cisplatin on migration and i As a potential inhibitor of Nrf2 signaling, we are wonder if the inhibition effect of stigmasterol could extend to other cancer malignant biological functions. Currently, it was found that stigmasterol not only facilitated cisplatin-driven migration inhibition but also invasion suppression (Fig.5A-D). Therefore, we further estimated the expression of migration or invasion related molecules. The joint treatment with both stigmasterol and cisplatin significantly decreased snail and vimentin expression in Ishikawa cells， however, reverse expression pattern in E-cadherin has been observed (Fig.5E). Stigmasterol is a potential inhibitor of Nrf2 Since Nrf2 involves in endometrial cancer chemotherapy resistance, it is an urgent to found a drug to inhibit this signal pathway. A potential candidate inhibitor called stigmasterol was selected by network pharmacology. The molecular structure of stigmasterol is shown in figure 3A. As showed in figure 3B&C, stigmasterol can enter into Nrf2 protein active pocket and the docking sites lie in this active pocket. A higher score indicated higher accuracy of the binding and we found these all had good binding property except 5F72 (Table 2). Yellow virtual bond is stable hydrogen bond formed between small molecule and protein. For the 5F72- stigmasterol complex, they interact with a hydrogen bond and the relevant Page 6/19 Page 6/19 aminoacid is Thr-69. Stigmasterol bound to the active site of 3WN7 via one hydrogen bond interaction, which were found to exist between the tested molecule and Arg-415. For 4ZY3- stigmasterol complex, which interacts through two hydrogen bonds and the key residues are Asn-414 and Ser-363 (Fig.D&E). Although the sores in mouse is higher than that in human (Table 2), considering it may exist a gap between theoretical prediction and physical truth, we decided to further verify the interactions through experiments. From above data, it suggests that stigmasterol is a potential candidate Nrf2 inhibitor. Stigmasterol sensitizes endometrial cancer cell to cisplatin by suppression of Nrf2 expression As shown in figure 4A, stigmasterol suppressed Nrf2, NQO1and HO1 expression with a dose dependent manner. Interesting is that there is no effect on cellular growth when stigmasterol treated Ishikawa cells alone. Cell growth assays showed that, in combination with cisplatin and stigmasterol treatment significantly inhibited the growth of endometrial cancer compared to treatment with the therapeutic drug alone (Fig.4B). Similarly, in Nrf2-overexpressed cells, stigmasterol also could enhance the inhibition effect of cisplatin (Fig.4C). The clonogenic assay produced a similar result; stigmasterol enhanced the inhibitory effect of cisplatin on endometrial cancer clonal growth (Fig. 4D & E). To determine whether combinational treatment with cisplatin and stigmasterol could affect the early apoptosis, the flow cytometry assay was performed. It was found that the early apoptosis rate is higher when Ishikawa cells were exposed to both stigmasterol and cisplatin compared to stigmasterol or cisplatin alone (Fig.4F &G), which suggests that stigmasterol is a potential inhibitor for Nrf2 and plays a critical role in enhancing chemotherapy sensitivity. Discussion Although many efforts and progresses in endometrial cancer therapy have been observed in the past, the five year survive rate has not been significantly improved [10, 19, 27, 28]. The high failure rate responding to chemotherapy in endometrial carcinoma attributes to the unclear molecular mechanisms of chemoresistance and there is no effective therapeutic strategy for overcoming the resistance. Therefore, it is urgent to discover the molecular mechanism and further find the potential candidate small molecule compound to facilitate the chemotherapy sensitivity. In previous study, it has been demonstrated that chemoresistance is associated with the crosstalk between leptin-induced Notch and IL-1 signaling in endometrial cancer. At the most recent, a study pointed that cancer stem cells are responsible for cancer proliferation and chemoresistance [20], it was found that SPARC-related modular calcium binding 2 (SMOC-2) drive the endometrial cancer chemoresistance functional as a endometrial cancer stem cell molecule. Similarly, Caner Saygin et.al reported that CD55, as unique signaling node, drives self-renewal and chemoresistance in endometrioid cancers [29]. Other study found that autophagy also involves in chemoresistance [30]. Despite these progresses have been achieved, the 5-years survival rate of those patients with endometrial cancer has not been remarkably improved. Thus, we carried out serial studies focusing on this issue. We found that IDH1 is overexpressed in endometrial cancer tissue and plays an essential role in chemoresistance. Moreover, we observed that chemoresistance was enhanced via a regulatory loop in which Nrf2 activated IDH1-ɑ-KG-TET1-Nrf2 signaling via binding to the ARE sites in the IDH1 promoter region [22]. This study highlights a critical role of IDH1-ɑ-KG-TET1-Nrf2 signaling in chemoresistance and suggests that rational combination therapy with metformin and chemotherapeutics has the potential to suppress chemoresistance. In addition, we also found that Nrf2 play an essential role in endometrial cancer development and chemoresistance. However, we did not further demonstrate how Nrf2 involves in chemoresistance and did not search for a potential medicine targeting Nrf2 for cancer therapy. Increasing evidence from recent studies showed that the transcription factor NF-E2-related factor 2 (Nrf2) plays a critical role in promoting cancer recurrence through increased tolerance to adjuvant chemo- and/or radiation therapies. It has been well addressed that Nrf2 involves in cancer chemoresistance [11, 19]. The mechanisms of Nrf2 mediated drug resistance involve multiple genes and details of the molecular pathways of such drug resistance have been summarized elsewhere. Stigmasterol enhances the efficacy of chemotherapeutic drugs through TET1-mediated hydroxymethylation via the Nrf2 signaling pathway Although stigmasterol could inhibit Nrf2 expression in both mRNA and protein levels, if the whole Nrf2 signaling and Nrf2-ARE activity have been inhibited is not clear. Currently, luciferase activity assay disclosed that stigmasterol significantly suppressed Nrf2-ARE activity (Fig.6A). Previous study demonstrated that Nrf2 is regulated by Tet1 via hydroxymethylation. Whether stimasterol affects the Tet1-driven hydroxymethylation has not been clarified. In this study, it was found that stimasterol inhibited Tet1 with a dose manner (Fig.6B). The dot bot demonstrated that stigmasterol obviously decreased the 5hmC level (Fig.6C). Moreover, we found that stigmasterol could block Tet1 induced Nrf2 expression (Fig. 6D). These results suggest that stigmasterol could inhibit Nrf2 by suppressing Tet1- Page 7/19 Page 7/19 drived hydroxymethylation. In addition, we decreased Nrf2 level by transfection with keap1 plasmid and found that low level of Nrf2 resulted in enhanced sensitive to cisplatin (Fig.6E). drived hydroxymethylation. In addition, we decreased Nrf2 level by transfection with keap1 plasmid and found that low level of Nrf2 resulted in enhanced sensitive to cisplatin (Fig.6E). Discussion This prompts us further investigate whether stigmasterol could repress human Nrf2 expression, especially in human endometrial cancer cells. As expected, we found that stigmasterol inhibited Nrf2 protein expression in human endometrial cancer with a dose manner. Most importantly, stigmasterol combine with cisplatin could significantly suppress Nrf2-ARE activity and enhance cisplatin-induced inhibitions on cellular growth, migration and invasion. In addition, stigmasterol enhanced cisplatin-induced early apoptosis. In previous study, most inhibitors of Nrf2 repress Nrf2 protein level through suppressing its transcription activation except of brusatol [14, 15, 17]. Here, we found that stigmasterol inhibited Tet1 expression and Tet1-induced hydroxymethylation, following in turn inhibit Nrf2 protein expression. So, the decline of Nrf2 protein by stigmasterol also attributes to decrease activity of transcription. A complete understanding of the role of Nrf2 will facilitate overcoming chemoresistance. The joint effects between stigmasterol and cisplatin observed in endometrial cancer cells provide the possibility of achieving improved therapeutic effects with lower chemotherapeutic doses. Our findings provide the foundation for the hypothesis that stigmasterol sensitizes endometrial cancer cells to chemotherapy by suppression of Nrf2 expression. Discussion Most recent, it has been reported that Nrf2 contributed to chemoresistance and was associated with a poor prognosis in pancreatic cancer patients. Wu J et.al also reported that Nrf2 induced cisplatin resistance via suppressing the iron export related gene SLC40A1 in ovarian cancer cells [31]. In our previous endometrial cancer studies, we showed that high level of Nrf2 expression is clearly responsible for chemoresistance [19]. More importantly, brusatol, a specific inhibitor of Nrf2, could reverse chemoresistance in multiple cancers including endometrial cancer, but the detail mechanism needs further study. In current study, it was found Page 8/19 Page 8/19 that Nrf2 is aberrantly expressed in endometrial cancer or pre-cancer tissues (Fig.1A). Stably transfection of Nrf2 resulted in cisplatin resistance accompanying with slightly decline of Nrf2 protein in Ishikawa- Nrf2 cells (Fig.2C & D). These data suggest that Nrf2 plays an essential role in chemoresistance in endometrial cancer. that Nrf2 is aberrantly expressed in endometrial cancer or pre-cancer tissues (Fig.1A). Stably transfection of Nrf2 resulted in cisplatin resistance accompanying with slightly decline of Nrf2 protein in Ishikawa- Nrf2 cells (Fig.2C & D). These data suggest that Nrf2 plays an essential role in chemoresistance in endometrial cancer. Since Nrf2 involves in chemoresistance, targeting this molecule maybe enhance the response to chemotherapy. Thus, downregulating Nrf2 protein level or repressing its transcription activation is a possible way to inhibit Nrf2 and Nrf2-dirven chemoresistance. Previous study showed that brusatol could reverse chemoresistance in several different kinds of cancer by downregulation of Nrf2 expression [21, 32, 33]. However，further study found that brusatol is not directly repress the Nrf2 pathway, it has broad- spectrum inhibition effect on the majority of detected proteins. So, this nonselective mechanism of brusatol limits its possibility of application in clinical practice. Since then, increasing potential candidate inhibitor of Nrf2 has been reported, including glucocorticoids, flavonoids, wogonin, dexamethasone, and luteolin. However, these compounds have relative weak inhibition effect on Nrf2 expression compared with brusatol. In current study, we found a possible inhibitor of Nrf2 by model analysis via network pharmacology. It is stigmasterol which distributes in many plants such as halodrymium. In this prediction model, stigmasterol matches more with mouse Nrf2 protein, rather the human Nrf2 protein (Fig.3). So, we compared the gene sequence of Nrf2 between mouse and human, it was found it is a highly conservative gene. Availability of data and materials Data files generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Funding This work was supported by grants from the National Natural Science Foundation of China [grant numbers 81973289]. Consent for publication Individual’s personal data details from donors are not mentioned in this manuscript. Individual’s personal data details from donors are not mentioned in this manuscript. Conclusion Our results showed that Nrf2 plays an essential role in chemoresistance in endometrial cancer. Moreover, stigmasterol, as a novel potential inhibitor of Nrf2 has been identified, has the potential to suppress chemoresistance in combination therapy with cisplatin. Page 9/19 Abbreviations 5hmc: 5-hydroxymethylcytosine ARE：Antioxidant response element IDH1: Isocitrate dehydrogenase [NADP] IHC: Immunohistochemical NQO1: NAD(P)H dehydrogenase [quinone] 1 NRF2: Nuclear factor erythroid 2-related factor 2 PBS: phosphate buffer saline PCR: Polymerase chain reaction TET1: Tet methylcytosine dioxygenase 1 Ethics approval and consent to participate Human samples were obtained under approval of the institutional review board from the Medical College, Tongji University, China. Competing interests The authors declare no conflicts of interest. Contributions Page 10/19 LH and FGR designed the study and wrote the manuscript, LH, CHF, BMZ, ZD and YSH performed the experiments and analyzed the data. ZD and YSH scanned the Nrf2 inhibitor, YJ, ZHT and ZWX read the pathological slides, CHF revised the manuscript. FGR supervised the study. Acknowledgements We thank all the numbers of our team for their help in this project. References 1. Satyaswaroop PG, Clarke CL, Zaino RJ, Mortel R. Apparent resistance in human endometrial carcinoma during combination treatment with tamoxifen and progestin may result from desensitization following downregulation of tumor progesterone receptor. Cancer Lett 1992, 62(2):107-114. 1. Satyaswaroop PG, Clarke CL, Zaino RJ, Mortel R. Apparent resistance in human endometrial carcinoma during combination treatment with tamoxifen and progestin may result from desensitization following downregulation of tumor progesterone receptor. Cancer Lett 1992, 62(2):107-114. 2. Perez-Medina T, Bajo J, Folgueira G, Haya J, Ortega P. Atypical endometrial hyperplasia treatment with progestogens and gonadotropin-releasing hormone analogues: long-term follow-up. 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References Targeting cancer stem cell signature gene SMOC-2 Overcomes chemoresistance and inhibits cell proliferation of endometrial carcinoma. EBioMedicine 2019, 40:276-289. 21. Ren D, Villeneuve NF, Jiang T, Wu T, Lau A, Toppin HA ,et al. Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. Proc Natl Acad Sci U S A 2011, 108(4):1433-1438. 22. Bai M, Yang L, Liao H, Liang X, Xie B, Xiong J ,et al. Metformin sensitizes endometrial cancer cells to chemotherapy through IDH1-induced Nrf2 expression via an epigenetic mechanism. Oncogene 2018, 37(42):5666-5681. 23. Tian D, Shi Y, Chen D, Liu Q, Fan F. The Wnt inhibitor LGK-974 enhances radiosensitivity of HepG2 cells by modulating Nrf2 signaling. Int J Oncol 2017, 51(2):545-554. 23. Tian D, Shi Y, Chen D, Liu Q, Fan F. The Wnt inhibitor LGK-974 enhances radiosensitivity of HepG2 cells by modulating Nrf2 signaling. Int J Oncol 2017, 51(2):545-554. 24. Limonciel A, Jennings P. A review of the evidence that ochratoxin A is an Nrf2 inhibitor: implications for nephrotoxicity and renal carcinogenicity. Toxins (Basel) 2014, 6(1):371-379. 24. Limonciel A, Jennings P. A review of the evidence that ochratoxin A is an Nrf2 inhibitor: implications for nephrotoxicity and renal carcinogenicity. Toxins (Basel) 2014, 6(1):371-379. Page 12/19 25. Tang X, Wang H, Fan L, Wu X, Xin A, Ren H ,et al. Luteolin inhibits Nrf2 leading to negative regulation of the Nrf2/ARE pathway and sensitization of human lung carcinoma A549 cells to therapeutic Page 12/19 25. Tang X, Wang H, Fan L, Wu X, Xin A, Ren H ,et al. Luteolin inhibits Nrf2 leading to negative regulation of the Nrf2/ARE pathway and sensitization of human lung carcinoma A549 cells to therapeutic Tables Table 1. Sequences of primers used for amplification of target genes Reverse: 5'- GGAAGATGGTGATGGGATT -3' Reverse: 5'- GGAAGATGGTGATGGGATT -3' Table 2. The predicted results of stigmasterol matches with Nrf2 protein Table 2. The predicted results of stigmasterol matches with Nrf2 protein Page 13/19 Page 13/19 Nrf2 active structural domain Human Mouse 2FLU 5F72 3WN7 4ZY3 Total score 6.7571 3.6141 6.695 6.1729 C score 2 1 3 4 Figures Figures Figure 1 Stigmasterol enhances the efficacy of cisplatin through TET1-mediated hydroxymethylation via the Nrf2 signaling pathway. Nrf2-ARE activity assay was performed by luciferase activity determination after stigmasterol treatment with indicate dose. p<0.05 compared with control (A). Western blot was used to estimate the effect of stigmasterol on Tet1 expression (B). Dot-blot assay results of EC cells with stimasterol treatment (C). Full length of Tet1 plasmid was tranfected in Ishikawa cells, then stimasterol was used to treat these cells. Western blot was carried out to determine whether tet1 overexpression could block stigmasterol-induced Nrf2 decline (D). CCK-8 was used to detect the effect of stigmasterol and cisplatin on the spec-2 cells with low level of Nrf2 due to stablely transfection with Keap1, p<0.05, ** p<0.01 compared with control (E). Figure 1 Figure 1 Figure 1 Figure 1 Stigmasterol enhances the efficacy of cisplatin through TET1-mediated hydroxymethylation via the Nrf2 signaling pathway. Nrf2-ARE activity assay was performed by luciferase activity determination after stigmasterol treatment with indicate dose. p<0.05 compared with control (A). Western blot was used to estimate the effect of stigmasterol on Tet1 expression (B). Dot-blot assay results of EC cells with stimasterol treatment (C). Full length of Tet1 plasmid was tranfected in Ishikawa cells, then stimasterol was used to treat these cells. Western blot was carried out to determine whether tet1 overexpression could block stigmasterol-induced Nrf2 decline (D). CCK-8 was used to detect the effect of stigmasterol and cisplatin on the spec-2 cells with low level of Nrf2 due to stablely transfection with Keap1, p<0.05, ** p<0.01 compared with control (E). Stigmasterol enhances the efficacy of cisplatin through TET1-mediated hydroxymethylation via the Nrf2 signaling pathway. Nrf2-ARE activity assay was performed by luciferase activity determination after stigmasterol treatment with indicate dose. p<0.05 compared with control (A). Western blot was used to estimate the effect of stigmasterol on Tet1 expression (B). Dot-blot assay results of EC cells with stimasterol treatment (C). Full length of Tet1 plasmid was tranfected in Ishikawa cells, then stimasterol was used to treat these cells. Western blot was carried out to determine whether tet1 overexpression could block stigmasterol-induced Nrf2 decline (D). CCK-8 was used to detect the effect of stigmasterol and cisplatin on the spec-2 cells with low level of Nrf2 due to stablely transfection with Keap1, p<0.05, ** p<0.01 compared with control (E). Page 14/19 Figure 2 Stigmasterol enhances the inhibition effect of cisplatin on migration and invasion activity. The cells were seed on the transwell chambers on the 24-well plate, after indicated treatments, the migration cells (A & B) and invasive cells (C & D) were counted, respectively. p<0.05 compared with control. The migration and invasion related molecules were detected by western blot (E). The drug concentrations are as follows: cisplatin, 10 μg/ml/ml; stigmasterol, 10 μg/ml. Figure 2 Figure 2 Stigmasterol enhances the inhibition effect of cisplatin on migration and invasion activity. The cells were seed on the transwell chambers on the 24-well plate, after indicated treatments, the migration cells (A & B) and invasive cells (C & D) were counted, respectively. p<0.05 compared with control. The migration and invasion related molecules were detected by western blot (E). The drug concentrations are as follows: cisplatin, 10 μg/ml/ml; stigmasterol, 10 μg/ml. Figure 1 Stigmasterol enhances the inhibition effect of cisplatin on migration and invasion activity. The cells were seed on the transwell chambers on the 24-well plate, after indicated treatments, the migration cells (A & B) and invasive cells (C & D) were counted, respectively. p<0.05 compared with control. The migration and invasion related molecules were detected by western blot (E). The drug concentrations are as follows: cisplatin, 10 μg/ml/ml; stigmasterol, 10 μg/ml. Page 15/19 Page 15/19 Figure 3 Stigmasterol sensitizes endometrial cancer cell to cisplatin by suppression of Nrf2 expression. Western blot was used to detect the effect of stigmasterol on Nrf2 expression (A). Stigmasterol enhances the inhibitory effect of cisplatin on EC cell proliferation. Stigmasterol was used at indicated doses; a CCK8 assay (B & C) and colony formation assay (D &E) were used to assess proliferation. The left panels show representative images of crystal violet-stained colonies. Right panels show comparison of colony numbers following indicated treatments. , p<0.05 compared with control (D &E). The early apoptosis was determined by FCM, p<0.05 compared with control (F & G). Figure 3 Stigmasterol sensitizes endometrial cancer cell to cisplatin by suppression of Nrf2 expression. Western blot was used to detect the effect of stigmasterol on Nrf2 expression (A). Stigmasterol enhances the inhibitory effect of cisplatin on EC cell proliferation. Stigmasterol was used at indicated doses; a CCK8 assay (B & C) and colony formation assay (D &E) were used to assess proliferation. The left panels show representative images of crystal violet-stained colonies. Right panels show comparison of colony numbers following indicated treatments. , p<0.05 compared with control (D &E). The early apoptosis was determined by FCM, p<0.05 compared with control (F & G). Page 16/19 Figure 4 Stigmasterol is the Nrf2 inhibitor candidate. Network candidate. (A) the molecular structure of stigmastero atom, red is oxygen atom. The predicted human Nrf2 Nrf2 domain 3WN7 and 4ZY3 (C) with which stigma Figure 4 Stigmasterol is the Nrf2 inhibitor candidate. Network pharmacology screens the possible inhibitor candidate. (A) the molecular structure of stigmasterol. White represents carbon atom, blue is hydrogen atom, red is oxygen atom. The predicted human Nrf2 structural domain 2FLU and 5F72 (B) and mouse Nrf2 domain 3WN7 and 4ZY3 (C) with which stigmasterol binds. It shows the key amino acid of above structural domain interacting with stigmasterol, including human (D) and mouse (E) Nrf2 protein. Figure 4 Stigmasterol is the Nrf2 inhibitor candidate. Networ ( ) f Figure 4 Figure 4 Figure 4 Stigmasterol is the Nrf2 inhibitor candidate. Network pharmacology screens the possible inhibitor candidate. (A) the molecular structure of stigmasterol. White represents carbon atom, blue is hydrogen atom, red is oxygen atom. The predicted human Nrf2 structural domain 2FLU and 5F72 (B) and mouse Nrf2 domain 3WN7 and 4ZY3 (C) with which stigmasterol binds. It shows the key amino acid of above structural domain interacting with stigmasterol, including human (D) and mouse (E) Nrf2 protein. Page 17/19 Page 17/19 Page 18/19 Figure 5 Nrf2 involves in chemotherapy resistance. Nrf2 is high expressed in Nrf2 transfected stable cell line in mRNA level (A) and protein level (B). CCK8 assay was performed to determine the inhibition effect of cisplatin on cell proliferative activity. p<0.05 compared with the control group (C). Western blot was used to detect the change of the Nrf2 protein after indicated dose of cisplatin treatment in both parental Ishikawa cells and Ishikawa-Nrf2 cells (D). Figure 6 Nrf2 is overexpressed in endometrial cancer. The expression pattern of Nrf2 in normal endometrium (n=10), hyperplasia (n=20) and endometrial cancer (n=90) was assessed using IHC staining. Representative images were captured at 100×magnification (A). The IHC scores in endometrial lesion tissues compared with normal endometrial tissues are presented (B). p<0.05 compared with normal endometrial tissues. The expression pattern of Nrf2 in endometrial fresh tissue was determined by western blot (C). Figure 5 Nrf2 involves in chemotherapy resistance. Nrf2 is high expressed in Nrf2 transfected stable cell line in mRNA level (A) and protein level (B). CCK8 assay was performed to determine the inhibition effect of cisplatin on cell proliferative activity. p<0.05 compared with the control group (C). Western blot was used to detect the change of the Nrf2 protein after indicated dose of cisplatin treatment in both parental Ishikawa cells and Ishikawa-Nrf2 cells (D). Page 18/19 Page 18/19 Figure 6 Nrf2 is overexpressed in endometrial cancer. The expression pattern of Nrf2 in normal endometrium (n=10), hyperplasia (n=20) and endometrial cancer (n=90) was assessed using IHC staining. Representative images were captured at 100×magnification (A). The IHC scores in endometrial lesion tissues compared with normal endometrial tissues are presented (B). *p<0.05 compared with normal endometrial tissues. The expression pattern of Nrf2 in endometrial fresh tissue was determined by Figure 6 Figure 6 supplementarytable1.xlsx Supplementary Files his is a list of supplementary files associated with this preprint. Click to dow supplementarytable1.xlsx Page 19/19 Page 19/19
https://openalex.org/W3144190777	https://hal.science/hal-02555983v2/document	Latin	null	Propagation Phenomena with Nonlocal Diffusion in Presence of an Obstacle	Journal of dynamics and differential equations	2,021	cc-by	45,081	To cite this version: Julien Brasseur, Jérôme Coville. Propagation phenomena with nonlocal diffusion in presence of an obstacle. Journal of Dynamics and Differential Equations, 2023, 35 (1), pp.237-301. 10.1007/s10884- 021-09988-y. hal-02555983v2 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02555983 https://hal.science/hal-02555983v2 Submitted on 14 May 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 PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION IN PRESENCE OF AN OBSTACLE JULIEN BRASSEUR AND J´ERˆOME COVILLE Abstract. We consider a nonlocal semi-linear parabolic equation on a connected exterior domain of the form RN \ K, where K ⊂RN is a compact “obstacle”. The model we study is motivated by applications in biology and takes into account long range dispersal events that may be anisotropic depending on how a given population perceives the environment. To formulate this in a meaningful manner, we introduce a new theoretical framework which is of both mathematical and biological interest. The main goal of this paper is to construct an entire solution that behaves like a planar travelling wave as t →−∞and to study how this solution propagates depending on the shape of the obstacle. We show that whether the solution recovers the shape of a planar front in the large time limit is equivalent to whether a certain Liouville type property is satisﬁed. We study the validity of this Liouville type property and we extend some previous results of Hamel, Valdinoci and the authors. Lastly, we show that the entire solution is a generalised transition front. 2010 Mathematics Subject Classiﬁcation. 35J60. 1. Introduction Since the seminal works of Fisher [40], Kolmogorov, Petrovskii and Piskunov [50] on the propagation of advantageous genes in an homogeneous population, reaction-diﬀusion models have been extensively used to study the complex dynamics arising in nature [11, 21, 47, 48, 58, 65]. One of the main success of this type of modelling is the notion of “travelling waves” that has emerged from it, which has provided a rich and ﬂexible theoretical framework to analyse the underlying dynamics of the problem considered. In the past two decades, reaction-diﬀusion models involving more realistic descriptions of spatial interactions as well as of the environment have been considered to analyse a wide range of problems from ecology [48, 49, 65, 77], combustion theory [45, 46, 78] or phase transition in heterogeneous medium [33, 35]. This has considerably increased our understanding of the impact of the time and spatial heterogeneities of the environment on propagation phenomena. In turn, this profusion of work has led to the introduction of new notions of travelling waves generalising the traditional notion of planar wave and reﬂecting the essential properties of the environment [8, 10, 12, 13, 14, 16, 49, 56, 59, 63, 72, 77, 83]. In particular, notions such as pulsating fronts, random fronts or conical (or curved) fronts have been introduced to analyse propagation phenomena occurring in time and/or space periodic environments [8, 13, 57, 59, 83], or random ergodic environments [56, 63, 72], or to study propagation phenomena with some geometrical constraints [6, 16, 62]. It turns out that almost all of these new notions fall into the deﬁnition of generalised transition wave recently introduced by Berestycki and Hamel in [9], see also [10, 43]. It is worth mentioning that the complexity of propagation phenomena may come from either heterogeneous interactions (heterogeneous diﬀusion and reaction) or the geometry of the domain where the equation is deﬁned (cylinder with rough boundary or domain with a complex structure). In the latter case, new phenomena are observed such as the pinning of fronts. We point the interested reader to [11, 60, 83] and references therein for a more thorough description of the state of the art on propagation phenomena in the context of reaction-diﬀusion equations. Propagation phenomena can also be observed using other types of models, in particular nonlocal models that take into account long range dispersal phenomena (which are commonly observed in ecology, see [3, 4, 20, 23]). JULIEN BRASSEUR AND J´ERˆOME COVILLE JULIEN BRASSEUR AND J´ERˆOME COVILLE 2 7. On the impact of the geometry 40 7.1. A Liouville type result 40 7.2. The stationary solution encodes the geometry 41 8. Large time behaviour of super-level sets 44 8.1. An upper bound: the super-level sets move at speed at most c 44 8.2. A lower bound: the super-level sets move asymptotically at speed c 46 8.3. Conclusion: the super-level sets move exactly at speed c 51 9. The entire solution u(t, x) is a generalised transition wave 55 Acknowledgments 57 Appendix A. The J-covering property 58 References 61 7. On the impact of the geometry 7. On the impact of the geometry 7.2. The stationary solution encodes the geometry Appendix A. The J-covering property References Contents Contents 1. Introduction 2 1.1. Biological motivation 4 1.2. Notations and deﬁnitions 6 1.3. Assumptions 8 2. Main results 9 2.1. General existence results 9 2.2. Large time behaviour 10 2.3. Global mean speed and transition fronts 12 2.4. Some open problems 13 2.5. Organization of the paper 14 3. The Cauchy problem 15 3.1. Some comparison principles 15 3.2. Existence of a unique solution 18 3.3. Parabolic type estimates 20 4. A priori bounds for entire solutions 22 5. Time before reaching the obstacle 23 5.1. Preliminaries 24 5.2. Construction of sub- and supersolutions 26 5.3. Construction of the entire solution 33 5.4. Further properties of the entire solution 35 6. Local behaviour after the encounter with K 38 6.1. Local uniform convergence to the stationary solution 38 6.2. Convergence near the horizon 39 2010 Mathematics Subject Classiﬁcation. 35J60. 1 1. Introduction 1.1. Biological motivation 1.2. Notations and deﬁnitions 1.3. Assumptions 2. Main results 2.1. General existence results 2.2. Large time behaviour 2.3. Global mean speed and transition fronts 2.4. Some open problems 2.5. Organization of the paper 3. The Cauchy problem 3.1. Some comparison principles 3.2. Existence of a unique solution 3.3. Parabolic type estimates 4. A priori bounds for entire solutions 5. Time before reaching the obstacle 5.1. Preliminaries 5.2. Construction of sub- and supersolutions 5.3. Construction of the entire solution 5.4. Further properties of the entire solution 6. Local behaviour after the encounter with K 6.1. Local uniform convergence to the stationary solution 6.2. Convergence near the horizon 2010 Mathematics Subject Classiﬁcation. 35J60. 1 1 1. Introduction For example, planar fronts [2, 5, 22, 25, 30, 31, 34], pulsating fronts [32, 38, 68, 76] and generalised transition waves [15, 54, 73, 74, 75] have PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 3 been constructed for integro-diﬀerential models of the form 1) ∂tu(t, x) = J ∗u(t, x) −u(t, x) + f(t, x, u(t, x)) for (t, x) ∈R × RN, (1.1) where f is a classical bistable or monostable nonlinearity, J is a nonnegative probability density function and ∗is the standard convolution operator given by where f is a classical bistable or monostable nonlinearity, J is a nonnegative probability density function and ∗is the standard convolution operator given by J ∗u(x) := ˆ RN J (x −y)u(y)dy. However, to the best of our knowledge, there are no results dealing with the impact of the geometry on the large time dynamics of such nonlocal semi-linear equation, and only linear versions of (1.1) seem to have been considered, see [28, 29]. However, to the best of our knowledge, there are no results dealing with the impact of the geometry on the large time dynamics of such nonlocal semi-linear equation, and only linear versions of (1.1) seem to have been considered, see [28, 29]. In the spirit of the pioneer work of Berestycki, Hamel and Matano [12], we analyse here the eﬀect of the geometry of the domain on the propagation phenomena for an adapted version of (1.1) on exterior domains. Precisely, given a compact set K ⊂RN with nonempty interior such that the exterior domain Ω:= RN \ K is connected, we are interested in the properties and large time behaviour of the entire solutions u to the following nonlocal semi-linear parabolic problem ∂tu = Lu + f(u) a.e. in R × Ω, (P) ∂tu = Lu + f(u) a.e. in R × Ω, ∂tu = Lu + f(u) a.e. in R × Ω, (P) where L is the nonlocal diﬀusion operator given by Lu(x) := ˆ RN\K J(δ(x, y))(u(y) −u(x))dy. Here, J is a nonnegative kernel, f is a “bistable” nonlinearity and δ : Ω× Ω→[0, ∞) is a distance on Ωthat behaves locally like the Euclidean distance (precise assumptions on J, f and δ will be given later on, see Subsection 1.3). 1. Introduction Here, J is a nonnegative kernel, f is a “bistable” nonlinearity and δ : Ω× Ω→[0, ∞) is a distance on Ωthat behaves locally like the Euclidean distance (precise assumptions on J, f and δ will be given later on, see Subsection 1.3). ) The problem (P) can be seen as a nonlocal version of the reaction-diﬀusion problem studied by Berestycki, Hamel and Matano in [12], namely ∂tu = ∆u + f(u) in R × Ω, ∇u · ν = 0 on R × ∂Ω. (1.2) (1.2) There, they show that for any unit vector e ∈SN−1 (where SN−1 denotes the unit sphere of RN), there exists a generalised transition wave in the direction e solution to (1.2), i.e. for any e ∈SN−1, there exists an entire solution, u(t, x), to (1.2) deﬁned for all t ∈R and all x ∈Ωthat satisﬁes 0 < u(t, x) < 1 for all (t, x) ∈R × Ωand such that \|u(t, x) −φ(x · e + ct)\| −→ t→−∞0 uniformly in x ∈Ω, where (φ, c) is a planar travelling wave of speed c > 0. That is, (φ, c) is the unique (up to shift) increasing solution to where (φ, c) is a planar travelling wave of speed c > 0. That is, (φ, c) is the unique (up to shift) increasing solution to ( c φ′ = φ′′ + f(φ) in R, lim z→+∞φ(z) = 1, lim z→−∞φ(z) = 0. ( c φ′ = φ′′ + f(φ) in R, ( lim z→+∞φ(z) = 1, lim z→−∞φ(z) = 0. 4 JULIEN BRASSEUR AND J´ERˆOME COVILLE JULIEN BRASSEUR AND J´ERˆOME COVILLE 4 Moreover, they prove that there exists a classical solution, u∞, to          ∆u∞+ f(u∞) = 0 in Ω, ∇u∞· ν = 0 on ∂Ω, 0 < u∞⩽1 in Ω, u∞(x) →1 as \|x\| →+∞, (1.3) (1.3) which they show corresponds to the large time limit of u(t, x) in the s which they show corresponds to the large time limit of u(t, x) in the sense that \|u(t, x) −u∞(x)φ(x · e + ct)\| −→ t 0 uniformly in x ∈Ω. 1. Introduction which they show corresponds to the large time limit of u(t, x) in the sense that which they show corresponds to the large time limit of u(t, x) in the sense that \|u(t x) −u (x)φ(x · e + ct)\| −→0 uniformly in x ∈Ω \|u(t, x) −u∞(x)φ(x · e + ct)\| −→ t→∞0 uniformly in x ∈Ω. In addition, they were able to classify the solutions u∞to (1.3) with respect to the geometry of K. Precisely, they proved that if the obstacle K is either starshaped or directionally convex (see [12, Deﬁnition 1.2]), then the solutions u∞to (1.3) are actually identically equal to 1 in the whole set Ω. This remarkable rigidity property was further extended to more complex obstacles by Bouhours in [17] who showed a sort of “stability” of this result with respect to small regular perturbations of the obstacle. Yet, this phenomenon does not hold in general. Indeed, Berestycki et al. [12] proved that when the domain is no longer starshaped nor directionally convex but merely simply connected (see [12]), then (1.3) admits nontrivial solutions with 0 < u∞< 1 in Ω, thus implying that the disturbance caused by the obstacle may remain forever depending on the geometry of K. Our main objective in this article is to construct such an entire solution for the problem (P) and to study its main properties with respect to the geometry of the domain. 1.1. Biological motivation. Before stating our main results, let us ﬁrst discuss the rele- vance of this type of model. To this end, let us go back to the very description of population dispersal. For it, let us denote by u(t, x) the density of the population at time t and lo- cation x. Moreover, let us discretize uniformly the domain Ωinto small cubes of volume \|∆xi\| centered at points xi ∈Ω, and the time into discrete time steps ∆t. Then, following Huston et al. [44], we can describe the evolution of the population in terms of the exchange of individuals between sites. Namely, for a site xi, the total number of individuals N(t, xi) will change during the time step ∆t according to N(t + ∆t, xi) −N(t, xi) ∆t = Ni←−Ni→ ∆t , where Ni←and Ni→denote the total number of individuals reaching and leaving the site xi, respectively. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 5 5 Figure 1. The geodesic distance (continuous line) and the Euclidean distance (dashed line) between xi, xj ∈RN \ K. Figure 1. The geodesic distance (continuous line) ( ) and the Euclidean distance (dashed line) between xi, xj ∈RN \ K. and the Euclidean distance (dashed line) between xi, xj ∈RN \ K. intrinsic description of the landscape such as its connectivity, fragmentation, anisotropy or other particular geometrical structure [1, 26, 36, 37, 69, 80, 81]. intrinsic description of the landscape such as its connectivity, fragmentation, anisotropy or other particular geometrical structure [1, 26, 36, 37, 69, 80, 81]. Here, we are particularly interested in the impact that the geometry of Ωcan have on this rate. A natural assumption is to consider that J (xi, xj) depends on the “eﬀective distance” between xi and xj. The perception of the environment being a characteristic trait of a given species (as observed in [41]), this notion of “eﬀective distance” will then change depending on the species considered. Let us consider, for instance, an habitat consisting of a uniform ﬁeld with, in the middle of it, a circular pond, e.g. Ω:= R2 \ B1 where B1 denotes the unit disk of R2. One can then imagine that, for some species having a high dispersal capacity (as, for example, bees [66]), the pond will not be considered as an obstacle in the sense that it does not aﬀect their displacement (since the individuals can easily “jump” from one side of the pond to another). On the contrary, for other species, such as many land animals, this pond will actually be seen as a physical dispersal barrier. Whence, to go from one side of the pond to another they will have to circumvent it. So, in this case, the metric considered to evaluate this “eﬀective distance” has to reﬂect such type of behaviour (see e.g. [71] for an illustrative example). ( [ ] ) A way to understand the impact of the landscape on the movement of the individuals is to use a “least cost path” modelling [1, 36, 79]. The metric related to this geographic concept can then serve as a prototype for the metric used to evaluate J (xi, xj). 1. Introduction Since N(t, xi) = u(t, xi)\|∆xi\|, this can be rewritten where Ni←and Ni→denote the total number of individuals reaching and leaving the site xi, respectively. Since N(t, xi) = u(t, xi)\|∆xi\|, this can be rewritten u(t + ∆t, xi) −u(t, xi) ∆t \|∆xi\| = γ \|∆xi\| +∞ X j=−∞ J (xi, xj)u(t, xj) −J (xj, xi)u(t, xi) \|∆xj\|, ∆t where J (xi, xj) denotes the rate of transfer of individuals from the site xi to the site xj and γ denotes a dispersal rate (or diﬀusion coeﬃcient). In ecology, understanding the structure of the rate of transfer J (xi, xj) is of prime interest as it is known to condition some important feature of the dispersal of the individuals [26, 51, 61, 70]. For example, this rate can reﬂect some constraints of the environment on the capacity of movement of the individuals [26, 27, 36, 42, 71] and/or incorporate important features that are biologically/ecologically relevant such as a dispersal budget [7, 44] or a more PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION The idea behind the “least cost path” concept is to assign to each path taken to join one site to another some costs related to a predetermined constraint and try to minimize the costs. This notion can then be related to the notion of geodesic path on a smooth surface where the costs then reﬂect some geometrical aspect of the landscape. Following this idea, it is then natural to consider the “eﬀective distance” as some geodesic distance δ reﬂecting how the geometry of the landscape is perceived by the species considered and to take J (xi, xj) = J(δ(xi, xj)), where J is a function encoding the probability to make a jump of length δ(xi, xj). In the above example, the appropriate distance will then be either the standard Euclidean distance (i.e. δ(xi, xj) = \|xi −xj\|) or the geodesic distance deﬁned in the perforated domain Ω. Since diﬀusion is classically accompanied by demographic variations (which we may sup- pose to be described by a nonlinear function f of the density of population), by letting \|∆xj\| →0+ and ∆t →0+, we then formally get ∂tu(t, x) = γ ˆ Ω J(δ(x, y))u(t, y)dy −u(t, x) ˆ Ω J(δ(y, x))dy + f(u(t, x)), which thereby yields equation (P), up to an immaterial constant γ. 6 JULIEN BRASSEUR AND J´ERˆOME COVILLE It is worth mentioning that, although the description of the rate of transfer using a geodesic distance is well-known in the ecology community [36, 79], to our knowledge, this the ﬁrst time that such concept has been formalised mathematically in the framework of nonlocal reaction-dispersal equations to describe the evolution of a population living in a domain and having a long distance dispersal strategy. The mathematical framework we propose goes far beyond the situation we analyse here. Indeed, the model (P) is quite natural and well-posed as soon as a geodesic-type distance, which we will refer to as “quasi-Euclidean” (see Deﬁnition 1.2), can be deﬁned on the domain Ωconsidered, allowing thus to handle domains with very complex geometrical structure (such graph trees, which are particularly pertinent in conservation biology for the help they can provide in the understanding of the impact of blue and green belts in urban landscapes [52, 84]). As we will see, our setting also allows to model an extremely wide class of biologically relevant “eﬀective distances” (see Remark 1.5). 1.2. Notations and deﬁnitions. Before we set our main assumptions, we need to introduce some necessary deﬁnitions. 1.2. Notations and deﬁnitions. Before we set our main assumptions, we need to introduce some necessary deﬁnitions. We begin with the metric framework on which we will work. We begin with the metric framework on which we will work. Deﬁnition 1.1. Let x, y ∈RN. We call a path connecting x to y any continuous piecewise C1 function γ : [0, 1] →RN with γ(0) = x and γ(1) = y and we denote by length(γ) its length. The set of all such paths is denoted by H(x, y). Deﬁnition 1.2. Let E ⊂RN. A quasi-Euclidean distance on E is a distance δ on E such that δ(x, y) = \|x −y\| if [x, y] ⊂E and δ(x, y) ⩾\|x −y\| for all x, y ∈E. We denote by Q(E) the set of all quasi-Euclidean distances on E. Example 1.3. The geodesic distance dE on a set E, deﬁned by Moreover, if E = RN \ K for some compact convex set K ⊂RN with C2 boundary and if supp(J) contains a nonempty open set (e.g. if J is continuous), then (E, δ) has the J-covering property for any δ ∈Q(E) (see Proposition A.2 in the Appendix). Let us also list in this subsection a few notations and deﬁnitions used in also list in this subsection a few notations and deﬁnitions used in the paper: \|E\| is the Lebesgue measure of the measurable set E; 1E is the characteristic function of the set E; SN−1 is the unit sphere of RN; BR is the open Euclidean ball of radius R > 0 centered at the origin; BR(x) is the open Euclidean ball of radius R > 0 centered at x ∈RN; A(R1, R2) is the open annulus BR2 \ BR1; A(x, R1, R2) is the open annulus x + A(R1, R2); g ∗h is the convolution of g and h; ∆2 h is the operator given by ∆2 hf(x) = f(x + h) −2f(x) + f(x −h); ⌊x⌋ is the integral part of x ∈R, i.e. ⌊x⌋= sup{k ∈Z; k ⩽x}. \|E\| is the Lebesgue measure of the measurable set E; 1E is the characteristic function of the set E; SN−1 is the unit sphere of RN; BR is the open Euclidean ball of radius R > 0 centered at the origin; BR is the open Euclidean ball of radius R > 0 centered at the origin; BR(x) is the open Euclidean ball of radius R > 0 centered at x ∈RN; A(R1, R2) is the open annulus BR2 \ BR1; ( ) 2 \ 1 A(x, R1, R2) is the open annulus x + A(R1, R2); g g ; ∆2 h is the operator given by ∆2 hf(x) = f(x + h) −2f(x) + f(x −h); ⌊x⌋ is the integral part of x ∈R, i.e. ⌊x⌋= sup{k ∈Z; k ⩽x}. Given E ⊂RN and p ∈[1, ∞], we denote by Lp(E) the Lebesgue space of (equivalence classes of) measurable functions g for which the p-th power of the absolute value is Lebesgue integrable when p < ∞(resp. essentially bounded when p = ∞). When the context is clear, we will write ∥g∥p instead of ∥g∥Lp(E). The set L∞(E) ∩C(E) of bounded continuous functions on E will be denoted by Cb(E). Example 1.3. The geodesic distance dE on a set E, deﬁned by Example 1.3. The geodesic distance dE on a set E, deﬁned by dE(x, y) :=    inf γ∈H(x,y) γ⊂E length(γ) if x, y belong to the same connected component, +∞ otherwise, dE(x, y) :=    inf γ∈H(x,y) γ⊂E length(γ) if x, y belong to the same connected component, +∞ otherwise, otherwise, is a nontrivial quasi-Euclidean distance. If dF is the geodesic distance on a set F ⊃E, then its restriction dF E to E × E is another nontrivial example of quasi-Euclidean distance on E. Moreover, since Q(E) is a convex set, one may obtain other examples of such distances by convex combination of the previous examples and/or the Euclidean distance. Remark 1.4. If E is convex, then the Euclidean distance is the only quasi-Euclidean distance. Remark 1.5. Roughly speaking, a quasi-Euclidean distance can be interpreted as the length of a path connecting two points and which behaves locally like the Euclidean distance. In fact, the condition δ(x, y) ⩾\|x −y\| can be equivalently rephrased by saying that, for any two points x, y ∈E, there exists a path γ ∈H(x, y) (which is not compelled to stay in E) connecting x to y and such that δ(x, y) = length(γ). Biologically speaking, it provides a natural and ﬂexible tool to model the “eﬀective distance” between two locations. It can account for a wide range of situations, for example it can model a population whose individuals can jump through some obstacles (say small ones) and not through others (say large ones), or through portions of an obstacle, as well as all the intermediary situations. 7 PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION Deﬁnition 1.6. Let E ⊂RN be a connected set and let δ ∈Q(E). Let J : [0, ∞) →[0, ∞) be a measurable function with \|supp(J)\| > 0. For any x ∈E, we deﬁne Π0(J, x) := {x} and Πj+1(J, x) := [ z∈Πj(J,x) supp (J(δ(·, z))) for any j ⩾0. We say that the metric space (E, δ) has the J-covering property if E = [ j⩾0 Πj(J, x) for every x ∈E. Remark 1.7. If E is a connected set and if δ is the Euclidean distance, then the above property is automatically satisﬁed (see Proposition A.1 in the Appendix). Example 1.3. The geodesic distance dE on a set E, deﬁned by Moreover, if δ is the Euclidean distance, then it also holds that Remark 1.9. If E = Ω= RN \ K for some compact set K ⊂RN, if δ ∈Q(Ω) and if g ∈Bα p,∞(Ω; δ) has compact support, then grad ∈Bα p,∞(RN). Moreover, if δ is the Euclidean distance, then it also holds that RBα p,∞(RN) := g s.t. grad ∈Bα p,∞(RN) ⊂Bα p,∞(Ω; δ). RBα p,∞(RN) := g s.t. grad ∈Bα p,∞(RN) ⊂Bα p,∞(Ω; δ). ral, Bα p,∞(Ω; δ) and RBα p,∞(RN) are distinct function space 1.3. Assumptions. Let us now specify the assumptions made all along this paper. Through- out the paper we will always assume that K ⊂RN is a compact set, that Ω:= RN \ K is connected and that δ ∈Q(Ω). (1.4) As already mentioned above, we will suppose that f is of “bistable” type. More precisely, we will assume that f : [0, 1] →R is such that (1.4) As already mentioned above, we will suppose that f is of “bistable” type. More precisely, we will assume that f : [0, 1] →R is such that ∃θ ∈(0, 1), f(0) = f(θ) = f(1) = 0, f < 0 in (0, θ), f > 0 in (θ, 1), f ∈C1,1([0, 1]), f ′(0) < 0, f ′(θ) > 0 and f ′(1) < 0. (1.5) ∃θ ∈(0, 1), f(0) = f(θ) = f(1) = 0, f < 0 in (0, θ), f > 0 in (θ, 1), f ∈C1,1([0, 1]), f ′(0) < 0, f ′(θ) > 0 and f ′(1) < 0. (1.5) ∃θ ∈(0, 1), f(0) = f(θ) = f(1) = 0, f < 0 in (0, θ), f > 0 in (θ, 1), f ∈C1,1([0, 1]), f ′(0) < 0, f ′(θ) > 0 and f ′(1) < 0. Example 1.3. The geodesic distance dE on a set E, deﬁned by Given α ∈(0, 1) and p ∈[1, ∞], Bα p,∞(RN) stands for the Besov-Nikol’skii space consisting in all measurable functions g ∈Lp(RN) such that [g]Bα p,∞(RN) := sup h̸=0 ∥g(· + h) −g∥Lp(RN) \|h\|α < ∞. Note that, when p = ∞, the space Bα ∞,∞(RN) coincides with the classical H¨older space C0,α(RN). For a set E ⊂RN and g : E →R, we set Note that, when p = ∞, the space Bα ∞,∞(RN) coincides with the classical H¨older space C0,α(RN). For a set E ⊂RN and g : E →R, we set [g]C0,α(E) := sup x,y∈E, x̸=y \|g(x) −g(y)\| \|x −y\|α . Moreover, given (k, α) ∈N × (0, 1), (E, F) ⊂R × RN and a function g : E × F →R, we say that g ∈Ck(E, C0,α(F)) if, for all (t, x) ∈E × F, it holds that Moreover, given (k, α) ∈N × (0, 1), (E, F) ⊂R × RN and a function g : E × F →R, we say that g ∈Ck(E, C0,α(F)) if, for all (t, x) ∈E × F, it holds that g(·, x) ∈Ck(E) and g(t, ·) ∈C0,α(F). For our purposes, we need to introduce a new function space, closely related to Bα p,∞(RN). For our purposes, we need to introduce a new function space, closely related to Bα p,∞(RN). 8 JULIEN BRASSEUR AND J´ERˆOME COVILLE Deﬁnition 1.8. Let E ⊂RN be a measurable set and let δ be a distance on E. Let α ∈(0, 1) and p ∈[1, ∞). We call Bα p,∞(E; δ) the space of functions g : R+ →R such that grad ∈Lp(RN) where grad(x) := g(\|x\|) and such that Deﬁnition 1.8. Let E ⊂RN be a measurable set and let δ be a distance on E. Let α ∈(0, 1) and p ∈[1, ∞). We call Bα p,∞(E; δ) the space of functions g : R+ →R such that grad ∈Lp(RN) where grad(x) := g(\|x\|) and such that x2 ∥g(δ(x1, ·)) −g(δ(x2, ·))∥Lp(E) \|x1 −x2\|α < ∞. ∥g(δ(x1, ·)) −g(δ(x2, ·))∥Lp(E) \|x1 −x2\|α < α p,∞(E;δ) := sup x1,x2∈E, x1̸=x2 ∥g(δ(x1, ·)) −g(δ(x2, ·))∥Lp(E) \|x1 −x2\|α < ∞. [g]Bα p,∞(E;δ) := sup x1,x2∈E, x1̸=x2 ∥ Remark 1.9. If E = Ω= RN \ K for some compact set K ⊂RN, if δ ∈Q(Ω) and if g ∈Bα p,∞(Ω; δ) has compact support, then grad ∈Bα p,∞(RN). Example 1.3. The geodesic distance dE on a set E, deﬁned by (1.5) (1.5) Also, we suppose that J : [0, ∞) →[0, ∞) is a compactly supported measurable function with \|supp(J)\| > 0 such that Also, we suppose that J : [0, ∞) →[0, ∞) is a compactly supported measurable function with \|supp(J)\| > 0 such that                  (Ω, δ) has the J-covering property, ˆ RN Jrad(z)dz = 1 where Jrad(z) := J(\|z\|), ∀x1 ∈Ω, lim x2→x1 ∥J(δ(x1, ·)) −J(δ(x2, ·))∥L1(Ω) = 0, J δ ∈L∞(Ω) where J δ(x) := ˆ Ω J(δ(x, z))dz. (1.6) (1.6) Biologically speaking, the ﬁrst assumption in (1.6) means that if δ reﬂects how the individuals of a given species move in the environment given by Ωand if J(δ(x, y)) represents their dispersal rate, then the individuals can reach any point of Ωin a ﬁnite number of “jumps” regardless of their initial position. Mathematically speaking, it ensures that the strong maximum principle holds (as will be made clear throughout the paper). As for the last two assumptions, they are essentially meant to ensure that J δ ∈Cb(Ω). They are satisﬁed if, for instance, either δ is the Euclidean distance or J is non-increasing and J ∈Bα 1,∞(Ω; δ). Biologically speaking, the ﬁrst assumption in (1.6) means that if δ reﬂects how the individuals of a given species move in the environment given by Ωand if J(δ(x, y)) represents their dispersal rate, then the individuals can reach any point of Ωin a ﬁnite number of “jumps” regardless of their initial position. Mathematically speaking, it ensures that the strong maximum principle holds (as will be made clear throughout the paper). As for the last two assumptions, they are essentially meant to ensure that J δ ∈Cb(Ω). They are satisﬁed if, for instance, either δ is the Euclidean distance or J is non-increasing and J ∈Bα 1,∞(Ω; δ). 1,∞( ) Lastly, we require the datum (J, f) to be such that there exist an increasing function φ ∈C(R) and a speed c > 0 satisfying ( c φ′ = J1 ∗φ −φ + f(φ) in R, lim z→+∞φ(z) = 1, lim z→−∞φ(z) = 0. (1.7) (1.7) where J1 is the nonnegative even kernel given by: where J1 is the nonnegative even kernel given by: J1(x) := ˆ RN−1 Jrad(x, y′)dy′. (1.8) (1.8) PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 9 Remark 1.10. Example 1.3. The geodesic distance dE on a set E, deﬁned by Notice that (1.7) implies that 0 < φ < 1 and that φ ∈C0,1(R). Actually, the fact that f ∈C1,1([0, 1]) (as imposed by assumption (1.5)) guarantees that φ ∈C2(R) (as can be seen by a classical bootstrap argument). Remark 1.11. Although this is well-known (see e.g. [5, 25, 30, 85]), it is worth mentioning that (1.7) is not an empty assumption. For example, it is satisﬁed if, in addition to (1.5) and (1.6), the following assumptions are made: Jrad ∈W 1,1(RN), max [0,1] f ′ < 1 and ˆ 1 0 f(s)ds > 0. (1.9) (1.9) See also [19, Section 2.4] for additional comments on the matter. See also [19, Section 2.4] for additional comments on the matter. 2. Main results The results of Berestycki, Hamel and Matano for the classical problem (1.2) say that there exists an entire solution u(t, x) that behaves like a planar wave as t →−∞and as a planar wave multiplied by u∞(x) as t →+∞, where u∞solves (1.3). Moreover, they were able to classify the solutions to (1.3) with respect to the geometry of K, providing us with a good insight on how the latter inﬂuences the large time dynamics. Our goal here is to obtain corresponding results for the nonlocal problem (P). In the ﬁrst place, we will prove that there exists an entire solution to (P) with analogous properties as in the classical case. Then, we will study more precisely how the geometry of K aﬀects its large time behaviour and we will prove that this question is equivalent to investigating under which circumstances a certain Liouville type property holds. 2.1. General existence results. Our ﬁrst main result deals with the existence and unique- ness of an entire (i.e. time-global) solution to problem (P). Theorem 2.1 (Existence of an entire solution). Assume (1.4), (1.5), (1.6), (1.7) and let (φ, c) be as in (1.7). Suppose that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1) and that max [0,1] f ′ < inf ΩJ δ, (2.1) (2.1) Then, there exists an entire solution u ∈C2(R, C0,α(Ω)) to (P) satisfying 0 < u < 1 and ∂tu > 0 in R × Ω. Moreover, Then, there exists an entire solution u ∈C2(R, C0,α(Ω)) to (P) satisfying 0 < u < 1 and ∂tu > 0 in R × Ω. Moreover, \|u(t, x) −φ(x1 + ct)\| −→ t→−∞0 uniformly in x ∈Ω. (2.2) \|u(t, x) −φ(x1 + ct)\| −→ t→−∞0 uniformly in x ∈Ω. (2.2) (2.2) Furthermore, (2.2) determines a unique bounded entire solution to (P). If, in addition, (1.9) holds, then there exists a continuous solution, u∞: Ω→(0, 1], to Furthermore, (2.2) determines a unique bounded entire solution to (P). If, in addition, (1.9) holds, then there exists a continuous solution, u∞: Ω→(0, 1], to Furthermore, (2.2) determines a unique bounded entire solution to (P). If, in addition, (1.9) holds, then there exists a continuous solution, u∞: Ω→(0, 1], to Lu∞+ f(u∞) = 0 in Ω, u∞(x) →1 as \|x\| →∞, (P∞) (P∞) such that \|u(t, x) −u∞(x)φ(x1 + ct)\| −→ t→+∞0 locally uniformly in x ∈Ω. 2. Main results (2.3) \|u(t, x) −u∞(x)φ(x1 + ct)\| −→ t→+∞0 locally uniformly in x ∈Ω (2.3) (2.3) Remark 2.2. We have stated, for simplicity, the existence of an entire solution that propagates in the direction e1 = (1, 0, · · · , 0). However, this restriction is immaterial and our arguments also yield that, for every e ∈SN−1, there exists an entire solution propagating in the direction e and satisfying the same properties as above. Remark 2.2. We have stated, for simplicity, the existence of an entire solution that propagates in the direction e1 = (1, 0, · · · , 0). However, this restriction is immaterial and our arguments also yield that, for every e ∈SN−1, there exists an entire solution propagating in the direction e and satisfying the same properties as above. JULIEN BRASSEUR AND J´ERˆOME COVILLE 10 10 (a) t = 50 (b) t = 100 (c) t = 200 (d) t = 300 (e) t = 400 (f) t = 450 (g) t = 500 (h) t = 550 (i) t = 600 (j) t = 650 (k) t = 700 (l) t = 750 Figure 2. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is the Euclidean distance and the obstacle K is the union of the unit disk and four ellipsoids. On the domain Ω:= [30, −50] × [−15, 15] \ K we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.075. We observe that the solution behaves like a generalised transition wave. (b) t = 100 (c) t = 200 (a) t = 50 (d) t = 300 (b) t = 100 (a) t = 50 (d) t = 300 (h) t = 550 (e) t = 400 (f) t = 450 (g) t = 500 (e) t = 400 (f) t = 450 (h) t = 550 (g) t = 500 (l) t = 750 (k) t = 700 (j) t = 650 (i) t = 600 (i) t = 600 (l) t = 750 (j) t = 650 (k) t = 700 Figure 2. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. 2. Main results For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is the Euclidean distance and the obstacle K is the union of the unit disk and four ellipsoids. On the domain Ω:= [30, −50] × [−15, 15] \ K we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.075. We observe that the solution behaves like a generalised transition wave. Remark 2.3. A consequence of the uniqueness part of Theorem 2.1 is that the entire solution u(t, x) shares the same symmetry as K in the hyperplane {x1} × RN−1. More precisely, if T is an isometry of RN−1 such that (x1, T x′) ∈Ωfor any (x1, x′) ∈Ω, then u(t, x1, T x′) = u(t, x1, x′) for all (t, x) ∈R × Ω. Remark 2.4. If (Ω, δ) does not have the J-covering property, then we still have the existence of an entire solution satisfying (2.2), but we only have that 0 ⩽u(t, x) ⩽1 and ∂tu(t, x) ⩾0 for any (t, x) ∈R × Ω(as opposed to the strict inequalities in Theorem 2.1). Moreover, the uniqueness may fail because the strong maximum principle does not hold in this case. Remark 2.4. If (Ω, δ) does not have the J-covering property, then we still have the existence of an entire solution satisfying (2.2), but we only have that 0 ⩽u(t, x) ⩽1 and ∂tu(t, x) ⩾0 for any (t, x) ∈R × Ω(as opposed to the strict inequalities in Theorem 2.1). Moreover, the uniqueness may fail because the strong maximum principle does not hold in this case. 2.2. Large time behaviour. As in the local case [12], the large time behaviour of u(t, x) depends on the geometry of K. Hamel, Valdinoci and the authors have shown in [19] that, if δ is the Euclidean distance and K is convex, then the problem (P∞) admits a Liouville type property: namely, the only possible solution to (P∞) is the trivial solution u∞≡1. We prove that this fact can be extended to arbitrary quasi-Euclidean distances (up to a slight additional assumption on J), which then results in the following theorem: 2.2. Large time behaviour. As in the local case [12], the large time behaviour of u(t, x) depends on the geometry of K. \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. In other words: if the obstacle K is convex, then the entire solution u(t, x) to (P) will eventually recover the shape of the planar travelling wave φ(x1 + ct) as t →+∞, i.e. the presence of an obstacle will not alter the large time behaviour of the solution u(t, x). This is a consequence of the fact that (P∞) satisﬁes a Liouville type property, see Figure 3. (a) t = 0 (b) t = 150 (c) t = 300 (d) t = 450 (e) t = 600 (f) t = 750 (g) t = 900 (h) t = 1000 Figure 3. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is the Euclidean distance and the obstacle K is a disk of radius 4. On the domain Ω:= [−15, 15]2 \ K we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.05. We observe that the solution converges to a trivial asymptotic proﬁle as t →∞, namely 1. (c) t = 300 (b) t = 150 (a) t = 0 (d) t = 450 (b) t = 150 (a) t = 0 (h) t = 1000 (e) t = 600 (f) t = 750 (g) t = 900 (f) t = 750 (e) t = 600 (g) t = 900 (h) t = 1000 Figure 3. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is the Euclidean distance and the obstacle K is a disk of radius 4. On the domain Ω:= [−15, 15]2 \ K we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.05. We observe that the solution converges to a trivial asymptotic proﬁle as t →∞, namely 1. However, the authors have shown in [18] that there exist obstacles K as well as a datum (J, f) for which this property is violated, i.e. such that (P∞) admits a non-trivial solution eu∞∈C(Ω) with 0 < eu∞< 1 in Ω. Hence, the picture described at Theorem 2.5 cannot be expected for general obstacles. 2. Main results Hamel, Valdinoci and the authors have shown in [19] that, if δ is the Euclidean distance and K is convex, then the problem (P∞) admits a Liouville type property: namely, the only possible solution to (P∞) is the trivial solution u∞≡1. We prove that this fact can be extended to arbitrary quasi-Euclidean distances (up to a slight additional assumption on J), which then results in the following theorem: 11 PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION Theorem 2.5. Suppose all the assumptions of Theorem 2.1 and that K ⊂RN is convex. If δ(x, y) ̸≡\|x −y\| suppose, in addition, that J is non-increasing. Then, there exists a unique entire solution u(t, x) to (P) in Ωsuch that 0 < u(t, x) < 1 and ∂tu(t, x) > 0 for all (t, x) ∈R × Ωand \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. Nevertheless, this does not immediately imply that the solution u∞to (P∞) arising in Theorem 2.1 is not constant. We prove that, whether the unique entire solution u(t, x) to (P) satisfying (2.2) recovers the shape of the planar travelling wave φ(x1+ct) as t →+∞is equivalent to the question of whether (P∞) satisﬁes the Liouville type property. Precisely, Theorem 2.6. Suppose all the assumptions of Theorem 2.1. Let u(t, x) be the unique bounded entire solution to (P) satisfying (2.2). Let u∞∈C(Ω) be the solution to (P∞) such that (2.3) holds, i.e. such that \|u(t, x) −u∞(x)φ(x1 + ct)\| −→ t→+∞0 locally uniformly in x ∈Ω. Then, u∞≡1 in Ωif, and only if, (P∞) satisﬁes the Liouville property. 12 JULIEN BRASSEUR AND J´ERˆOME COVILLE JULIEN BRASSEUR A JULIEN BRASSEUR AND J´ERˆOME COVILLE 12 q [ , , ] Corollary 2.7. There exist a smooth, simply connected, non-starshaped compact set K ⊂ RN, a quasi-Euclidean distance δ ∈Q(Ω) and a datum (J, f) satisfying all the assumptions of Theorem 2.1, such that the unique bounded entire solution u(t, x) to (P) satisfying (2.2) does not recover the shape of a planar travelling wave in the large time limit, that is \|u(t, x) −u∞(x)φ(x1 + ct)\| −→ t→+∞0 locally uniformly in x ∈Ω, \|u(t, x) −u∞(x)φ(x1 + ct)\| −→ t→+∞0 locally uniformly in x ∈Ω, where u∞∈C(Ω) is a solution to (P∞) such that 0 < u∞< 1 in Ω. where u∞∈C(Ω) is a solution to (P∞) such that 0 < u∞< 1 in Ω. Remark 2.8. The distance δ ∈Q(Ω) in Corollary 2.7 may be chosen to be either the Euclidean or the geodesic distance, see [18]. See Figure 4 for an example illustrating the conclusion of Corollary 2.7. The obstacle that is pictured is the same as the one we constructed in [18]. (a) t = 0 (b) t = 40 (c) t = 80 (d) t = 120 (e) t = 160 (f) t = 200 (g) t = 240 (h) t = 280 Figure 4. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is the Euclidean distance and the obstacle K is the annulus A(2, 5) to which we have removed a small channel to make its complement connected. Theorem 2.10. Assume all the assumptions of Theorem 2.1. Let u(t, x) be the unique entire solution to (P) satisfying (2.2) and let u∞∈C(Ω) be the solution to (P∞) such that (2.3) \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. Then, u(t, x) is a generalised transition almost-planar invasion front between 0 and u∞with global mean speed c, in the sense that sup (t,x)∈R×Ω, x1+ct⩾A \|u(t, x) −u∞(x)\| −→ A→∞0 and sup (t,x)∈R×Ω, x1+ct⩽−A u(t, x) −→ A→∞0. Remark 2.11. Notice that the presence of an arbitrary quasi-Euclidean distance, which in- troduces anisotropic features to the diﬀusion as well as a higher sensitivity to the geometry, does not slow down the propagation. 2.4. Some open problems. Prior to proving our main results, let us mention some open questions which, we believe, are of interest. We have considered, for simplicity, the case of compactly supported kernels only. It would be of interest to investigate the validity of our results without this assumption. Although this remains an open question, we believe that our results remain true at least if J decays faster than any exponential. However, if J is too heavily tailed, then the asymptotic growth or decay of φ(x1 + ct) may no longer be of exponential type and new tools would be needed to conclude. Nevertheless, let us mention that Hamel, Valdinoci and the authors proved in [19] that the Liouville type property holds for convex obstacles and the Euclidean distance as long as there exists an increasing subsolution to J1 ∗φ −φ + f(φ) = 0 in R connecting the two stable states 0 and 1, which is known to be the case even if J decays slower than any exponential (e.g. if (1.9) holds and J1 admits a ﬁrst order moment). ( ( ) ) Another interesting problem, which would be of both mathematical and biological interest, is the study of the inﬂuence of the quasi-Euclidean distance on the large time dynamics. For example: given an obstacle K and a datum (J, f), may it happen that the solution successfully invades the whole of Ωwhen δ is the Euclidean distance but fails to do so when δ is the geodesic distance? Numerical simulations suggest that this phenomenon could indeed occur (see Figure 5), which leads us to formulate the following conjecture: Conjecture 2.12. \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. On the domain Ω:= [−11, 11]2 \ K, we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.1. We observe that the solution converges to a non-trivial asymptotic proﬁle as t →∞. (b) t = 40 (a) t = 0 (c) t = 80 (d) t = 120 (a) t = 0 (d) (h) t = 280 (g) t = 240 (e) t = 160 (f) t = 200 (f) t = 200 (e) t = 160 (h) t = 280 (g) t = 240 Figure 4. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is the Euclidean distance and the obstacle K is the annulus A(2, 5) to which we have removed a small channel to make its complement connected. On the domain Ω:= [−11, 11]2 \ K, we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.1. We observe that the solution converges to a non-trivial asymptotic proﬁle as t →∞. Remark 2.9. If the convergence in (2.3) was known to be uniform in space, then the local uniform convergence in Theorems 2.5-2.6 and in Corollary 2.7 could be replaced by a uniform convergence without modiﬁcation in the proofs. 2.3. Global mean speed and transition fronts. As we have stated in the previous sub- section, the propagation may not always be complete depending on the shape of the obstacle. That is, it may happen that the solution u∞to the stationary problem (P∞) arising in (2.3) is not identically 1. An important question that remains to be addressed is the character- isation of the speed at which this solution propagates. We prove that the entire solution u(t, x) to (P) is a generalised transition front in the sense of Berestycki-Hamel [9] and that its global mean speed coincides with that of the planar front given by (1.7). PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 13 holds. Then, u(t, x) is a generalised transition almost-planar invasion front between 0 and u∞with global mean speed c, in the sense that holds. \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. There exists a compact obstacle K ⊂RN such that RN \ K is connected, as well as a datum (J, f) satisfying all the assumptions of Theorem 2.1 such that u∞≡1 in Ωif δ is the Euclidean distance and such that 0 < u∞< 1 in Ωif δ is the geodesic distance (or any other quasi-Euclidean distance distinct from the Euclidean distance). Note that, besides being supported by our numerical simulations, this conjecture is fairly reasonable. Indeed, it is quite natural to expect the solution to (P) to be more sensitive to the geometry of K when δ is the geodesic distance. In the case of the obstacle that is pictured in Figure 5, the individuals do not reach the rectangle inscribed by the obstacle in the same fashion: when δ is the Euclidean distance, they can simply “jump” through K, but, when δ is the geodesic distance, they have no choice but to go through a small channel which considerably penalises the propagation. So the success of the invasion may not depend on the interplay between K, J and f only but on the interplay between K, J, f and δ. A rigorous proof of Conjecture 2.12 would provide us with a very interesting result from the point of view of ecology. Indeed, it would suggest that, for a given ecological niche, the success of an invasion may crucially depend on the characteristic trait of the species determining its perception of the environment, namely the “eﬀective distance” between locations. Lastly, as in [12], it would be interesting to determine whether the convergence in (2.3) is uniform, namely whether it holds that limt→+∞supx∈Ω\|u(t, x) −u∞(x)φ(x1 + ct)\| = 0. Based on our numerical simulations and on the analogy with the local case, we conjecture JULIEN BRASSEUR AND J´ERˆOME COVILLE 14 (a) t = 300 (b) t = 700 (c) t = 300 (d) t = 700 (e) t = 1100 (f) t = 1200 (g) t = 1100 (h) t = 1200 (i) t = 1300 (j) t = 1500 (k) t = 1300 (l) t = 1500 (m) t = 1900 (n) t = 2000 (o) t = 1900 (p) t = 2000 Figure 5. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is either the geodesic distance (left rows (A, B, E, F, I, J, M, N)) or the Euclidean distance (right rows (C, D, G, H, K, L, O, P)) and the obstacle K is a “square annulus” (the diﬀerence between two axis-parallel rectangles) to which we have removed a small channel to make its complement connected. On the domain Ω:= [−5, 5]2 \ K, we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.1. The scheme is implemented in Python relying on the FEM library DOLFIN [55] and the VisiLibity library [64] in order to evaluate the geodesic distance. We observe that the respective solutions converge to diﬀerent asymptotic proﬁles as t →∞with a signiﬁcative diﬀerence in the qualitative behaviour of their dynamics. These simulations clearly highlight the importance of the distance in the ﬁnal outcome of the propagation and on the transition behaviour. (d) t = 700 (k) t = 1300 (l) t = 1500 (i) t = 1300 (n) t = 2000 (o) t = 1900 (m) t = 1900 (p) t = 2000 (m) t = 1900 (m) t = 1900 (n) t = 2000 (p) t = 2000 (o) t = 1900 (n) t = 2000 Figure 5. Numerical approximation of the solution of problem (P) at diﬀerent times, starting from a Heaviside type initial density. For the simulation, J(x) ∼e−\|x\|21B1(x), the distance δ is either the geodesic distance (left rows (A, B, E, F, I, J, M, N)) or the Euclidean distance (right rows (C, D, G, H, K, L, O, P)) and the obstacle K is a “square annulus” (the diﬀerence between two axis-parallel rectangles) to which we have removed a small channel to make its complement connected. On the domain Ω:= [−5, 5]2 \ K, we perform an IMEX Euler scheme in time combined with a ﬁnite element method in space with a time step of 0.1. The scheme is implemented in Python relying on the FEM library DOLFIN [55] and the VisiLibity library [64] in order to evaluate the geodesic distance. We observe that the respective solutions converge to diﬀerent asymptotic proﬁles as t →∞with a signiﬁcative diﬀerence in the qualitative behaviour of their dynamics. \|u(t, x) −φ(x1 + ct)\| −→ t→±∞0 locally uniformly in x ∈Ω. These simulations clearly highlight the importance of the distance in the ﬁnal outcome of the propagation and on the transition behaviour. that this holds true in general. We have been recently aware of some recent works [67] in this direction dealing with convex obstacles in the particular case where δ is the Euclidean distance. 2.5. Organization of the paper. In the following Section 3, we focus on the properties of the Cauchy problem associated to (P). This will pave the way towards the construction of 15 PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION an entire solution to (P). There, we will establish various comparison principles, existence and uniqueness results as well as some parabolic-type estimates. Section 4 deals with the a priori regularity of entire solutions. Indeed, it is not clear whether parabolic-type estimates hold for entire solutions, but we prove that, in some circumstances, such estimates can be shown to hold. In Section 5, relying on the results collected in the previous sections and on a sub- and super-solution technique, we prove the existence and uniqueness of an entire solution converging uniformly to φ(x1 + ct) as t →−∞. Next, in Section 6, we study the local behaviour of the entire solution in the large time limit. In Section 7, we study the inﬂuence of the geometry of K on the large time behaviour of the entire solution. Finally, in Sections 8 and 9 we respectively study the long time behaviour of the entire solution and prove that this entire solution is actually a generalised transition wave. 3. The Cauchy problem This section is devoted to the study of the Cauchy problem ∂tu = Lu + f(u) a.e. in (t0, ∞) × Ω, u(t0, ·) = u0(·) a.e. in Ω, (3.1) (3.1) where t0 ∈R and u0 is a given data. The study of (3.1) is essential to our purposes in that it shall pave the way towards the construction of an entire solution to (P). where t0 ∈R and u0 is a given data. The study of (3.1) is essential to our purposes in that it shall pave the way towards the construction of an entire solution to (P). We will establish various comparison principles, existence and uniqueness results for (3.1) as well as some a priori estimates under appropriate assumptions on the datum (J, f) and the initial datum u0. 3.1. Some comparison principles. In this section, we prove several comparison principle that ﬁt for our purposes. 3.1. Some comparison principles. In this section, we prove several comparison principle that ﬁt for our purposes. Next, we let κ > 0 be so large that κ ⩾∥µ∥∞+ ∥J δ∥∞+ 1, and we let ew be the function given by ew(t, x) := eκ(t−t0)w(t, x) for all (t, x) ∈[t0, t1] × Ω. By a straightforward calculation, we have that and we let ew be the function given by ew(t, x) := eκ(t−t0)w(t, x) for all (t, x) ∈[t0, t1] × Ω. By a straightforward calculation, we have that ∂t ew(t, x) = eκt∂tw(t, w) + κ ew(t, x) ⩾eκtLw(t, x) + (µ(t, x) + κ) ew(t, x) = ˆ Ω J(δ(x, y)) ew(t, y) dy + (µ(t, x) + κ −J δ) ew(t, x). (3.6) (3.6) Furthermore, recalling (3.5) and using that w(·, x) ∈C([t0, t1]) (remember (3.2)), we h \| ew(t, x) −ew(t′, x)\| = \|eκ(t−t0)w(t, x) −eκ(t′−t0)w(t′, x)\| = \|(eκ(t−t0) −eκ(t′−t0))w(t, x) + eκ(t′−t0)(w(t, x) −w(t′, x))\| ⩽C1 \|eκ(t−t0) −eκ(t′−t0)\| + eκ(t′−t)\|t −t′\| ⩽C1(κ + 1)eκ(t1−t0)\|t −t′\|, .7) ⩽C1(κ + 1)eκ(t1−t0)\|t −t′\|, (3.7) (3.7) for all t, t′ ∈[t0, t1] and all x ∈Ω. for all t, t′ ∈[t0, t1] and all x ∈Ω. Now, for all s ⩾0, we deﬁne the perturbation ews of ew given by ews(t, x) = ew(t, x)+se2κ(t−t0) for all (t, x) ∈[t0, t1] × Ω. Observe that ∂t ews(t, x) = ∂t ew(t, x) + 2κse2κ(t−t0). So, using (3.6), by a short computation we ﬁnd that ∂t ews(t, x) ⩾ ˆ Ω J(δ(x, y)) ews(t, y)dy + γ1(t, x) ews(t, x) + γ2(t, x)se2κ(t−t0). where γ1 and γ2 denote the following expressions where γ1 and γ2 denote the following expressions γ1(t, x) := µ(t, x) + κ −J δ(x) and γ2(t, x) := κ −µ(t, x). γ1(t, x) := µ(t, x) + κ −J δ(x) and γ2(t, x) := κ −µ(t, x). Observe that, by construction of κ, we have γ1(t, x) > 0 and γ2(t, x) > 0 for all (t, x) ∈ [t0, t1] × Ω. In particular, we have Observe that, by construction of κ, we have γ1(t, x) > 0 and γ2(t, x) > 0 for all (t, x) ∈ [t0, t1] × Ω. In particular, we have ∂t ews(t, x) > 0 for all x ∈Ω, as soon as ews(t, x) > 0 for all x ∈Ω. 3.1. Some comparison principles. In this section, we prove several comparison principle that ﬁt for our purposes. Lemma 3.1 (Comparison principle). Assume (1.4), (1.6) and suppose that f ∈C0,1 loc(R). Let t0, t1 ∈R with t0 < t1 and let u1 and u2 be two bounded measurable functions deﬁned in [t0, t1] × Ωand such that, for all i ∈{1, 2}, ui(t, ·), ∂tui(t, ·) ∈C(Ω) for all t ∈(t0, t1] and ui(t0, ·) ∈C(Ω), that ui(·, x) ∈C([t0, t1]) ∩C1((t0, t1]) for all x ∈Ω, (3.2) (3.2) sup (t,x)∈(t0,t1]×Ω \|∂tui(t, x)\| < ∞. (3.3) (3.3) sup (t,x)∈(t0,t1]×Ω \|∂tui(t, x)\| < ∞. (3.3) Suppose that Suppose that ∂tu1 −Lu1 −f(u1) ⩾∂tu2 −Lu2 −f(u2) in (t0, t1] × Ω, u1(t0, ·) ⩾u2(t0, ·) in Ω. (3.4) ∂tu1 −Lu1 −f(u1) ⩾∂tu2 −Lu2 −f(u2) in (t0, t1] × Ω, u1(t0, ·) ⩾u2(t0, ·) in Ω. (3.4) Then, (3.4) u1(t, x) ⩾u2(t, x) for all (t, x) ∈[t0, t1] × Ω. u1(t, x) ⩾u2(t, x) for all (t, x) ∈[t0, t1] × Ω. Remark 3.2. For related results in similar contexts, the reader may consult [15, 24]. JULIEN BRASSEUR AND J´ERˆOME COVILLE 16 Proof. We set w := u1 −u2. Readily, we notice that Proof. We set w := u1 −u2. Readily, we notice that Proof. We set w := u1 −u2. Readily, we notice that C1 := sup (t,x)∈(t0,t1]×Ω \|w(t, x)\| + \|∂tw(t, x)\| < ∞. (3.5) C1 := sup (t,x)∈(t0,t1]×Ω \|w(t, x)\| + \|∂tw(t, x)\| < ∞. (3.5) (3.5) (Remember (3.3) and the boundedness assumption on u1 and u2.) ( ( ) p Moreover, we let µ ∈L∞([t0, t1] × Ω) be any function so that (u1(t, x)) −f(u1(t, x)) = µ(t, x)(u1(t, x) −u2(t, x)) for all (t, x) ∈[t0, t1] × Ω. f(u1(t, x)) −f(u1(t, x)) = µ(t, x)(u1(t, x) −u2(t, x)) for all (t, x) ∈[t0, t1] × Ω. Note that such a function always exists since u1 and u2 are bounded and since f ∈C0,1 loc(R). Now, using the hypotheses made on u1 and u2, we have Note that such a function always exists since u1 and u2 are bounded and since f ∈C0,1 loc(R). Now, using the hypotheses made on u1 and u2, we have ∂tw(t, x) −Lw(t, x) ⩾f(u1(t, x)) −f(u2(t, x)) = µ(t, x)w(t, x), for any (t, x) ∈[t1, t2] × Ω. Next, we let κ > 0 be so large that for any (t, x) ∈[t1, t2] × Ω. 3.1. Some comparison principles. In this section, we prove several comparison principle that ﬁt for our purposes. Recalling (3.8), we further obtain that ∂t ews(t, x) > 0 for all (t, x) ∈(t0, t1] × Ω, so that ews is an increasing function of time for all x ∈Ω. In particular, we have for all 0 < ε < t1 −t∗, where we have used (3.7). This implies that ews(t∗+ ε, x) > 0 for all x ∈Ωand all 0 < ε < min{t1 −t∗, 3s/(4C2)}, which contradicts the maximality of t∗. Therefore, t∗= t1 which enforces that ews(t, x) > 0 for all (t, x) ∈(t0, t1]×Ω. Recalling (3.8), we further obtain that ∂t ews(t, x) > 0 for all (t, x) ∈(t0, t1] × Ω, so that ews is an increasing function of time for all x ∈Ω. In particular, we have ews(t, x) > ews(t0, x) = ew(t0, x) + s for all (t, x) ∈(t0, t1] × Ω. ews(t, x) > ews(t0, x) = ew(t0, x) + s for all (t, x) ∈(t0, t1] × Ω. Letting now s →0+, we obtain that Letting now s →0+, we obtain that eκ(t−t0)w(t, x) = ew(t, x) ⩾ew(t0, x) ⩾0 for all (t, x) ∈(t0, t1] × Ω. Therefore, we have w(t, x) ⩾0 for all (t, x) ∈[t0, t1] × Ω, as desired. □ eκ(t−t0)w(t, x) = ew(t, x) ⩾ew(t0, x) ⩾0 for all (t, x) ∈(t0, Therefore, we have w(t, x) ⩾0 for all (t, x) ∈[t0, t1] × Ω, as desired. Therefore, we have w(t, x) ⩾0 for all (t, x) ∈[t0, t1] × Ω, as desired. □ Remark 3.3. It turns out that our proof also yield a version of Lemma 3.1 on half-spaces H ⊂Ω. Namely, if RJ > 0 is such that supp(J) ⊂[0, RJ], if one replaces (3.4) by ∂tu1 −Lu1 −f(u1) ⩾∂tu2 −Lu2 −f(u2) in (t0, t1] × H, u1(t0, ·) ⩾u2(t0, ·) in H. and if one further assume that u1 ⩾u2 in [t0, t1] × x ∈Ω\ H; inf y∈H δ(x, y) ⩽RJ , (3.9) (3.9) then, it still holds that u1(t, x) ⩾u2(t, x) for all (t, x) ∈[t0, t1]×H. Notice that it is because of the nonlocality of the operator L that we need to assume (3.9). then, it still holds that u1(t, x) ⩾u2(t, x) for all (t, x) ∈[t0, t1]×H. Notice that it is because of the nonlocality of the operator L that we need to assume (3.9). 3.1. Some comparison principles. In this section, we prove several comparison principle that ﬁt for our purposes. (3.8) Since ews(t, x) = ew(t, x) + se2κ(t−t0) and since ew(t0, x) = w(t0, x) ⩾0, we have ews(t, x) ⩾ew(t, x) −ew(t0, x) + ew(t0, x) + se2κ(t−t0) ⩾−\| ew(t, x) −ew(t0, x)\| + s. (3.8) PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 17 Using (3.7) with t′ = t0, we obtain Using (3.7) with t′ = t0, we obtain ews(t, x) ⩾−C2\|t −t0\| + s, where C2 := C1(κ + 1)eκ(t−t0). In turn, this implies that ews(t, x) > 0 for all (t, x) ∈ t0, t0 + s 2C2 × Ω. In particular, the following quantity is well-deﬁned t∗:= sup n t ∈(t0, t1) ; ews(τ, x) > 0 for all (τ, x) ∈(t0, t) × Ω o . t∗:= sup n t ∈(t0, t1) ; ews(τ, x) > 0 for all (τ, x) ∈(t0, t) × Ω o . Clearly, t∗> t0 + s/(4C2). Suppose, by contradiction, that t∗< t1. Then, by deﬁnition of t∗, we must have ews(t∗, x) ⩾0 and ews(t, x) > 0 for all t ∈(t0, t∗) and all x ∈Ω. From the latter and (3.8), we deduce that ews(t, x) is monotone increasing in (t0, t∗). Hence, we have ews(t, x) ⩾ews t0 + s 4C2 , x ⩾3s 4 > 0 for all (t, x) ∈ t0 + s 4C2 , t∗ × Ω. Letting t →t− ∗, we get ews(t∗, x) ⩾3s/4. Thus, recalling the deﬁnition of ews, we have ews(t∗+ ε, x) ⩾ew(t∗+ ε, x) −ew(t∗, x) + ews(t∗, x) ⩾−C2ε + 3s 4 , ews(t, x) ⩾ews t0 + s 4C2 , x ⩾3s 4 > 0 for all (t, x) ∈ t0 + s 4C2 , t∗ × Ω. →t− ∗, we get ews(t∗, x) ⩾3s/4. Thus, recalling the deﬁnition of ews, we have ews(t∗+ ε, x) ⩾ew(t∗+ ε, x) −ew(t∗, x) + ews(t∗, x) ⩾−C2ε + 3s 4 , for all 0 < ε < t1 −t∗, where we have used (3.7). This implies that ews(t∗+ ε, x) > 0 for all x ∈Ωand all 0 < ε < min{t1 −t∗, 3s/(4C2)}, which contradicts the maximality of t∗. Therefore, t∗= t1 which enforces that ews(t, x) > 0 for all (t, x) ∈(t0, t1]×Ω. (3.12) (3.12) Observe that, thanks to (1.6), we have that J δ ∈Cb(Ω) and that the operator L[·] maps Cb(Ω) into itself. In fact, by our assumptions on J, L[·] is a well-deﬁned continuous linear operator in Cb(Ω) (endowed with the sup-norm) and we have ∥L∥⩽∥J δ∥∞. Next, multiplying (3.1) by eωτ, where ω is given by (3.10), and integrating over τ ∈[t0, t], we arrive at the following integral equation u(t, x)=e−ω(t−t0)u0(x)+ ˆ t t0 e−ω(t−τ)L[u](τ, x)+(ω−J δ(x))u(τ, x)+f(u(τ, x)) dτ. (3.13) Since (3.1) and (3.13) are equivalent, it suﬃces to establish the existence and uniqueness of a solution to (3.13). For the sake of clarity, we subdivide the proof of this into three steps. Since (3.1) and (3.13) are equivalent, it suﬃces to establish the existence and uniqueness of a solution to (3.13). For the sake of clarity, we subdivide the proof of this into three steps. 3.1. Some comparison principles. In this section, we prove several comparison principle that ﬁt for our purposes. Lemma 3.4. Assume (1.4), (1.6) and suppose that f ∈C1(R). Let t0, t1 ∈R with t0 < t1 and let u : [t0, t1] × Ω→R be a measurable function such that u(t, ·) ∈C(Ω) for each ﬁxed JULIEN BRASSEUR AND J´ERˆOME COVILLE 18 t ∈[t0, t1], and that u(·, x) ∈C1([t0, t1]) ∩C2((t0, t1]) for each ﬁxed x ∈Ω. Suppose, in addition, that u, ∂tu and ∂2 t u are uniformly bounded (in x and t) and that ∂tu = Lu + f(u) in (t0, t1] × Ω, ∂tu = Lu + f(u) in (t0, t1] × Ω, ∂tu(t0, ·) ⩾0 in Ω. Then, ∂tu(t, x) ⩾0 in [t0, t1] × Ω. Then, ∂tu(t, x) ⩾0 in [t0, t1] × Ω. Proof. Letting v(t, x) := ∂tu(t, x) we have v(t0, ·) ⩾0 in Ωand ∂tv(t, x) −Lv(t, x) = v(t, x)f ′(u(t, x)) =: µ(t, x)v(t, x) in (t0, t1] × Ω, ∂tv(t, x) −Lv(t, x) = v(t, x)f ′(u(t, x)) =: µ(t, x)v(t, x) in (t0, t1] where µ(t, x) is a bounded function (because f ∈C1(R) and u is bounded). From here, we may apply the same strategy as in Lemma 3.1. □ where µ(t, x) is a bounded function (because f ∈C1(R) and u is bounded). From here, we may apply the same strategy as in Lemma 3.1. □ where µ(t, x) is a bounded function (because f ∈C1(R) and u is bounded). From here, we may apply the same strategy as in Lemma 3.1. □ 3.2. Existence of a unique solution. In this section, we will establish the existence and uniqueness of a solution to (3.1). For the sake of convenience, for f ∈C0,1 ∩C1(R), we set 3.2. Existence of a unique solution. In this section, we will establish the existence and uniqueness of a solution to (3.1). For the sake of convenience, for f ∈C0,1 ∩C1(R), we set ω := sup \|f ′\| + 2 sup J δ. (3.10) ω := sup R \|f ′\| + 2 sup Ω J δ. 3.10) (3.10) Then, we have the following result: Then, we have the following result: Proposition 3.5 (Existence and uniqueness). Let t0 ∈R and let u0 ∈Cb(Ω). Assume (1.4), (1.6) and suppose that f ∈C0,1 ∩C1(R). Then, there exists a unique solution u ∈ C2([t0, ∞), C(Ω)) to (3.1). 3.1. Some comparison principles. In this section, we prove several comparison principle that ﬁt for our purposes. Moreover, for all T > t0, the following estimates hold: ω−1∥∂ttu∥L∞([t0,T]×Ω) ⩽∥∂tu∥L∞([t0,T]×Ω) ⩽ ω + \|f(0)\| ∥u∥L∞([t0,T]×Ω). (3.11) ω−1∥∂ttu∥L∞([t0,T]×Ω) ⩽∥∂tu∥L∞([t0,T]×Ω) ⩽ ω + \|f(0)\| ∥u∥L∞([t0,T]×Ω). (3.11) Proof. The proof is rather standard but we nevertheless outline the main ingredients. First of all, we observe that the a priori estimates (3.11) follow directly by using (3.1) and the equation obtained when diﬀerentiating (3.1) with respect to t. Now, let us deﬁne L[u](t, x) := ˆ Ω J(δ(x, y))u(t, y)dy. (3.12) 2. Construction of a micro-solution in a small window of time (Notice that, since f ∈C1(R), a straightforward bootstrap argument shows that uT0 ∈C2([t0, T0], C(Ω)).) Using (3.14) together with (3.11) we may apply the comparison principle Lemma 3.1 to deduce that uT0 is the unique solution to (3.1) in [t0, T0]. Hence (un)n⩾0 is a Cauchy sequence in the topology of Cb([t0, T0] × Ω) (equipped with the sup-norm). Since (Cb([t0, T0] × Ω), ∥·∥∞) is complete, it follows that un converges towards a function uT0 ∈Cb([t0, T0] × Ω) which, by dominated convergence, solves the equation on [t0, T0]×Ω. (Notice that, since f ∈C1(R), a straightforward bootstrap argument shows that uT0 ∈C2([t0, T0], C(Ω)).) Using (3.14) together with (3.11) we may apply the comparison principle Lemma 3.1 to deduce that uT0 is the unique solution to (3.1) in [t0, T0]. 2. Construction of a micro-solution in a small window of time Let T0 ∈(t0, t0 + ω−1 log(2)) be arbitrary and let (un)n⩾0 be the sequence of functions deﬁned on (t, x) ∈[t0, T0] × Ωby u0(t, x) = e−ω(t−t0)u0(x), and, for n ⩾0, and, for n ⩾0, un+1(t, x) = u0(t, x)+ ˆ t t0 e−ω(t−τ)L[un](τ, x)+(ω−J δ(x))un(τ, x)+f(un(τ, x)) dτ. Remark that, since f is continuous, J δ ∈Cb(Ω), u0 ∈Cb([t0, T0]×Ω) and L[·] is a continuous linear operator in Cb(Ω), it follows that (un)n⩾0 ⊂Cb([t0, T0] × Ω). Now for any n ⩾1 it holds that Remark that, since f is continuous, J δ ∈Cb(Ω), u0 ∈Cb([t0, T0]×Ω) and L[·] is a continuous linear operator in Cb(Ω), it follows that (un)n⩾0 ⊂Cb([t0, T0] × Ω). ( ) Now, for any n ⩾1, it holds that ( ) ( ) ⩾ ([ ] Now, for any n ⩾1, it holds that \|un+1(t, x) −un(t, x)\| ⩽2ω ˆ t t0 e−ω(t−τ)dτ sup (τ,x)∈[t0,T0]×Ω \|un(τ, x) −un−1(τ, x)\| ⩽2 1 −e−ω(T0−t0) sup (τ,x)∈[t0,T0]×Ω \|un(τ, x) −un−1(τ, x)\|, where we have used the deﬁnition of ω. We therefore arrive at sup (t,x)∈[t0,T0]×Ω \|un+1(t, x) −un(t, x)\| ⩽H sup (t,x)∈[t0,T0]×Ω \|un(t, x) −un−1(t, x) where we have set H := 2 1 −e−ω(T0−t0) . Notice that, since T0 < t0 + ω−1 log(2), we have that H ∈(0, 1). Thus, Notice that, since T0 < t0 + ω−1 log(2), we have that H ∈(0, 1). Thus, sup (t,x)∈[t0,T0]×Ω \|un+1(t, x) −un(t, x)\| ⩽Hn sup (t,x)∈[t0,T0]×Ω \|u1(t, x) −u0(t, x)\| →0 as n t, since T0 < t0 + ω−1 log(2), we have that H ∈(0, 1). Thus, Notice that, since T0 < t0 + ω log(2), we have that H ∈(0, 1). Thus, sup (t,x)∈[t0,T0]×Ω \|un+1(t, x) −un(t, x)\| ⩽Hn sup (t,x)∈[t0,T0]×Ω \|u1(t, x) −u0(t, x)\| →0 as n →∞. \|un+1(t, x) −un(t, x)\| ⩽Hn sup (t,x)∈[t0,T0]×Ω \|u1(t, x) −u0(t, x)\| →0 as n →∞. sup (t,x)∈[t0,T0]×Ω \|un+1(t, x) −un(t, x)\| ⩽Hn sup (t,x)∈[t0,T0]×Ω \|u1(t, x) −u0(t, x)\| → Hence (un)n⩾0 is a Cauchy sequence in the topology of Cb([t0, T0] × Ω) (equipped with the sup-norm). Since (Cb([t0, T0] × Ω), ∥·∥∞) is complete, it follows that un converges towards a function uT0 ∈Cb([t0, T0] × Ω) which, by dominated convergence, solves the equation on [t0, T0]×Ω. Step 1. A preliminary a priori bound on ∥u(t, ·)∥∞ Step 1. A preliminary a priori bound on ∥u(t, ·)∥∞ Prior to proving the existence of a solution u to (3.1) (or, equivalently, to (3.13)), let us ﬁrst establish a preliminary a priori bound on ∥u(t, ·)∥∞. For it, we observe that L[u](τ, x) + (ω −J δ(x))u(τ, x) + f(u(τ, x)) ⩽2ω∥u(τ, ·)∥∞+ \|f(0)\|. ugging this into (3.13), we obtain L[u](τ, x) + (ω −J δ(x))u(τ, x) + f(u(τ, x)) ⩽2ω∥u(τ, ·)∥∞+ \|f(0)\|. Now, plugging this into (3.13), we obtain eωt∥u(t, ·)∥∞⩽eωt0∥u0∥∞+ 2ω ˆ t t0 eωτ ∥u(τ, ·)∥∞dτ + \|f(0)\| ˆ t t0 eωτ dτ. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 19 Letting v(t) := eωt∥u(t, ·)∥∞and g(t) := \|f(0)\| ´ t t0 eωτ dτ, this becomes ( ) ( ) ˆ t ( )d ( ) Letting v(t) := eωt∥u(t, ·)∥∞and g(t) := \|f(0)\| ´ t t0 eωτ dτ, this becomes v(t) ⩽v(t0) + 2ω ˆ t t0 v(τ)dτ + g(t). Applying now Gr¨onwall’s lemma, we arrive at v(t) ⩽(g(t) + v(t0))eω(t−t0). Developping this expression using the deﬁnition of v and g, we obtain Applying now Gr¨onwall’s lemma, we arrive at v(t) ⩽(g(t) + v(t0))eω(t−t0). Developping this expression using the deﬁnition of v and g, we obtain ∥u(t, ·)∥∞⩽eω(t−t0) \|f(0)\| ω (eω(t−t0) −1) + ∥u0∥∞ , (3.14) (3.14) for any t ⩾t0. In particular, ∥u(t, ·)∥∞is locally bounded in t ∈[t0, ∞). for any t ⩾t0. In particular, ∥u(t, ·)∥∞is locally bounded in t ∈[t0, ∞). Step 2. Construction of a micro-solution in a small window of time Step 3. Conclusion The solution to (3.1) in the whole [t0, ∞) is obtained by a classical “analytic continuation” type argument by concatenating micro-solutions uTk on time intervals of the form [Tk−1, Tk] JULIEN BRASSEUR A JULIEN BRASSEUR AND J´ERˆOME COVILLE 20 with k ⩾0, where Tk := T0 + k(T0 −t0) for any −1 ⩽k ∈Z. This is indeed possible because the micro-solutions uTk are uniquely determined, continuous up to Tk and they satisfy ∂tuTk(T − k , ·) = ∂tuTk+1(T + k , ·). Hence, using again (3.11), the comparison principle Lemma 3.1, the fact that f is C1 and that uTk is bounded for any k ⩾0, we may easily check that the so-constructed solution is unique and has the claimed regularity in both space and time. The proof is thereby complete. □ Remark 3.6. Although this is a standard fact, we recall that a micro-solution on a time interval of length at most ω−1 log(2) is necessarily continuous in space provided the initial data is continuous (the proof of this fact follows closely the arguments of Step 2). By induction, it follows that a solution to the Cauchy problem (3.1) is also necessarily space continuous provided u0 ∈C(Ω). In particular, this justiﬁes why we could use the comparison principle Lemma 3.1 (that requires space continuity) to derive the uniqueness of the solution. Remark 3.7. If the initial datum u0 can be extended as a continuous function up to the boundary (for example if it is uniformly continuous), then the solution to the Cauchy problem (3.1) can also be extended so that u ∈C2([t0, ∞), C(Ω)). Moreover, this extension is a solution of the equation in Ω. 3.3. Parabolic type estimates. Let us now complete this section with a time-global para- bolic estimate for the Cauchy problem (3.1). For it, we will require the additional assumption 3.3. Parabolic type estimates. Let us now complete this section with a time-global para- bolic estimate for the Cauchy problem (3.1). For it, we will require the additional assumption max f ′ < inf J δ. (3.15) max R f ′ < inf ΩJ δ. 3.15) max R f ′ < inf ΩJ δ. (3.15) Precisely, we prove Proposition 3.8 (Parabolic estimates). Assume (1.4) and (1.6). Suppose, in addition, that f ∈C0,1 ∩C1(RN), that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1) and that (3.15) holds. Let t0 ∈R and let u0 ∈C0,α(Ω). Let u ∈C2([t0, ∞), C(Ω, [0, 1])) be the unique solution to (3.1). Suppose that u is uniformly bounded by some constant M0 > 0. Then, there exists a constant M > 0 (depending on J, f ′, M0, [u0]C0,α(Ω), Ωand δ) such that Proposition 3.8 (Parabolic estimates). Assume (1.4) and (1.6). Suppose, in addition, that f ∈C0,1 ∩C1(RN), that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1) and that (3.15) holds. Let t0 ∈R and let u0 ∈C0,α(Ω). Let u ∈C2([t0, ∞), C(Ω, [0, 1])) be the unique solution to (3.1). Suppose that u is uniformly bounded by some constant M0 > 0. Then, there exists a constant M > 0 (depending on J, f ′, M0, [u0]C0,α(Ω), Ωand δ) such that sup t⩾t0 [u(t, ·)]C0,α(Ω) + [∂tu(t, ·)]C0,α(Ω) ⩽M. Remark 3.9. Notice that, in addition to (1.4) and (1.6), it is further required that J ∈ Bα 1,∞(Ω; δ) and that (J, f) satisﬁes (3.15). These extra assumptions are essentially the same as those which were shown in [19, Lemma 3.2] (see also [18, Remark 2.5]) to be suﬃcient for the stationary solution to be (at least) H¨older continuous (remember Remark 1.9). The estimate we derive for [u(t, ·)]C0,α(Ω) (see (3.17) below) is actually very similar to the one obtained in [19, Lemma 3.2] for the stationary problem. Also, as we already pointed out in [18], this is a sort of “nondegeneracy condition” which is somehow necessary to ensure global parabolic regularity. Indeed, if δ is the Euclidean distance and K = ∅, this condition reads maxR f ′ < 1 and, when this condition is not satisﬁed, it is known that there exists kernels J ∈L1(RN) such that the equation ∂tu = J ∗u −u + f(u) admits discontinuous standing fronts [5, 82]. In this situation, the solution of the Cauchy problem (3.1) starting from a smooth Heaviside type initial datum is expected to converge towards a discontinuous front (in some weak topology), making thus the above estimate impossible. Remark 3.9. Notice that, in addition to (1.4) and (1.6), it is further required that J ∈ Bα 1,∞(Ω; δ) and that (J, f) satisﬁes (3.15). (3.15) These extra assumptions are essentially the same as those which were shown in [19, Lemma 3.2] (see also [18, Remark 2.5]) to be suﬃcient for the stationary solution to be (at least) H¨older continuous (remember Remark 1.9). The estimate we derive for [u(t, ·)]C0,α(Ω) (see (3.17) below) is actually very similar to the one obtained in [19, Lemma 3.2] for the stationary problem. Also, as we already pointed out in [18], this is a sort of “nondegeneracy condition” which is somehow necessary to ensure global parabolic regularity. Indeed, if δ is the Euclidean distance and K = ∅, this condition reads maxR f ′ < 1 and, when this condition is not satisﬁed, it is known that there exists kernels J ∈L1(RN) such that the equation ∂tu = J ∗u −u + f(u) admits discontinuous standing fronts [5, 82]. In this situation, the solution of the Cauchy problem (3.1) starting from a smooth Heaviside type initial datum is expected to converge towards a discontinuous front (in some weak topology), making thus the above estimate impossible. Proof. Let u be a solution of (3.1). By Proposition 3.5, we know that u is continuous, therefore it is well-deﬁned for all t ∈[t0, ∞) and all x ∈Ω. Actually, since u0 ∈C0,α(Ω), the PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 21 function u is also space continuous in the whole of Ω(remember Remark 3.7) and, hence, is also well-deﬁned for all t ∈[t0, ∞) and all x ∈Ω. Let us ﬁx some x1, x2 ∈Ωwith x1 ̸= x2, deﬁne Ψu(t) := u(t, x1) −u(t, x2) and set function u is also space continuous in the whole of Ω(remember Remark 3.7) and, hence, is also well-deﬁned for all t ∈[t0, ∞) and all x ∈Ω. Let us ﬁx some x1, x2 ∈Ωwith x1 ̸= x2, deﬁne Ψu(t) := u(t, x1) −u(t, x2) and set H(t, x1, x2) := ˆ Ω (u(t, y) −u(t, x1))(J(δ(x1, y)) −J(δ(x2, y)))dy. Observe immediately that, since \|u\| ⩽M0 and since J ∈Bα 1,∞(Ω; δ), we have \|H(t, x1, x2)\| ⩽2M0[J]Bα 1,∞(Ω;δ)\|x1 −x2\|α =: β. Observe immediately that, since \|u\| ⩽M0 and since J ∈Bα 1,∞(Ω; δ), we have \|H(t, x1, x2)\| ⩽2M0[J]Bα 1,∞(Ω;δ)\|x1 −x2\|α =: β. (3.15) Since f ∈C1(R) and u(·, x) ∈C(R) for all x ∈Ω, it follows from the mean value theorem that there exists a function Λ, ranging between u(t, x1) and u(t, x2), such that f ′(Λ(t))Ψu(t) = f(u(t, x1)) −f(u(t, x2)) and that f ′(Λ) is continuous. Letting γ(t) := J δ(x2) −f ′(Λ(t)) and using the function H, we can write the equation satisﬁed by Ψu as Since f ∈C1(R) and u(·, x) ∈C(R) for all x ∈Ω, it follows from the mean value theorem that there exists a function Λ, ranging between u(t, x1) and u(t, x2), such that f ′(Λ(t))Ψu(t) = f(u(t, x1)) −f(u(t, x2)) and that f ′(Λ) is continuous. Letting γ(t) := J δ(x2) −f ′(Λ(t)) and using the function H, we can write the equation satisﬁed by Ψu as Ψ′ u(t) = H(t, x1, x2) + γ(t)Ψu(t) for t > t0, Ψu(t0) = u0(x1) −u0(x2), Observe that, since f ′(Λ) is continuous, γ is also continuous. Next, we let v(t) be the unique solution of v′(t) = β −γ(t)v(t) for t > t0, v(t0) = d0, (3.16) (3.16) where we have set d0 := [u0]C0,α(Ω)\|x1−x2\|α. Now, since (3.16) is a linear ordinary diﬀerential linear equation, we can compute v explicitly. Namely, we have where we have set d0 := [u0]C0,α(Ω)\|x1−x2\|α. Now, since (3.16) is a linear ordinary diﬀerential linear equation, we can compute v explicitly. Namely, we have v(t) = d0 exp − ˆ t t0 γ(τ)dτ + β ˆ t t0 exp − ˆ t T γ(τ)dτ dT. umption (3.15), we have γ ⩾infΩJ δ −maxR f ′ =: γ∗> 0. In particular, ˆ t v(t) ⩽d0e−γ∗(t−t0) + β ˆ t t0 e−γ∗(t−T)dT = d0e−γ∗(t−t0) + βγ−1 ∗(1 −e−γ∗(t−t0)). Recalling the deﬁnition of β and d0, we obtain that 0 < v(t) ⩽ [u0]C0,α(Ω) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) \|x1 −x2\|α. rmore, that if ψ is either Ψu or −Ψu, then we have v′(t) −β + γ(t)v(t) ⩾ψ′(t) −β + γ(t)ψ(t) for t > t0, v(t0) ⩾ψ(t0). v′(t) −β + γ(t)v(t) ⩾ψ′(t) −β + γ(t)ψ(t) for t > t0, v(t0) ⩾ψ(t0). Hence, by the comparison principle for ordinary diﬀerential equations, we have \|u(t, x1) −u(t, x2)\| = \|Ψu(t)\| ⩽v(t) ⩽ [u0]C0,α(Ω) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) \|x1 −x2\|α. (3.17) Thus, [u(t, ·)]C0,α(Ω) ⩽([u0]C0,α(Ω) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ)). (3.15) Let us now establish the corre- sponding inequality for ∂tu. Using (3.1), we have 1) −u(t, x2)\| = \|Ψu(t)\| ⩽v(t) ⩽ [u0]C0,α(Ω) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) \|x1 −x2\|α. Thus, [u(t, ·)]C0,α(Ω) ⩽([u0]C0,α(Ω) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ)). Let us now establish the corre- sponding inequality for ∂tu. Using (3.1), we have Thus, [u(t, ·)]C0,α(Ω) ⩽([u0]C0,α(Ω) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ)). Let us now establish the corre- sponding inequality for ∂tu. Using (3.1), we have \|∂tu(t, x1) −∂tu(t, x2)\| ⩽∥u(t, ·)∥∞ ˆ Ω \|J(δ(x1, y)) −J(δ(x2, y))\|dy \|∂tu(t, x1) −∂tu(t, x2)\| ⩽∥u(t, ·)∥∞ ˆ Ω \|J(δ(x1, y)) −J(δ(x2, y))\|dy + \|J δ(x1)u(t, x1) −J δ(x2)u(t, x2)\| + \|f(u(t, x1)) −f(u(t, x2))\| =: A + B + C. (3.18) (3.18) JULIEN BRASSEUR AND J´ERˆOME COVILLE 22 Since J ∈Bα 1,∞(Ω; δ) and \|u\| ⩽M0 we have Since J ∈Bα 1,∞(Ω; δ) and \|u\| ⩽M0 we have Since J ∈Bα 1,∞(Ω; δ) and \|u\| ⩽M0 we have A ⩽M0[J]Bα 1,∞(Ω;δ)\|x1 −x2\|α. (3.19) A ⩽M0[J]Bα 1,∞(Ω;δ)\|x1 −x2\|α. (3.19) Now, using the trivial relation J δ(x1)u(t, x1)−J δ(x2)u(t, x2) = J δ(x1)(u(t, x1)−u(t, x2))+u(t, x2) J δ(x1)−J δ(x2) , together with the fact that J ∈Bα 1,∞(Ω; δ) and that \|u\| ⩽M0, we further have B ⩽∥J δ∥∞\|u(t, x1) −u(t, x2)\| + M0[J]Bα 1,∞(Ω;δ)\|x1 −x2\|α. (3.20) Plugging (3.19) and (3.20) in (3.18), we get Plugging (3.19) and (3.20) in (3.18), we get \|∂tu(t, x1) −∂tu(t, x2)\| ⩽eω\|u(t, x1) −u(t, x2)\| + 2M0[J]Bα 1,∞(Ω;δ)\|x1 −x2\|α, where we have set eω := ∥f ′∥∞+ ∥J δ∥∞. Recalling (3.17), we thus obtain \|∂tu(t, x1) −∂tu(t, x2)\| \|x1 −x2\|α ⩽eω [u0]C0,α(Ω)+2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) +2M0[J]Bα 1,∞(Ω;δ). □ The proof is thereby complete. The proof is thereby complete. Remark 3.10. If the datum (J, f) satisﬁes (1.5) and (1.6) (with f being deﬁned only on [0, 1]), then Proposition 3.5 guarantees the existence of a unique solution, u(t, x), to the Cauchy problem (3.1) for an initial datum ranging in [0, 1]. Indeed, it suﬃces to apply Proposition 3.5 to ef, where ef ∈C0,1 ∩C1(R) is the extension of f given by ef(s) :=      f ′(0)s if s < 0, f(s) if 0 ⩽s ⩽1, f ′(1)(s −1) if s > 1. (3.21) (3.21) The comparison principle Lemma 3.1 then guarantees that 0 ⩽u(t, x) ⩽1 so that (3.1) (with f being deﬁned only on [0, 1]) makes sense. (3.15) Moreover, if (J, f) also satisﬁes (2.1), then (J, ef) satisﬁes (3.15). Indeed, this is because inf ΩJ δ −max R ef ′ = inf ΩJ δ −max [0,1] f ′ > 0. In particular, Proposition 3.8 applies. Therefore, the unique solution to the Cauchy problem (3.1) with (J, f) satisfying (1.5), (1.6), (2.1) and J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1) enjoys parabolic type estimates. In particular, Proposition 3.8 applies. Therefore, the unique solution to the Cauchy problem (3.1) with (J, f) satisfying (1.5), (1.6), (2.1) and J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1) enjoys parabolic type estimates. ∂tu(t, x) is well-deﬁned whenever u(t, x) is, (4.3) The corresponding inequality for ∂tu follows from the same arguments as in the proof of Proposition 3.8. □ Remark 4.3. If (J, f) satisfy (1.5), (1.6), (1.7) and (2.1), then Lemma 4.1 implies that every solution to (4.1) ranging in [0, 1] and satisfying (4.2) (where (φ, c) is as in (1.7)) satisfy parabolic type estimates. To see this it suﬃces to argue as in Remark 3.10 by extending f linearly outside [0, 1] and to recall that φ ∈C2(R) (remember Remark 1.10). ∂tu(t, x) is well-deﬁned whenever u(t, x) is, (4.3) ∂tu(t, x) is well-deﬁned whenever u(t, x) is, ∂tu(t, x) is well-deﬁned whenever u(t, x) is, (4.3) ∂tu(t, x) is well-deﬁned whenever u(t, x) is, (4.3) (4.3) as follows from the equation satisﬁed by u. Let (tn)n⩾0 ⊂(−∞, 0) be a decreasing sequence with tn →−∞as n →∞. Let us now ﬁx some n ⩾0, let t > tn, let z, z′ ∈Ω\ N with z ̸= z′ and deﬁne Ψu(t) := u(t, z) −u(t, z′). At this stage, using (3.15) and recalling (4.3), we may apply the same trick as in Proposition 3.8, to get as follows from the equation satisﬁed by u. Let (tn)n⩾0 ⊂(−∞, 0) be a decreasing sequence with tn →−∞as n →∞. Let us now ﬁx some n ⩾0, let t > tn, let z, z′ ∈Ω\ N with z ̸= z′ and deﬁne Ψu(t) := u(t, z) −u(t, z′). At this stage, using (3.15) and recalling (4.3), we may apply the same trick as in Proposition 3.8, to get \|Ψu(t)\| ⩽ \|u(tn, z) −u(tn, z′)\| \|z −z′\|α + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) \|z −z′\|α, (4.4) for all t > tn, where γ∗:= infΩJ δ −maxR f ′ > 0. Next, using (4.2), we have lim sup n→∞ \|u(tn, z) −u(tn, z′)\| \|z −z′\|α = lim sup n→∞ \|φ(z1 + ctn) −φ(z′ 1 + ctn)\| \|z −z′\|α ⩽[φ]C0,α(R Th f l tti i (4 4) d lli th t Ψ (t) (t ) (t ′) \|Ψu(t)\| ⩽ \|u(tn, z) −u(tn, z′)\| \|z −z′\|α + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) \|z −z′\|α, (4.4) (4.4) for all t > tn, where γ∗:= infΩJ δ −maxR f ′ > 0. Next, using (4.2), we have lim sup n→∞ \|u(tn, z) −u(tn, z′)\| \|z −z′\|α = lim sup n→∞ \|φ(z1 + ctn) −φ(z′ 1 + ctn)\| \|z −z′\|α ⩽[φ]C0,α(R). lim sup n→∞ \|u(tn, z) −u(tn, z′)\| \|z −z′\|α = lim sup n→∞ \|φ(z1 + ctn) −φ(z′ 1 + ctn)\| \|z −z′\|α ⩽[φ]C0,α(R). Therefore, letting n →∞in (4.4) and recalling that Ψu(t) = u(t, z) −u(t, z′), we obtain \|u(t, z) −u(t, z′)\| ⩽ [φ]C0,α(R) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) \|z −z′\|α. Therefore, letting n →∞in (4.4) and recalling that Ψu(t) = u(t, z) −u(t, z′), we obtain \|u(t, z) −u(t, z′)\| ⩽ [φ]C0,α(R) + 2M0γ−1 ∗[J]Bα 1,∞(Ω;δ) \|z −z′\|α. Hence, (u(t, ·))t∈R is uniformly H¨older continuous. 4. A priori bounds for entire solutions There are no a priori regularity estimates for entire solutions to (P). In absence of speciﬁc assumptions on the datum (f, J), entire solutions may even not be continuous at all. In this section, we provide some results which show that, under some circumstances, a parabolic- type estimate holds true. Lemma 4.1 (A priori estimates). Assume (1.4) and (1.6). Suppose that f ∈C0,1 ∩C1(RN), that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1) and that (3.15) holds. Let φ ∈C0,α(R) and c > 0. Suppose that there exists an uniformly bounded measurable function u : R×Ω→R satisfying    ∂tu = Lu + f(u) for a.e. (t, x) ∈R × Ω, (4.1) lim t→−∞ess sup x∈Ω \|u(t, x) −φ(x1 + ct)\| = 0. (4.2) (4.1) (4.2) PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 23 Then, there exists a constant M > 0 (depending on J, f ′, φ, ∥u∥L∞(R×Ω), Ωand δ) such that sup x∈Ω ∥u(·, x)∥C1,1(R) + sup t∈R [u(t, ·)]C0,α(Ω) + [∂tu(t, ·)]C0,α(Ω) ⩽M. sup x∈Ω ∥u(·, x)∥C1,1(R) + sup t∈R [u(t, ·)]C0,α(Ω) + [∂tu(t, ·)]C0,α(Ω) ⩽M. Remark 4.2. As it was already observed by Berestycki, Hamel and Matano in the local case [12], the condition (4.2) plays the role of an “initial condition” at −∞. Remark 4.2. As it was already observed by Berestycki, Hamel and Matano in the local case [12], the condition (4.2) plays the role of an “initial condition” at −∞. Proof. Let u : R × Ω→R be an uniformly bounded solution of (4.1) with (4.2), and let M0 > 0 be such that ess sup(t,x)∈R×Ω\|u(t, x)\| ⩽M0. Using the equation (4.1) satisﬁed by u, the fact that f is C1 and the boundedness assumption on u, it follows directly using the equation (4.1) satisﬁed by u and the one obtained by diﬀerentiating (4.1) with respect to t, that ess supx∈Ω∥u(·, x)∥C1,1(R) ⩽M0(1 + ω + \|f(0)\| + ω(ω + \|f(0)\|)), where ω is as in (3.10). Thus, up to redeﬁne u in a set of measure zero, we may assume that u(·, x) is a C1,1(R) function for a.e. x ∈Ω. Then, u is deﬁned for all x ∈Ω\ N and for all t ∈R where N ⊂Ω is a set of Lebesgue measure zero. Notice that and (5.2) determines a unique bounded entire solution to (P). and (5.2) determines a unique bounded entire solution to (P) We will rely on a strategy already used in [12]. That is, we will construct a continuous subsolution w−and a continuous supersolution w+ to (P) satisfying w−⩽w+ and we will use these functions to construct an entire solution to (P) satisfying the desired requirements. 5.1. Preliminaries. Let us start by collecting some known facts on the travelling waves deﬁned at (1.7). Let (φ, c) be the unique (up to shifts) increasing solution of ( c φ′ = J1 ∗φ −φ + f(φ) in R, lim z→+∞φ(z) = 1, lim z→−∞φ(z) = 0, (5.3) ( c φ′ = J1 ∗φ −φ + f(φ) in R, lim z→+∞φ(z) = 1, lim z→−∞φ(z) = 0, (5.3) (5.3) where J1 is given by (1.8). In the remaining part of the paper we shall assume, for simplicity, that the function φ is normalized by where J1 is given by (1.8). In the remaining part of the paper we shall assume, for simplicity, that the function φ is normalized by φ(0) = θ. (5.4) φ(0) = θ. Notice that (5.3) and (5.4) determine φ uniquely. Let us now introduce two numbers which will play an important role in the sequel. We deﬁne λ, µ > 0 as the respective positive solutions of ˆ R J1(h)eλhdh −1 −cλ + f ′(0) = 0, (5.5) (5.5) and ˆ R J1(h)eµhdh −1 −cµ + f ′(1) = 0. (5.6) (5.6) Since f and J satisfy (1.5) and (1.6), respectively, and since J is compactly supported, the existence of such λ and µ is a simple exercise (see e.g. [53, Lemma 2.5]). We will sometimes refer to (5.5) and (5.6) as the characteristic equation satisﬁed by λ and µ. Since f and J satisfy (1.5) and (1.6), respectively, and since J is compactly supported, the existence of such λ and µ is a simple exercise (see e.g. [53, Lemma 2.5]). We will sometimes refer to (5.5) and (5.6) as the characteristic equation satisﬁed by λ and µ. An important property of λ and µ is that they “encode” the asymptotic behaviour of φ and φ′. More precisely: Lemma 5.2. Assume (1.5), (1.6) and (1.7). Let (φ, c) be a solution to (5.3) and let λ, µ > 0 be the respective positive solutions to (5.5) and (5.6). 5. Time before reaching the obstacle In this section we prove the existence of an entire solution to (P) that is monotone in- creasing with t and which converges to a planar wave φ(x1 + ct) as t →−∞. In addition, we show that this limit condition at −∞is somehow comparable to an initial value problem in that it determines a unique bounded entire solution. More precisely, we prove the following 24 JULIEN BRASSEUR AND J´ERˆOME COVILLE JULIEN BRASSEUR AND J´ERˆOME COVILLE 24 Theorem 5.1. Assume (1.4), (1.5), (1.6), (1.7) and (2.1). Suppose that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1). Then, there exists an entire solution u ∈C2(R, C0,α(Ω)) to (P) such that 0 < u(t, x) < 1 and ∂tu(t, x) > 0 for all (t, x) ∈R × Ω. (5.1) (5.1) Moreover, lim t→−∞\|u(t, x) −φ(x1 + ct)\| = 0 uniformly in x ∈Ω, (5.2) and (5.2) determines a unique bounded entire solution to (P). Then, it holds that A0 := lim z→−∞e−λzφ(z) = lim z→−∞ e−λzφ′(z) λ ∈(0, ∞), and A1 := lim z→∞eµz(1 −φ(z)) = lim z→∞ eµzφ′(z) µ ∈(0, ∞). Moreover, lim z→−∞e−λzJ1 ∗φ(z) = A0 ˆ R J(h)eλhdh. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 25 Proof. See e.g. Li et al. [53, Theorem 2.7] for the proof of the behaviour of φ and φ′. To obtain the asymptotic of J1 ∗φ(z), it suﬃces to observe that Proof. See e.g. Li et al. [53, Theorem 2.7] for the proof of the behaviour of φ and φ′. To obtain the asymptotic of J1 ∗φ(z), it suﬃces to observe that e−λzJ1 ∗φ(z) = ˆ R J1(h)eλhe−λ(z+h)φ(z + h)dh. Now, since, for all h ∈R, we have e−λ(z+h)φ(z + h) →A0 as z →−∞and since J1 is compactly supported, the asymptotic behaviour of J1 ∗φ(z) follows by a simple application of the Lebesgue dominated convergence theorem. □ A rather direct consequence of Lemma 5.2 is that it ensures the existence of numbers α0, β0, γ0, δ0 > 0 such that α0eλz ⩽φ(z) ⩽β0eλz and γ0eλz ⩽φ′(z) ⩽δ0eλz if z ⩽0, (5.7) and numbers α1, β1, γ1, δ1 > 0 such that α1e−µz ⩽1 −φ(z) ⩽β1e−µz and γ1e−µz ⩽φ′(z) ⩽δ1e−µz if z > 0. (5.8) (5.7) Finally, let us state a lemma that guarantees that φ is convex near −∞. Finally, let us state a lemma that guarantees that φ is convex near −∞. .3. Let (φ, c) be a solution to (5.3). Then, there exists some z∗< 0 such that Lemma 5.3. Let (φ, c) be a solution to (5.3). Then, there exists some z∗ φ′′(z) ⩾λ 8 φ′(z) for any z ⩽z∗, where λ is the positive solution to (5.5). In particular, φ is convex in (−∞, z∗] and we have φ( ) φ( ) where λ is the positive solution to (5.5). In particular, φ is convex in (−∞, z∗] and we have φ z1 + z2 2 ⩽φ(z1) + φ(z2) 2 for any z1, z2 ⩽z∗. Proof. Let us ﬁrst observe that, since f ∈C1,1([0, 1]), by a classical bootstrap argument we automatically get that φ ∈C2(R) and that c φ′′(z) = J1 ∗φ′(z) −φ′(z) + φ′(z)f ′(φ(z)) for any z ∈R. (5.9) c φ′′(z) = J1 ∗φ′(z) −φ′(z) + φ′(z)f ′(φ(z)) for any z ∈R. (5.9) c φ′′(z) = J1 ∗φ′(z) −φ′(z) + φ′(z)f ′(φ(z)) for any z ∈R. and (5.2) determines a unique bounded entire solution to (P). (5.9) (5.9) The assumption that f ∈C1,1([0, 1]) further gives that \|f ′(φ(z)) −f ′(0)\| ⩽C φ(z) for some C > 0 (depending on f) and for any z ∈R. In particular, we have The assumption that f ∈C1,1([0, 1]) further gives that \|f ′(φ(z)) −f ′(0)\| ⩽C φ(z) for some C > 0 (depending on f) and for any z ∈R. In particular, we have f ′(φ(z)) ⩾f ′(0) −C φ(z) for any z ∈R. (5.10) f ′(φ(z)) ⩾f ′(0) −C φ(z) for any z ∈R. f ′(φ(z)) ⩾f ′(0) −C φ(z) for any z ∈R. 5.10) By Lemma 5.2, we know that, for all ε > 0, there exists Rε > 0 such that By Lemma 5.2, we know that, for all ε > 0, there exists Rε > 0 such that By Lemma 5.2, we know that, for all ε > 0, there exists Rε > 0 such tha λ(A0 −ε)eλz ⩽φ′(z) ⩽λ(A0 + ε)eλz, (5.11) λ(A0 −ε)eλz ⩽φ′(z) ⩽λ(A0 + ε)eλz, 5.11) λ(A0 −ε)eλz ⩽φ′(z) ⩽λ(A0 + ε)eλz, for all z ⩽−Rε. Hence, using (5.9), (5.10) and (5.11), we obtain that c φ′′(z) ⩾λ(A0 −ε)eλz ˆ R J1(h)eλhdh + λ(A0 + ε)eλz f ′(0) −C(A0 + ε) eλz −1 , ′(z) ⩾λ(A0 −ε)eλz ˆ R J1(h)eλhdh + λ(A0 + ε)eλz f ′(0) −C(A0 + ε) eλz −1 e have used that J1 is even. By rearranging the terms we may rewrite this as c φ′′(z) ⩾λA0eλz f ′(0) −1 + ˆ R J1(h)eλhdh + λεeλz f ′(0) −1 − ˆ R J1(h)eλhdh −Cλ(A0 + ε)2e2λz. Using now the characteristic equation (5.5), we ﬁnd that c φ′′(z) ⩾cλ2A0eλz + λεeλz cλ −2 ˆ R J1(h)eλhdh −Cλ(A0 + ε)2e2λz. JULIEN BRASSEUR AND J´ERˆOME COVILLE 26 JULIEN BRASSEUR AND J´ERˆOME COVILLE 26 Choosing ε small enough, say 0 < ε < ε0, where ε0 := A0 min ( 1, cλ 2 cλ −2 ˆ R J1(h)eλhdh −1) , we obtain that c φ′′(z) ⩾cλ2 2 A0eλz −Cλ(A0 + ε)2e2λz ⩾λ(A0 + ε)eλzcλ 4 −2CA0eλz , c φ′′(z) ⩾cλ2 2 A0eλz −Cλ(A0 + ε)2e2λz ⩾λ(A0 + ε)eλzcλ 4 −2CA0eλz , or all z ⩽−Rε. Up to choose Rε > 0 larger, we may assume that 2CA0eλz ⩽cλ/8 for all ⩽−Rε. Therefore, recalling (5.11), we ﬁnally obtain that for all z ⩽−Rε. and (5.2) determines a unique bounded entire solution to (P). Up to choose Rε > 0 larger, we may assume that 2CA0eλz ⩽cλ/8 for all z ⩽−Rε. Therefore, recalling (5.11), we ﬁnally obtain that φ′′(z) ⩾λ2 8 (A0 + ε)eλz ⩾λ 8 φ′(z) for any z ⩽−Rε, □ which thereby completes the proof. □ which thereby completes the proof. Remark 5.4. Observe that the same arguments also yield the existence of some z∗> 0 such that φ is concave in [z∗, ∞). Remark 5.4. Observe that the same arguments also yield the existence of some z∗> 0 such that φ is concave in [z∗, ∞). 5.2. Construction of sub- and supersolutions. Let us introduce some necessary nota- tions. Let k > 0 be a positive number to be ﬁxed later on. We set ξ(t) := 1 λ log 1 1 −c−1keλct for t ∈(−∞, T), ξ(t) := 1 λ log 1 1 −c−1keλct for t ∈(−∞, T), (5.12) ξ(t) := 1 λ log 1 1 −c−1keλct for t ∈(−∞, T), (5.12) (5.12) where c is the speed of the travelling wave φ, λ is given by (5.5) and T := 1 λc log c k . (5.13) T := 1 λc log c k . T := 1 λc log c k . (5.13) To shorten our notations it will be convenient to set To shorten our notations it will be convenient to set To shorten our notations it will be convenient to set M ±(t) := ct ± ξ(t). (5.14) M ±(t) := ct ± ξ(t). (5.14) M ±(t) := ct ± ξ(t). (5.14) Readily, we observe that ξ(−∞) = 0 and ˙ξ(t) = keλM+(t). We now deﬁne two functions, w+ and w−, in RN × (−∞, T1] for some T1 ∈(−∞, T), by Readily, we observe that ξ(−∞) = 0 and ˙ξ(t) = keλM+(t). We now deﬁne two functions, w+ and w−, in RN × (−∞, T1] for some T1 ∈(−∞, T), by Readily, we observe that ξ(−∞) = 0 and ˙ξ(t) = keλM+(t). Step 1. Supersolution We aim to prove that the function w+ given by (5.15) is a supersolution to (P). More precisely, we want to show that P[w+](t, x) ⩾0 for any (t, x) ∈(−∞, T1] × Ω, P[w+](t, x) ⩾0 for any (t, x) ∈(−∞, T1] × Ω, and some T1 ∈(−∞, T]. We consider the cases x ∈{x1 ⩾0} and x ∈{x1 < 0} separately. Case x1 ⩾0. A straightforward calculation gives (5.18) ∂tw+(t, x) −f(w+(t, x)) = (c + ˙ξ(t))(φ′(z+) + φ′(z−)) −f φ(z+) + φ(z−) , where z+ := x1 + M +(t) and z−:= −x1 + M +(t). Furthermore, using (5.17) and the fac that supp(J) ⊂[0, RJ], we have (5.18) ∂tw+(t, x) −f(w+(t, x)) = (c + ˙ξ(t))(φ′(z+) + φ′(z−)) −f φ(z+) + φ(z−) , where z+ := x1 + M +(t) and z−:= −x1 + M +(t). Furthermore, using (5.17) and the fact that supp(J) ⊂[0, RJ], we have ∂tw+(t, x) −f(w+(t, x)) = (c + ˙ξ(t))(φ′(z+) + φ′(z−)) −f φ(z+) + φ(z−) , where z+ := x1 + M +(t) and z−:= −x1 + M +(t). Furthermore, using (5.17) and the fact that supp(J) ⊂[0, RJ], we have Lw+(t, x) = ˆ Ω J(δ(x, y))(w+(t, y) −w+(t, x))dy = ˆ RN J(\|x −y\|)(w+(t, y) −w+(t, x))dy. Consequently, −Lw+(t, x) = − ˆ RN J(\|x −y\|) φ(y1 + M +(t)) −φ(x1 + M +(t)) dy − ˆ RN J(\|x −y\|) φ(−y1 + M +(t)) −φ(−x1 + M +(t)) dy + I0(t, x), where we have set where we have set I0(t, x) := − ˆ {y1<0} J(\|x −y\|)∆2 y1φ(M +(t))dy, where the operator ∆2 y1 is as deﬁned in Section 1.2. Notice that, since x1 ⩾0 and since supp(J) ⊂[0, RJ], the integral over {y1 < 0} can be replaced by an integral over {−RJ ⩽ y1 < 0}. But given that M +(t) →−∞as t →−∞and that φ is convex near −∞(by Lemma 5.3), we have ∆2 y1φ(M +(t)) ⩽0 for all t ⩽T1 and all −RJ ⩽y1 ⩽0 (up to take T1 suﬃciently negative). Thus, we have that where the operator ∆2 y1 is as deﬁned in Section 1.2. Notice that, since x1 ⩾0 and since supp(J) ⊂[0, RJ], the integral over {y1 < 0} can be replaced by an integral over {−RJ ⩽ y1 < 0}. and (5.2) determines a unique bounded entire solution to (P). In particular, this still holds when K is, say, an inﬁnite wall with one or several holes pierced in it. Proof. For the sake of convenience, we introduce the operator P given by P[w](t, x) := ∂tw(t, x) −Lw(t, x) −f(w(t, x)). Notice that if T1 ∈(−∞, T) is suﬃciently negative, then M ±(t) < 0 for any t ∈(−∞, T1]. and (5.2) determines a unique bounded entire solution to (P). We now deﬁne two functions, w+ and w−, in RN × (−∞, T1] for some T1 ∈(−∞, T), by w+(t, x) = φ(x1 + M +(t)) + φ(−x1 + M +(t)) (x1 ⩾0), 2φ(M +(t)) (x1 < 0), w+(t, x) = φ(x1 + M +(t)) + φ(−x1 + M +(t)) (x1 ⩾0), 2φ(M +(t)) (x1 < 0), (5.15) and (5.15) w (t, x) 2φ(M +(t)) (x1 < 0 (5.15) and w−(t, x) = φ(x1 + M −(t)) −φ(−x1 + M −(t)) (x1 ⩾0), 0 (x1 < 0). (5.16) (5.16) (5.16) Notice that, if −∞< T1 ≪T, then w+ and w−satisfy Notice that, if −∞< T1 ≪T, then w+ and w−satisfy 0 ⩽w−< w+ ⩽1 for any (t, x) ∈(−∞, T1] × RN, Notice that, if −∞< T1 ≪T, then w+ and w−satisfy 0 ⩽w−< w+ ⩽1 for any (t, x) ∈(−∞, T1] × RN, the last inequality being a consequence of the weak maximum principle [19, Lemma 4.1]. We now claim the following the last inequality being a consequence of the weak maximum principle [19, Lemma 4.1]. We now claim the following Lemma 5.5. Let RJ > 0 be such that supp(J) ⊂[0, RJ]. Assume (1.4), (1.5), (1.6), (1.7) and suppose that K ⊂RN is such that Lemma 5.5. Let RJ > 0 be such that supp(J) ⊂[0, RJ]. Assume (1.4), (1.5), (1.6), (1.7) and suppose that K ⊂RN is such that K ⊂{x1 < −RJ} . (5.17) K ⊂{x1 < −RJ} . (5.17) Then, for k > 0 suﬃciently large, w+ and w−are, respectively, a supersolution and a subsolution to (P) in the time range t ∈(−∞, T1] for some T1 ∈(−∞, T). Then, for k > 0 suﬃciently large, w+ and w−are, respectively, a supersolution and a subsolution to (P) in the time range t ∈(−∞, T1] for some T1 ∈(−∞, T). 27 PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION Remark 5.6. Just as in the local case, the boundedness assumption on K in (1.4) can be relaxed since one only need (5.17) to hold. In particular, this still holds when K is, say, an inﬁnite wall with one or several holes pierced in it. Remark 5.6. Just as in the local case, the boundedness assumption on K in (1.4) can be relaxed since one only need (5.17) to hold. Step 1. Supersolution But given that M +(t) →−∞as t →−∞and that φ is convex near −∞(by Lemma 5.3), we have ∆2 y1φ(M +(t)) ⩽0 for all t ⩽T1 and all −RJ ⩽y1 ⩽0 (up to take T1 suﬃciently negative). Thus, we have that I0(t, x) ⩾0. Hence, using the equation satisﬁed by φ, we obtain Hence, using the equation satisﬁed by φ, we obtain −Lw+(t, x) ⩾−c(φ′(z+) + φ′(z−)) + f(φ(z+)) + f(φ(z−)). Plugging this in (5.18), we get Plugging this in (5.18), we get (5.19) P[w+](t, x) ⩾keλM+(t) (φ′(z+) + φ′(z−)) + f(φ(z+)) + f(φ(z−)) −f(φ(z+) + φ(z−)). Using the fact that f is of class C1,1, we may ﬁnd a constant ϱ > 0 such that (5.20) \|f(a) + f(b) −f(a + b)\| ⩽ϱ ab. (5.20) \|f(a) + f(b) −f(a + b)\| ⩽ϱ ab. (5.20) \|f(a) + f(b) −f(a + b)\| ⩽ϱ ab. JULIEN BRASSEUR AND J´ERˆOME COVILLE 28 Hence, (5.19) becomes (5.21) P[w+](t, x) ⩾keλM+(t) (φ′(z+) + φ′(z−)) −ϱ φ(z+)φ(z−). (5.21) Let us now treat the cases x ∈{x1 > −M +(t)} and x ∈{0 ⩽x1 ⩽−M +(t)} separately. In the latter case, we have z−⩽z+ ⩽0. Hence, using (5.7), (5.21) and the fact that φ′ > 0, we get P[w+](t, x) ⩾γ0keλx1+2λM+(t) −ϱβ2 0 e2λM+(t) ⩾e2λM+(t) γ0keλx1 −ϱβ2 0 . Thus, we have P[w+](t, x) ⩾0 for all x ∈{0 ⩽x1 ⩽−M +(t)} as soon as k is chosen so th 2 P[w+](t, x) ⩾γ0keλx1+2λM+(t) −ϱβ2 0 e2λM+(t) ⩾e2λM+(t) γ0keλx1 −ϱβ2 0 . P[w+](t, x) ⩾γ0keλx1+2λM+(t) −ϱβ2 0 e2λM+(t) ⩾e2λM+(t) γ0keλx1 −ϱβ2 0 . Thus, we have P[w+](t, x) ⩾0 for all x ∈{0 ⩽x1 ⩽−M +(t)} as soon as k is chosen so that k ⩾ϱβ2 0 γ0 . (5.22) (5.22) Let us now treat the case x ∈{x1 > −M +(t)}. In this case, we have z−< 0 < z+ and, again, we treat two situations independently, depending on whether λ < µ or λ ⩾µ. Let us now treat the case x ∈{x1 > −M +(t)}. In this case, we have z−< 0 < z+ and, again, we treat two situations independently, depending on whether λ < µ or λ ⩾µ. Assume ﬁrst that λ ⩾µ. Then, using (5.7), (5.8), (5.21) and the fact that φ′ > 0 and φ ⩽1, we deduce that P[w+](t, x) ⩾kγ1eλM+(t)e−µz+ −ϱβ0eλz− ⩾eλM+(t)kγ1e−λ(x1+M+(t)) −ϱβ0e−λx1+λM+(t) ⩾e−λx1kγ1 −ϱβ0eλM+(t) . PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION Now since K ⊂{x1 < −RJ}, we have Ω∩H+ = H+ \ K = H+, and so Now since K ⊂{x1 < −RJ}, we have Ω∩H+ = H+ \ K = H+, and so (5.26) Lw+(t, x) = ˆ H+ J(δ(x, y))(w+(t, y) −2φ(M +(t)))dy. Now since K ⊂{x1 < −RJ}, we have Ω∩H+ = H+ \ K = H+, and so (5.26) Lw+(t, x) = ˆ H+ J(δ(x, y))(w+(t, y) −2φ(M +(t)))dy. (5.26) Lw+(t, x) = ˆ H+ J(δ(x, y))(w+(t, y) −2φ(M +(t)))dy. (5.26) Observe that δ(x, y) ⩾RJ for all x ∈H− RJ := {x1 < −RJ} and all y ∈H+. But since supp(J) ⊂[0, RJ], we then have that J(δ(x, y)) = 0 for all (x, y) ∈H− RJ \K ×H+. Therefore, recalling (5.26), we have Lw+(t, x) = 0 for all (t, x) ∈(−∞, T1] × H− RJ \ K. Combining this with (5.25), we obtain that P[w+](t, x) ⩾0 for all (t, x) ∈(−∞, T1] × (H− RJ \ K). Observe that δ(x, y) ⩾RJ for all x ∈H− RJ := {x1 < −RJ} and all y ∈H+. But since supp(J) ⊂[0, RJ], we then have that J(δ(x, y)) = 0 for all (x, y) ∈H− RJ \K ×H+. Therefore, recalling (5.26), we have Lw+(t, x) = 0 for all (t, x) ∈(−∞, T1] × H− RJ \ K. Combining this with (5.25), we obtain that P[w+](t, x) ⩾0 for all (t, x) ∈(−∞, T1] × (H− RJ \ K). ( ), [ ]( , ) ⩾ ( , ) ( , 1] ( RJ \ ) Let us now treat the case x ∈{−RJ ⩽x1 < 0}. For it, we observe that δ(x, y) = \|x −y\| for all (x, y) ∈[−RJ, 0) × H+. Consequently, (5.26) rewrites ( ), [ ]( , ) ( , ) ( , Let us now treat the case x ∈{−RJ ⩽x1 < 0}. For it, we obse for all (x, y) ∈[−RJ, 0) × H+. Consequently, (5.26) rewrites Lw+(t, x) = ˆ H+ J(\|x −y\|) φ(y1 + M +(t)) + φ(−y1 + M +(t)) −2φ(M +(t)) dy = ˆ +∞ 0 J1(x1 −y1)∆2 y1φ(M +(t))dy1. = ˆ +∞ 0 J1(x1 −y1)∆2 y1φ(M +(t))dy1. Since supp(J1) ⊂[0, RJ] and −RJ ⩽x1 < 0, the above equality may be rewritten as Lw+(t, x) = ˆ RJ 0 J1(x1 −y1)∆2 y1φ(M +(t))dy1. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 29 Finally, by (5.22), (5.23), and (5.24), we have P[w+](t, x) ⩾0 whenever k ⩾max ϱβ2 0 γ0 , ϱβ0 γ1 eµL0 , in the set (t, x) ∈(−∞, T1] × {x1 ⩾0}, provided T1 is suﬃciently negative. in the set (t, x) ∈(−∞, T1] × {x1 ⩾0}, provided T1 is suﬃciently negative. Case x1 < 0. Readily, we see that Case x1 < 0. Readily, we see that ∂tw+(t, x) −f(w+(t, x)) = 2(c + ˙ξ(t))φ′(M +(t)) −f 2φ(M +(t)) . Now, since φ(0) = θ and φ′ > 0, we have f(2φ(M +(t))) ⩽0 as soon as φ(M +(t)) ⩽θ/2. Thus, since M +(t) is increasing, since limt→−∞M(t) = −∞and since limz→−∞φ(z) = 0, up to decrease further T1, we can assume that φ(M +(t)) ⩽θ/2 for all t ⩽T1. Hence, we have Now, since φ(0) = θ and φ′ > 0, we have f(2φ(M +(t))) ⩽0 as soon as φ(M +(t)) ⩽θ/2. Thus, since M +(t) is increasing, since limt→−∞M(t) = −∞and since limz→−∞φ(z) = 0, up to decrease further T1, we can assume that φ(M +(t)) ⩽θ/2 for all t ⩽T1. Hence, we have (5.25) ∂tw+(t, x) −f(w+(t, x)) ⩾2(c + ˙ξ(t))φ′(M +(t)) ⩾0. (5.25) ∂tw+(t, x) −f(w+(t, x)) ⩾2(c + ˙ξ(t))φ′(M +(t)) ⩾0. Let us now estimate Lw+(t, x). For it, let us denote by H+ and H−the half-spaces given by H+ := {x ∈RN; x1 > 0} and H−:= {x ∈RN; x1 ⩽0}, (5.25) (5 5) ∂t ( , ) f( ( , )) ⩾ ( + ξ( ))φ ( ( )) ⩾0 Let us now estimate Lw+(t, x). For it, let us denote by H+ and H−the half-spaces given by H+ := {x ∈RN; x1 > 0} and H−:= {x ∈RN; x1 ⩽0}, respectively. By deﬁnition of w+(t, x) we have respectively. By deﬁnition of w+(t, x) we have Lw+(t, x) = ˆ Ω J(δ(x, y))(w+(t, y) −w+(t, x))dy = ˆ Ω∩H−J(δ(x, y))(2φ(M +(t)) −2φ(M +(t)))dy + ˆ Ω∩H+ J(δ(x, y))(w+(t, y) −2φ(M +(t)))dy = ˆ Ω∩H+ J(δ(x, y))(w+(t, y) −2φ(M +(t)))dy. Lw+(t, x) = ˆ Ω J(δ(x, y))(w+(t, y) −w+(t, x))dy = ˆ Ω∩H−J(δ(x, y))(2φ(M +(t)) −2φ(M +(t)))dy + ˆ Ω∩H+ J(δ(x, y))(w+(t, y) −2φ(M +(t)))dy = ˆ Ω∩H+ J(δ(x, y))(w+(t, y) −2φ(M +(t)))dy. Step 1. Supersolution Since M +(t) ⩽0 for all t ⩽T1, we then have P[w+](t, x) ⩾0 as soon as k is chosen so that Since M +(t) ⩽0 for all t ⩽T1, we then have P[w+](t, x) ⩾0 as soon as k is chosen so that k ⩾ϱβ0 γ1 . k ⩾ϱβ0 γ1 . (5.23) (5.23) The remaining case λ < µ is treated using the same trick as in [12]. Namely, we notice that, if λ < µ, then, thanks to the characteristic equations (5.5) and (5.6), we must necessarily have f ′(0) > f ′(1) and The remaining case λ < µ is treated using the same trick as in [12]. Namely, we notice that, if λ < µ, then, thanks to the characteristic equations (5.5) and (5.6), we must necessarily have f ′(0) > f ′(1) and f(a) + f(b) −f(a + b) = (f ′(0) −f ′(1))b + O(b2) + O \|b(1 −a)\| , close to 1 and 0, respectively. In particular, if z+ ≫1 and z−≪−1, then f(φ(z+)) + f(φ(z−)) −f(φ(z+) + φ(z−)) ⩾0. Now, by deﬁnition of z+ and z−, there is some L0 > 0 such that the above inequality holds true for all t ⩽T1 and all x1 ∈[−M +(t) + L0, ∞) (up to take T1 suﬃciently negative). Consequently, using (5.19) and the fact that ξ′ and φ′ are positive quantities, we infer that P[w+](t, x) ⩾0 for all t ⩽T1 and all x ∈{x1 ⩾−M +(t) + L0}. [ ]( ) { ( ) } Lastly, let us treat the case x ∈{−M +(t) < x1 < −M +(t)+L0}. Using again (5.7), (5.8), (5.21) and the fact that φ′ > 0 and φ ⩽1, we obtain that P[w+](t, x) ⩾kγ1eλM+(t)e−µz+ −ϱβ0eλz− ⩾eλM+(t)kγ1e−µL0 −ϱβ0e−λx1 . Therefore, we have P[w+](t, x) ⩾0 as soon as k is chosen so that Therefore, we have P[w+](t, x) ⩾0 as soon as k is chosen so that k ⩾ϱβ0 γ1 eµL0. (5.24) k ⩾ϱβ0 γ1 eµL0. (5.24) PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION Since supp(J1) ⊂[0, RJ] and −RJ ⩽x1 < 0, the above equality may be rewritten as Lw+(t, x) = ˆ RJ 0 J1(x1 −y1)∆2 y1φ(M +(t))dy1. Since supp(J1) ⊂[0, RJ] and −RJ ⩽x1 < 0, the above equality may be rewritten as Lw+(t, x) = ˆ RJ 0 J1(x1 −y1)∆2 y1φ(M +(t))dy1. Lw+(t, x) = ˆ RJ 0 J1(x1 −y1)∆2 y1φ(M +(t))dy1. JULIEN BRASSEUR AND J´ERˆOME COVILLE 30 But given that M +(t) →−∞as t →−∞and that φ is convex near −∞(by Lemma 5.3), we have ∆2 y1φ(M +(t)) ⩽0 for all t ⩽T1 and all 0 ⩽y1 ⩽RJ (up to take T1 suﬃciently negative). Thus, we have Lw+(t, x) = ˆ RJ 0 J1(x1 −y1)∆2 y1φ(M +(t))dy1 ⩽0, for all t ⩽T1 and all x ∈{−RJ ⩽x1 < 0}. Hence, recalling (5.25), P[w+](t, x) ⩾2(c + ˙ξ(t))φ′(M +(t)) ⩾0, P[w+](t, x) ⩾2(c + ˙ξ(t))φ′(M +(t)) ⩾0, for all t ⩽T1 and all x ∈{−RJ ⩽x1 < 0}. Summing up, we have shown that, for every (t, x) ∈(−∞, T1] × Ωand T1 ∈(−∞, T) suﬃciently negative, it holds that for all t ⩽T1 and all x ∈{−RJ ⩽x1 < 0}. Summing up, we have shown that, for every (t, x) ∈(−∞, T1] × Ωand T1 ∈(−∞, T) suﬃciently negative, it holds that P[w+](t, x) ⩾0 whenever k ⩾max ϱβ2 0 γ0 , ϱβ0 γ1 eµL0 . This proves that w+ is indeed a supersolution to (P). This proves that w+ is indeed a supersolution to (P). Step 2. Subsolution We will follow the same strategy as above. We aim to prove that the function w−given by (5.16) is a subsolution to (P). More precisely, we want to show that P[w−](t, x) ⩽0 for any (t, x) ∈(−∞, T1] × Ω, and some T1 ∈(−∞, T). A direct calculation gives and some T1 ∈(−∞, T). A direct calculation gives and some T1 ∈(−∞, T). A direct calculation gives ∂tw−(t, x)−f(w−(t, x))= ( (c−˙ξ(t))(φ′(ζ+)−φ′(ζ−))−f φ(ζ+)−φ(ζ−) (x1 ⩾0), 0 (x1 < 0). (5.27) where ζ+ = x1 + M −(t), ζ−= −x1 + M −(t). Let us now estimate Lw−(t, x). ∂tw−(t, x)−f(w−(t, x))= ( (c−˙ξ(t))(φ′(ζ+)−φ′(ζ−))−f φ(ζ+)−φ(ζ−) (x1 ⩾0), 0 (x1 < 0). (5.27) where ζ+ = x1 + M −(t), ζ−= −x1 + M −(t). Let us now estimate Lw−(t, x). t, x)−f(w−(t, x))= ( (c−˙ξ(t))(φ′(ζ+)−φ′(ζ−))−f φ(ζ+)−φ(ζ−) (x1 ⩾0), 0 (x1 < 0). where ζ+ = x1 + M −(t), ζ−= −x1 + M −(t). Let us now estimate Lw−(t, x). Case x1 < 0. This case is straightforward. Indeed, as above, we can check tha Lw−(t, x) = ˆ H+ J(δ(x, y)) φ(y1 + M −(t)) −φ(−y1 + M −(t)) dy. But, since φ is increasing, the integrand above is nonnegative, and so Lw−(t, x) ⩾0. Hence, recalling (5.27), we ﬁnd that P[w−](t, x) ⩽0 for any x ∈{x1 < 0}. Case x1 ⩾0. Observe that, since supp(J) ⊂[0, RJ] and since K ⊂{x1 ⩽−RJ}, we have Lw−(t, x) = ˆ RN J(\|x −y\|)(w−(t, y) −w−(t, x)))dy, for all x ∈{x1 ⩾0}. Using the deﬁnition of w−, we have Lw−(t, x) = ˆ RN J(\|x −y\|) φ(y1 + M −(t)) −φ(x1 + M −(t)) dy − ˆ RN J(\|x −y\|) φ(−y1 + M −(t)) −φ(−x1 + M −(t)) dy −I1(t, x), Lw−(t, x) = ˆ RN J(\|x −y\|) φ(y1 + M −(t)) −φ(x1 + M −(t)) dy − ˆ RN J(\|x −y\|) φ(−y1 + M −(t)) −φ(−x1 + −(t, x) = ˆ RN J(\|x −y\|) φ(y1 + M −(t)) −φ(x1 + M −(t)) dy where we have set where we have set I1(t, x) := ˆ {−RJ⩽y1⩽0} J(\|x −y\|) φ(y1 + M −(t)) −φ(−y1 + M −(t)) dy. I1(t, x) := ˆ {−RJ⩽y1⩽0} J(\|x −y\|) φ(y1 + M −(t)) −φ(−y1 + M −(t)) dy. Step 2. Subsolution PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 31 Since y1 + M −(t) ⩽−y1 + M −(t) for all −RJ ⩽y1 ⩽0 and since φ is increasing, it holds that −I1(t, x) ⩾0. Therefore, by using (5.3), we get Lw−(t, x) ⩾c(φ′(ζ+) −φ′(ζ−)) −(f(φ(ζ+)) −f(φ(ζ−))). Recalling (5.27), we obtain Recalling (5.27), we obtain (5.28) P[w−](t, x) ⩽−˙ξ(t)(φ′(ζ+) −φ′(ζ−)) + f(φ(ζ+)) −f(φ(ζ−)) −f(φ(ζ+) −φ(ζ−)). Let us suppose that x ∈{x1 ⩾−M −(t)}. Then, using (5.20) and (5.28), we have (5.29) P[w−](t, x) ⩽−˙ξ(t)(φ′(ζ+) −φ′(ζ−)) + ϱ φ(ζ−)(φ(ζ+) −φ(ζ−)). (5.29) We consider the cases λ ⩾µ and λ < µ separately. Let us suppose that λ ⩾µ. Then, since ζ−⩽0 ⩽ζ+, using (5.7) and (5.8), we deduce from (5.29) that We consider the cases λ ⩾µ and λ < µ separately. Let us suppose that λ ⩾µ. Then, since ζ−⩽0 ⩽ζ+, using (5.7) and (5.8), we deduce from (5.29) that P[w−](t, x) ⩽−keλM+(t)γ0e−µ(x1+M−(t)) −δ0eλ(−x1+M−(t)) + ϱβ0eλ(−x1+M−(t)) = −eλ(−x1+M+(t))kγ0e−µM−(t)+(λ−µ)x1 −δ0eλM−(t) −ϱβ0e−2λξ(t) (5.30) ⩽−eλ(−x1+M+(t))kγ0 −δ0 −ϱβ0 , (5.30) since λ, µ > 0, M −(t) ⩽0 and ξ(t) ⩾0 for all t ⩽T1. Whence, P[w−](t, x) ⩽0 for x ∈{x1 ⩾−M −(t)} as soon as k is chosen so that since λ, µ > 0, M −(t) ⩽0 and ξ(t) ⩾0 for all t ⩽T1. Whence, P[w−](t, x) ⩽0 for x ∈{x1 ⩾−M −(t)} as soon as k is chosen so that k ⩾δ0 + ϱβ0 γ0 . Let us now consider the case λ < µ. Arguing as in the Step 1, i.e. using the characteristic equations (5.5) and (5.6), we deduce that f ′(0) > f ′(1) and that f(a + b) −f(a) −f(b) = −(f ′(0) −f ′(1)) b + O(b2) + O \|b(1 −a)\| , f(a + b) −f(a) −f(b) = −(f ′(0) −f ′(1)) b + O(b2) + O \|b(1 −a)\| , for a and b close to 1 and 0, respectively. Hence, we have f(φ(ζ+)) −f(φ(ζ−))−f φ(ζ+) −φ(ζ−) f(φ(ζ+)) −f(φ(ζ−))−f φ(ζ+) −φ(ζ−) = −(f ′(0) −f ′(1))φ(ζ−) + O φ2(ζ−) + O φ(ζ−)(1 −φ(ζ+)) , −f φ(ζ+) −φ(ζ−) = −(f ′(0) −f ′(1))φ(ζ−) + O φ2(ζ−) + O φ(ζ−)(1 −φ(ζ+)) , provided ζ−≪−1 and ζ+ ≫1. Thanks to the deﬁnition of ζ± and since φ satisﬁes (5.3), we can then ﬁnd a constant L1 > 0 such that provided ζ−≪−1 and ζ+ ≫1. Step 2. Subsolution Thanks to the deﬁnition of ζ± and since φ satisﬁes (5.3), we can then ﬁnd a constant L1 > 0 such that f(φ(ζ+)) −f(φ(ζ−)) −f φ(ζ+) −φ(ζ−) ⩽−κφ(ζ−), for all x ∈{x1 ⩾−M −(t) + L1}, where we have set κ := (f ′(0) −f ′(1))/2. This, together with (5.29) and (5.7), implies that P[w−](t, x) ⩽eλζ−(kδ0eλM+(t) −κα0). It follows that P[w−](t, x) ⩽0 in the set {x1 ⩾−M −(t) + L1} provided that T1 ∈(−∞, T] is chosen suﬃciently negative so that kδ0eλM+(t) ⩽κα0 for any −∞< t ⩽T1. Now, suppose that x ∈{−M −(t) ⩽x1 < −M −(t) + L1}. Then, it follows from (5.30) that P[w−](t, x) ⩽−eλ(−x1+M+(t))kγ0e−µM−(t)−(µ−λ)L1 −δ0eλM−(t) −ϱβ0e−2λξ(t) ⩽−eλ(−x1+M+(t))kγ0e−µM−(t)−(µ−λ)L1 −δ0 −ϱβ0 . JULIEN BRASSEUR AND J´ERˆOME COVILLE 32 Thus, P[w−](t, x) ⩽0 in the set x1 ∈{−M −(t) ⩽x1 < −M −(t) + L1} provided that T1 ∈(−∞, T] is chosen suﬃciently negative so that Thus, P[w−](t, x) ⩽0 in the set x1 ∈{−M −(t) ⩽x1 < −M −(t) + L1} provided that T1 ∈(−∞, T] is chosen suﬃciently negative so that γ0ke−µM−(t)−(µ−λ)L0 −δ0 −ϱβ0 ⩾0 for −∞< t ⩽T1. Next, suppose that x ∈{x1 < −M −(t)}. Then, ζ−⩽ζ+ ⩽0 and by (5.7), (5.8) and (5.29) we have that Next, suppose that x ∈{x1 < −M −(t)}. Then, ζ−⩽ζ+ ⩽0 and by (5.7), (5.8) and (5.29) we have that Next, suppose that x ∈{x1 < −M −(t)}. Then, ζ−⩽ζ+ ⩽0 and by (5.7), (5.8) and (5.29) we have that P[w−](t, x) ⩽−keλM+(t)γ0eλζ+ −δ0eλζ− + ϱβ2 0 eλζ−eλζ+ ⩽−keλM+(t)γ0eλ(x1+M−(t)) −δ0eλ(−x1+M−(t) + ϱβ2 0eλ(−x1+M−(t))eλ(x1+M−(t)) ⩽eλ(M+(t)+M−(t))−k γ0eλx1 −δ0e−λx1 + ϱβ2 0 e2λM−(t) ⩽e2λct −k γ0eλx1 −δ0e−λx1 + ϱβ2 0 e−2λξ(t) ⩽e2λct −kγ0eλx1 + kδ0 + ϱβ2 0 . 5.31) (5.31) Let R0 > 0 be the number given by Let R0 > 0 be the number given by R0 := 1 λ log δ0 γ0 + 2 . Choosing k large enough so that k ⩾ϱβ2 0/γ0, we have Choosing k large enough so that k ⩾ϱβ2 0/γ0, we have (5.32) −kγ0eλR0 + kδ0 + ϱβ2 0 ⩽−ϱβ2 0 < 0. (5.32) Now, since limt→−∞M −(t) = −∞, up to decrease further T1 if necessary, we may assume that −M −(t) > R0 + 1. Step 1. Construction of an entire solution Step 1. Construction of an entire solution Let w+ and w−be the functions deﬁned by (5.15) and (5.16), respectively. By Lemma 5.5, we know that w+ and w−are respectively a supersolution and a subsolution to (P) in the range (t, x) ∈(−∞, T1] × Ωfor some T1 ∈(−∞, T) where T is given by (5.13). We will construct a solution to (P) using a monotone iterative scheme starting from w−and using w+ as a barrier. Let n ⩾0 be so large that −n < T1 −1. By Proposition 3.5 and Remark 3.7, we know that there exists a unique solution un(t, x) ∈C1([−n, ∞), C(Ω)) to ( ∂tun = Lun + f(un) in (−n, ∞) × Ω, u(−n, ·) = w−(−n, ·) in Ω. In particular, we have w−(−n, x) = un(−n, x) ⩽w+(−n, x) for any x ∈Ω. In virtue of Proposition 3.5, the functions un, w−and w+ satisfy the regularity requirements of Lemma 3.1 in the time segment [−n, T1]. Therefore, by the comparison principle (Lemma 3.1), we deduce that In virtue of Proposition 3.5, the functions un, w−and w+ satisfy the regularity requirements of Lemma 3.1 in the time segment [−n, T1]. Therefore, by the comparison principle (Lemma 3.1), we deduce that w−(t, x) ⩽un(t, x) ⩽w+(t, x) for any (t, x) ∈(−n, T1) × Ω. (5.33) Note that, by assumption, −n + 1 ∈(−n, T1). In particular, u ( n + 1 x) : w−( n + 1 x) ⩽u ( n + 1 x) ⩽w+( n + 1 x) for any x w−(t, x) ⩽un(t, x) ⩽w+(t, x) for any (t, x) ∈(−n, T1) × Ω. (5.33) (5.33) Note that, by assumption, −n + 1 ∈(−n, T1). In particular, (−n + 1, x) := w−(−n + 1, x) ⩽un(−n + 1, x) ⩽w+(−n + 1, x) for any x ∈Ω Let τ > T1 be arbitrary. Using again the comparison principle Lemma 3.1, we obtain Let τ > T1 be arbitrary. Using again the comparison principle Lemma 3.1, we obtain (5.34) 0 ⩽un−1(t, x) ⩽un(t, x) ⩽1 for any (t, x) ∈(1 −n, τ) × Ω. 0 ⩽un−1(t, x) ⩽un(t, x) ⩽1 for any (t, x) ∈(1 −n, τ) × Ω. 0 ⩽un−1(t, x) ⩽un(t, x) ⩽1 for any (t, x) ∈(1 −n, τ) × Ω. 4) Since τ is arbitrary this still holds for any (t, x) ∈(1−n, ∞)×Ω. Step 2. Subsolution Hence, recalling (5.31) and (5.32), we have Now, since limt→−∞M −(t) = −∞, up to decrease further T1 if necessary, we may assume that −M −(t) > R0 + 1. Hence, recalling (5.31) and (5.32), we have P[w−](t, x) ⩽e2λct −kγ0eλR0 + kδ0 + ϱβ2 0 ⩽−β2 0ϱe2λct < 0. for all x ∈{R0 ⩽x1 < −M −(t)} and all t ⩽T1. for all x ∈{R0 ⩽x1 < −M −(t)} and all t ⩽T1. { ( )} Lastly, let us consider the case x ∈{0 ⩽x1 < R0}. Then, up to take T1 suﬃciently negative, we have ζ−< ζ+ ⩽z∗(where z∗is as in Lemma 5.3), which then gives φ′(ζ+) −φ′(ζ−) = ˆ ζ+ ζ− φ′′(z)dz ⩾λ 8 ˆ ζ+ ζ− φ′(z)dz = λ 8 φ(ζ+) −φ(ζ−) . Going back to (5.29) and recalling that ˙ξ(t) = keλM+(t), we obtain P[w−](t, x) ⩽ ϱφ(ζ−) −λk 8 eλM+(t) φ(ζ+) −φ(ζ−) ⩽ ϱβ0e−λx1+λM−(t) −λk 8 eλM+(t) φ(ζ+) −φ(ζ−) ⩽eλM+(t) ϱβ0e−λx1−2λξ(t) −λk 8 φ(ζ+) −φ(ζ−) ⩽eλM+(t) ϱβ0 −λk 8 φ(ζ+) −φ(ζ−) . Therefore, P[w−](t, x) ⩽0 in the set {0 ⩽x1 < R0} provided that k ⩾8λ−1ϱβ0 and that T1 is suﬃciently negative. This completes the proof. □ T1 is suﬃciently negative. This completes the proof. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 33 5.3. Construction of the entire solution. In this subsection, we will use the subsolution and the supersolution constructed above to prove Theorem 5.1. Proof of Theorem 5.1. For the clarity of the exposure, we split the proof into four steps. Step 1. Construction of an entire solution Step 1. Construction of an entire solution Notice that a consequence of this and the fact that f ∈C1([0, 1]) is that ¯u ∈C1,1(R, C0,α(Ω)) ∩C2(R, C0,α(Ω)), (5.39) ¯u ∈C1,1(R, C0,α(Ω)) ∩C2(R, C0,α(Ω)), (5.39) (5.39) as can be seen by a standard bootstrap argument. as can be seen by a standard bootstrap argument. Step 2. Asymptotic behaviour as t →−∞ Letting n →∞in (5.33) we obtain Step 2. Asymptotic behaviour as t →−∞ Letting n →∞in (5.33) we obtain Step 2. Asymptotic behaviour as t →−∞ Letting n →∞in (5.33) we obtain w−(t, x) ⩽¯u(t, x) ⩽w+(t, x) for any (t, x) ∈(−∞, T1] × Ω. (5.40) Consequently, if x1 < 0 and t ⩽T1, we have \|¯u(t, x)−φ(x1+ct)\|⩽\|¯u(t, x)−2φ(M +(t))\|+\|2φ(M +(t))−φ(x1+ct)\| ⩽4φ(M +(t)), (5.41) where M ±(t) has the same meaning as in (5.14). Similarly, if x1 ⩾0 and t ⩽T1, then \|¯u(t, x)−φ(x1+ct)\| ⩽\|w+(t, x) −φ(x1+ct)\| + \|w+(t, x) −¯u(t, x)\|. ( ) \|¯u(t, x)−φ(x1+ct)\|⩽\|¯u(t, x)−2φ(M +(t))\|+\|2φ(M +(t))−φ(x1+ct)\| ⩽4φ(M +(t)), (5.41) where M ±(t) has the same meaning as in (5.14). Similarly, if x1 ⩾0 and t ⩽T1, then \|¯u(t, x)−φ(x1+ct)\| ⩽\|w+(t, x) −φ(x1+ct)\| + \|w+(t, x) −¯u(t, x)\|. U i (5 40) t Using (5.40) we get \|¯u(t, x) −φ(x1 + ct)\| ⩽\|w+(t, x) −φ(x1 + ct)\| + \|w+(t, x) −w−(t, x)\| ⩽ ∥φ′∥∞ξ(t) + φ(M +(t)) + φ(M +(t)) + φ(M −(t)) + 2∥φ′∥∞ξ(t) = 3∥φ′∥∞ξ(t) + 2φ(M +(t)) + φ(M −(t)). (5.42) (5.42) By (5.41) and (5.42), we obtain By (5.41) and (5.42), we obtain \|¯u(t, x) −φ(x1 + ct)\| −→ t→−∞0 uniformly in x ∈Ω, since ξ(t) →0 and φ(M ±(t)) →0 as t →−∞. \|¯u(t, x) −φ(x1 + ct)\| −→ t→−∞0 uniformly in x ∈Ω, φ(M ±(t)) →0 as t →−∞. Step 3. Monotonicity of the entire solution Step 1. Construction of an entire solution In particular, (un)n>⌊1−T1⌋ is monotone increasing with n. Hence, un converges pointwise to some entire function ¯u(t, x) deﬁned in R × Ω. Moreover, by (5.34) and estimate (3.11) in Proposition 3.5, we have ∥un(·, x)∥C1,1([−n,∞)) ⩽1 + ω + ω2 =: C0 for any x ∈Ω, (5.35) (5.35) where ω = sup[0,1] \|f ′\| + 2 supΩJ δ. Also, given (5.34) and since [w−(−n, ·)]C0,α(Ω) is inde- pendent of n, we may apply Proposition 3.8 and deduce that [un(t, ·)]C0,α(Ω) + [∂tun(t, ·)]C0,α(Ω) ⩽C1 for any t ⩾−n, for some constant C1 > 0. Passing to the limit as n →∞we obtain that for some constant C1 > 0. Passing to the limit as n →∞we obtain that sup x∈Ω ∥¯u(·, x)∥C1,1(R) + sup t∈R [¯u(t, ·)]C0,α(Ω) + [∂t¯u(t, ·)]C0,α(Ω) ⩽C2, (5.36) sup x∈Ω ∥¯u(·, x)∥C1,1(R) + sup t∈R [¯u(t, ·)]C0,α(Ω) + [∂t¯u(t, ·)]C0,α(Ω) ⩽C2, (5.36) (5.36) where C2 := C0 + C1. Therefore, ¯u ∈C1,1(R, C0,α(Ω)). Furthermore, by (5.34), we have 0 ⩽¯u(t, x) ⩽1 for all (t, x) ∈R × Ω. (5.37) where C2 := C0 + C1. Therefore, ¯u ∈C1,1(R, C0,α(Ω)). Furthermore, by (5.34), we have 0 ⩽¯u(t, x) ⩽1 for all (t, x) ∈R × Ω. (5.37) where C2 := C0 + C1. Therefore, ¯u ∈C1,1(R, C0,α(Ω)). Furthermore, by (5.34), we have 0 ⩽¯u(t, x) ⩽1 for all (t, x) ∈R × Ω. (5.37) 0 ⩽¯u(t, x) ⩽1 for all (t, x) ∈R × Ω. (5.37) JULIEN BRASSEUR AND J´ERˆOME COVILLE 34 Let us now check that ¯u solves (P). Clearly, f(un) →f(u) as n →∞. Now, let k ⩾−T1 + 1 and n ⩾k. Then, by Dini’s theorem, for any (t, x) ∈(−k, ∞] × Ω, we have \|Lun(t, x) −L¯u(t, x)\| ⩽2∥J δ∥∞ sup z∈BRJ (x) \|¯u(t, z) −un(t, z)\| −→ n→∞0, (5.38) \|Lun(t, x) −L¯u(t, x)\| ⩽2∥J δ∥∞ sup z∈BRJ (x) \|¯u(t, z) −un(t, z)\| −→ n→∞0, (5.38) where J δ is as in (1.6). Furthermore, using (5.35) we obtain that, up to extract a subse- quence, ∂tun(·, x) →∂t¯u(·, x) in C1,α loc (R) for any α ∈(0, 1). Therefore, recalling (5.38) and since k can be taken arbitrarily large, we deduce that ¯u is indeed an entire solution to (P) in Ω× R. Step 3. Monotonicity of the entire solution Multiplying (5.44) by eλτ and integrating over τ ∈[t, T0], we come up with eλT0∂t¯u(T0, x0) ⩾eλt∂t¯u(t, x0) + ˆ T0 t eλτ L [∂t¯u] (τ, x0) + (λ −J δ(x0) + µ)∂t¯u(τ, x0) dτ, where the operator L[·] is given by (3.12). We now choose λ > 0 large enough so that λ > ∥J δ∥∞−µ. Then, on account of (5.43), we obtain where the operator L[·] is given by (3.12). We now choose λ > 0 large enough so that λ > ∥J δ∥∞−µ. Then, on account of (5.43), we obtain 0 = ∂t¯u(T0, x0) ⩾eλ(t−T0)∂t¯u(t, x0) ⩾0 for any t ⩽T0. As a result we infer that ∂t¯u(t, x0) = 0 for any t ⩽T0. In particular, L¯u(t, x0)+f(¯u(t, x0)) = 0 for any t ∈(−∞, T0]. Diﬀerentiating this with respect to t and using again that ∂t¯u(t, x0) = 0 for any t ⩽T0 together with the dominated convergence theorem, we arrive at ˆ Ω J(δ(x0, y))∂t¯u(t, y)dy = 0 for any t ∈(−∞, T0]. In turn this implies that ∂t¯u(t, y) = 0 for all (t, y) ∈(−∞, T0] × Π1(J, x0) where Π1(J, x0) is as in Deﬁnition 1.6. Applying the same arguments to the new set of stationary points Π1(J, x0), we obtain that ∂t¯u(t, y) = 0 for all (t, y) ∈(−∞, T0] × Π2(J, x0). Iterating this procedure over again implies that ∂t¯u(t, y) = 0 for all (t, y) ∈(−∞, T0] × Πj(J, x0) and all j ∈N. Since (Ω, δ) has the J-covering property, we therefore obtain that ∂t¯u(t, y) = 0 for every (t, y) ∈(−∞, T0] × Ω. In particular, this is true for every y ∈Ωwith y1 = y · e1 > 0 and, for any such ﬁxed y and any t < min{T0, T1}, it holds 0 < w−(t, y) ⩽¯u(t, y) ≡lim τ→−∞¯u(τ, y) = lim τ→−∞φ(y1 + cτ) = 0, a contradiction. Therefore, ∂t¯u(t, x) > 0 for all (t, x) ∈R × Ω. Step 3. Monotonicity of the entire solution Let us now prove that ¯u is monotone increasing in t ∈R. Note that, once this is done, we automatically get the following sharpening of (5.37): 0 < u(t, x) < 1 for any (t, x) ∈R × Ω. To show that ∂t¯u(t, x) > 0 we ﬁrst notice that      un(t, x) ⩾w−(t, x) in (−n, T1] × Ω, un(−n, ·) = w−(−n, ·) in Ω, ∂tw−(−n, ·) ⩾0 in Ω.      un(t, x) ⩾w−(t, x) in (−n, T1] × Ω, un(−n, ·) = w−(−n, ·) in Ω, ∂tw−(−n, ·) ⩾0 in Ω. In particular, we obtain that ∂tun(−n, x) ⩾0. By (5.39), we may apply Lemma 3.4 to obtain that ∂tun ⩾0 in t ∈[−n, ∞). By the uniform boundedness of ∂tun(·, x) in C0,1([−n, ∞)) (remember (5.35)), we may take the limit as n →∞to obtain In particular, we obtain that ∂tun(−n, x) ⩾0. By (5.39), we may apply Lemma 3.4 to obtain that ∂tun ⩾0 in t ∈[−n, ∞). By the uniform boundedness of ∂tun(·, x) in C0,1([−n, ∞)) (remember (5.35)), we may take the limit as n →∞to obtain ∂t¯u(t, x) ⩾0 for all (t, x) ∈R × Ω. (5.43) (5.43) PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 35 Let us now set µ := infs∈[0,1] f ′(s). By (5.39), we can diﬀerentiate with respect to t the equation satisﬁed by ¯u to get Let us now set µ := infs∈[0,1] f ′(s). By (5.39), we can diﬀerentiate with respect to t the equation satisﬁed by ¯u to get ∂2 t ¯u = L (∂t¯u) + f ′(¯u)∂t¯u ⩾L (∂t¯u) + µ ∂t¯u, (5.44) ∂2 t ¯u = L (∂t¯u) + f ′(¯u)∂t¯u ⩾L (∂t¯u) + µ ∂t¯u, ∂2 t ¯u = L (∂t¯u) + f ′(¯u)∂t¯u ⩾L (∂t¯u) + µ ∂t¯u, (5.44) which makes sense everywhere. We conclude by contradiction. Suppose that there exists (T0, x0) ∈R × Ωsuch that ∂t¯u(T0, x0) = 0. Choose any t ⩽T0 and let λ > 0 be some large number to be ﬁxed later on. Multiplying (5.44) by eλτ and integrating over τ ∈[t, T0], we come up with which makes sense everywhere. We conclude by contradiction. Suppose that there exists (T0, x0) ∈R × Ωsuch that ∂t¯u(T0, x0) = 0. Choose any t ⩽T0 and let λ > 0 be some large number to be ﬁxed later on. Step 4. Uniqueness of the entire solution Now, since ∂tu(t, x) > 0 for all (t, x) ∈R × Ω, we have 1 ⩾lim sup x1→∞u(t, x) ⩾lim inf x1→∞u(t, x) ⩾lim x1→∞u(T1, x) = 1, ) ∈(T1, ∞) × RN−1. Therefore, limx1→∞u(t, x) = 1 for all (t, x′) ∈R × RN−1 for all (t, x′) ∈(T1, ∞) × RN−1. Therefore, limx1→∞u(t, x) = 1 for all (t, x′) ∈R × RN−1. ( ) ( ) To complete the proof, it remains to show that limx1→−∞u(t, x) = 0 for all (t, x′) ∈ R × RN−1. The proof of this is slightly more involved and we need to compare u with the solution of an auxiliary problem. To this end, we let g ∈C1([0, 2]) be a nonlinearity of “ignition” type, namely such that the following properties hold: g [0,θ/4] ≡0, g (θ/4,2) > 0, g(2) = 0 and g′(2) < 0. Let us assume, in addition, that g(s) ⩾max[0,1] f for all s ∈[θ/2, 1 + θ/2]. Now, using the existence result [30, Theorems 1.2-1.3] (see in particular [30, Lemma 5.1] and the remarks in [30, Section 1.2] on page 5), we know that there exists a unique monotone increasing front ϕ ∈C(R) with speed c′ > 0, satisfying ϕ(0) = 1 and such that ( c′ ϕ′ = J1 ∗ϕ −ϕ + g(ϕ) in R, lim z→+∞ϕ(z) = 2, lim z→−∞ϕ(z) = 0, (5.45) (5.45) where J1 is as in (1.8). Now, let us deﬁne gϱ(s) := g(s−ϱ), for all ϱ > 0 and all s ∈[ϱ, 2+ϱ]. By deﬁnition of gϱ, we can check that the function ϕϱ(x) := ϱ + ϕ(x) solves where J1 is as in (1.8). Now, let us deﬁne gϱ(s) := g(s−ϱ), for all ϱ > 0 and all s ∈[ϱ, 2+ϱ]. By deﬁnition of gϱ, we can check that the function ϕϱ(x) := ϱ + ϕ(x) solves ( c′ ϕ′ ϱ = J1 ∗ϕϱ −ϕϱ + gϱ(ϕϱ) in R, lim z→+∞ϕϱ(z) = 2 + ϱ, lim z→−∞ϕϱ(z) = ϱ. (5.46) (5.46) Next, for all ϱ ∈(0, θ/4] and all A > 0, we let wϱ,A(t, x) := ϕϱ(x1 + A + c′t). We claim that Claim 5.8. For all ϱ ∈(0, θ/4], there exist Aϱ > 0 and tϱ ∈R such that u(t, x) ⩽wϱ,Aϱ(t, x) for all (t, x) ∈[tϱ, ∞) × Ω. u(t, x) ⩽wϱ,Aϱ(t, x) for all (t, x) ∈[tϱ, ∞) × Ω. Step 4. Uniqueness of the entire solution The proof is almost identical to that given in [12, Section 3]. The only diﬀerence with the local case is that the solution does no longer satisfy parabolic estimates. However, this is compensated by Lemma 4.1 and (5.36). □ 5.4. Further properties of the entire solution. In this section, we prove that the unique entire solution to (P) satisfying (5.1) and (5.2) shares the same limit as x1 →±∞than the planar wave φ(x1 + ct). Precisely, Proposition 5.7. Assume (1.4), (1.5), (1.6), (1.7) and (2.1). Suppose that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1). Let u(t, x) be the unique entire solution to (P) satisfying (5.1) and (5.2). Then, denoting a point x ∈Ωby x = (x1, x′) ∈R × RN−1, we have Proposition 5.7. Assume (1.4), (1.5), (1.6), (1.7) and (2.1). Suppose that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1). Let u(t, x) be the unique entire solution to (P) satisfying (5.1) and (5.2). Then, denoting a point x ∈Ωby x = (x1, x′) ∈R × RN−1, we have lim x1→−∞u(t, x) = 0 and lim x1→∞u(t, x) = 1 for all (t, x′) ∈R × RN−1. lim x1→−∞u(t, x) = 0 and lim x1→∞u(t, x) = 1 for all (t, x′) ∈R × RN−1. JULIEN BRASSEUR AND J´ERˆOME COVILLE 36 Proof. Let us ﬁrst prove that limx1→∞u(t, x) = 1 for all (t, x′) ∈R × RN−1. To see this, it suﬃces to observe that u(t, x) ⩾w−(t, x) for all (t, x) ∈(−∞, T1] × Ω. Hence, using (5.3) and the deﬁnition of w−(remember (5.16)), we deduce that 1 ⩾lim sup x1→∞u(t, x) ⩾lim inf x1→∞u(t, x) ⩾lim x1→∞ φ(x1 + M −(t)) −φ(−x1 + M −(t)) = 1, for all (t, x′) ∈(−∞, T1] × RN−1, where M −(t) has the same meaning as in (5.14). Now, since ∂tu(t, x) > 0 for all (t, x) ∈R × Ω, we have for all (t, x′) ∈(−∞, T1] × RN−1, where M −(t) has the same meaning as in (5.14). Now, since ∂tu(t, x) > 0 for all (t, x) ∈R × Ω, we have for all (t, x′) ∈(−∞, T1] × RN−1, where M −(t) has the same meaning as in (5.14). Step 4. Uniqueness of the entire solution Note that, by proving Claim 5.8, we end the proof of Proposition 5.7. To see this, ﬁx some ε > 0 and let ϱ = ε/2. Also, for R > 0, let H+ R and H− R be the half-spaces given by H+ R := x ∈RN; x1 > −R and H− R := x ∈RN; x1 ⩽−R , (5.47) (5.47) respectively. Assume, for the moment, that t ∈[tϱ, ∞). By (5.45), we know that there exists some Rϱ > 0 such that ϕ(z + Aϱ) ⩽ϱ for all z ⩽−Rϱ. In particular, we have respectively. Assume, for the moment, that t ∈[tϱ, ∞). By (5.45), we know that there exists some Rϱ > 0 such that ϕ(z + Aϱ) ⩽ϱ for all z ⩽−Rϱ. In particular, we have respectively. Assume, for the moment, that t ∈[tϱ, ∞). By (5.45), we know that there exists some Rϱ > 0 such that ϕ(z + Aϱ) ⩽ϱ for all z ⩽−Rϱ. In particular, we have wϱ,Aϱ(t, x) = ϱ + ϕ(x1 + Aϱ + c′t) ⩽2ϱ = ε for all (t, x) ∈[tϱ, ∞) × H− R wϱ,Aϱ(t, x) = ϱ + ϕ(x1 + Aϱ + c′t) ⩽2ϱ = ε for all (t, x) ∈[tϱ, ∞) × H− Rϱ+c′t. Applying now Claim 5.8, we then deduce that u(t, x) ⩽ε for all (t, x) ∈[tϱ, ∞)×Ω∩H− Rϱ+c′t, which in turn automatically implies that Applying now Claim 5.8, we then deduce that u(t, x) ⩽ε for all (t, x) ∈[tϱ, ∞)×Ω∩H− Rϱ+c′t, which, in turn, automatically implies that lim sup x1→−∞u(t, x) ⩽ε for all (t, x′) ∈[tϱ, ∞) × RN−1. (5.48) lim sup x1→−∞u(t, x) ⩽ε for all (t, x′) ∈[tϱ, ∞) × RN−1. (5.48) (5.48) PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 37 The analogue of this for t ∈(−∞, tϱ) is a simple consequence of the monotonicity of u(t, x). Indeed, using that u(·, x) is increasing for all x ∈Ω, we obtain u(t, x) ⩽u(tϱ, x) ⩽ wϱ,Aϱ(tϱ, x) ⩽ε, for all (t, x) ∈(−∞, tϱ) × Ω∩H− Rϱ+c′t, which, again, implies that lim sup x1→∞u(t, x) ⩽ε for all (t, x′) ∈(−∞, tϱ) × RN−1. (5.50) (5.50) Now, let R1 ⩾RJ, where RJ > 0 is any number such that supp(J) ⊂[0, RJ]. Since u(t, x) satisﬁes (2.2), there is then some tϱ ∈R such that Now, let R1 ⩾RJ, where RJ > 0 is any number such that supp(J) ⊂[0, RJ]. Since u(t, x) satisﬁes (2.2), there is then some tϱ ∈R such that u(t, x) ⩽φ(x1 + ct) + ϱ 2 for all (t, x) ∈(−∞, tϱ] × Ω. Since φ is increasing, we may assume that φ(−R1 −RK + ctϱ) ⩽ϱ/2 (up to take R1 larger if necessary). Consequently, for all A > 0, we have Since φ is increasing, we may assume that φ(−R1 −RK + ctϱ) ⩽ϱ/2 (up to take R1 larger if necessary). Consequently, for all A > 0, we have (5.51) u(tϱ, x) ⩽ϱ ⩽wϱ,A(tϱ, x) for all x ∈H− R1+RK. (5.51) On the other hand, since ϕ(0) = 1 and ϕ′ > 0, by taking Aϱ = R1 + RK −c′tϱ, we get wϱ,Aϱ(tϱ, x) = ϱ + ϕ(x1 + R1 + RK) > ϱ + ϕ(0) = ϱ + 1 for all x ∈H+ R1+RK. Since u < 1 in R × Ωand since wϱ,Aϱ(·, x) is increasing for all x ∈Ω, we deduce that Since u < 1 in R × Ωand since wϱ,Aϱ(·, x) is increasing for all x ∈Ω, we deduce that wϱ,Aϱ(t, x) > ϱ + 1 > u(t, x) for all (t, x) ∈[tϱ, ∞) × Ω∩H+ R1+RK. (5.52) On the other hand, since K ⊂H+ RK and since supp(J) ⊂[0, RJ], it follows tha wϱ,Aϱ(t, x) > ϱ + 1 > u(t, x) for all (t, x) ∈[tϱ, ∞) × Ω∩H+ R1+RK. (5.52) On the other hand, since K ⊂H+ RK and since supp(J) ⊂[0, RJ], it follows that ∂tu = Jrad ∗u −u + ef(u) in R × H− R +R . (5.53) wϱ,Aϱ(t, x) > ϱ + 1 > u(t, x) for all (t, x) ∈[tϱ, ∞) × Ω∩H+ R1+RK. (5.52) On the other hand, since K ⊂H+ RK and since supp(J) ⊂[0, RJ], it follows that ∂tu = Jrad ∗u −u + ef(u) in R × H− R1+RK. Step 4. Uniqueness of the entire solution (5.49) (5.49) Hence, collecting (5.48), (5.49), recalling that u(t, x) > 0 for all (t, x) ∈R × Ωand that ε > 0 is arbitrary, we conclude that Hence, collecting (5.48), (5.49), recalling that u(t, x) > 0 for all (t, x) ∈R × Ωand that ε > 0 is arbitrary, we conclude that lim x1→−∞u(t, x) = 0 for all (t, x′) ∈R × RN−1, □ which thereby proves Proposition 5.7. which thereby proves Proposition 5.7. Proof of Claim 5.8. First of all, we notice that, since K ⊂RN is compact, we may always ﬁnd some RK > 0 so that K ⊂H+ RK (we use the same notation as in (5.47)). Furthermore, we observe that, by construction of gϱ, there holds gϱ ⩾ef + ⩾ef for all s ∈[ϱ, 2 + ϱ] and all 0 < ϱ ⩽θ/4, where ef ∈C1(R) is the extension of f given by (3.21). In particular, this implies that the function wϱ,A satisﬁes ∂twϱ,A ⩾Jrad ∗wϱ,A −wϱ,A + ef(wϱ,A) in R × RN. (5.50) (5.50) (5.53) Hence, collecting (5.50), (5.51), (5.52) and (5.53), we ﬁnd that Hence, collecting (5.50), (5.51), (5.52) and (5.53), we ﬁnd that            ∂twϱ,Aϱ ⩾Jrad ∗wϱ,Aϱ −wϱ,Aϱ + ef(wϱ,Aϱ) in (tϱ, ∞) × H− R1+RK, ∂tu = Jrad ∗u −u + ef(u) in (tϱ, ∞) × H− R1+RK, wϱ,Aϱ > u in [tϱ, ∞) × Ω∩H+ R1+RK, wϱ,Aϱ(tϱ, ·) ⩾u(tϱ, ·) in H− R1+RK. ∂twϱ,Aϱ ⩾Jrad ∗wϱ,Aϱ −wϱ,Aϱ + ef(wϱ,Aϱ) in (tϱ, ∞) × H− R1+RK, ∂tu = Jrad ∗u −u + ef(u) in (tϱ, ∞) × H− R1+RK, > i [t ) Ω∩H+       ∂twϱ,Aϱ ⩾Jrad ∗wϱ,Aϱ −wϱ,Aϱ + ef(wϱ,Aϱ) in (tϱ, ∞) ∂tu = Jrad ∗u −u + ef(u) in (tϱ, ∞) ϱ ⩾ rad ϱ,Aϱ ϱ,Aϱ f( ∂tu = Jrad ∗u −u + ef(u) in (tϱ, ∞) × H− R1+RK, in [tϱ, ∞) × Ω∩H+ R1+RK, in H− R1+RK. By a straightforward adaptation of the parabolic comparison principle Lemma 3.1, we deduce that u(t, x) ⩽wϱ,Aϱ(t, x) for all (t, x) ∈[tϱ, ∞) × H− R1+RK and, hence, this holds for all By a straightforward adaptation of the parabolic comparison principle Lemma 3.1, we deduce that u(t, x) ⩽wϱ,Aϱ(t, x) for all (t, x) ∈[tϱ, ∞) × H− R1+RK and, hence, this holds for all (t, x) ∈[tϱ, ∞) × Ω, which thereby establishes Claim 5.8. □ By a straightforward adaptation of the parabolic comparison principle Lemma 3.1, we deduce that u(t, x) ⩽wϱ,Aϱ(t, x) for all (t, x) ∈[tϱ, ∞) × H− R1+RK and, hence, this holds for all (t, x) ∈[tϱ, ∞) × Ω, which thereby establishes Claim 5.8. □ JULIEN BRASSEUR AND J´ERˆOME COVILLE 38 6. Local behaviour after the encounter with K In this section, we study how the entire solution u(t, x) to (P) with (5.1) and (5.2) behaves after hitting the obstacle K. We will ﬁrst show that it converges to u∞(x)φ(x1 + ct), locally uniformly in x ∈Ωas t →∞, where u∞∈C(Ω) solves      Lu∞+ f(u∞) = 0 in Ω, 0 ⩽u∞⩽1 in Ω, u∞(x) →1 as \|x\| →∞. What is more, we will prove that u(t, x) converges to the planar wave φ(x1 +ct) as \|x′\| →∞ when (t, x1) stays in some compact set or, otherwise said, that the encounter with the obstacle does not much deform u(t, x) in hyperplanes which are orthogonal to the x1-direction. What is more, we will prove that u(t, x) converges to the planar wave φ(x1 +ct) as \|x′\| →∞ when (t, x1) stays in some compact set or, otherwise said, that the encounter with the obstacle does not much deform u(t, x) in hyperplanes which are orthogonal to the x1-direction. The results in this section are somehow independent of the geometry of K. The inﬂuence of the latter is in fact “encoded” in the function u∞as will be shown in the next section. 6.1. Local uniform convergence to the stationary solution. In this sub-section, we prove the local uniform convergence of u(t, x) towards u∞(x)φ(x1 + ct) as t →∞. Proposition 6.1. Assume (1.4), (1.5), (1.6), (1.9) and (2.1). Suppose that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1). Let u(t, x) be the unique entire solution to (P) satisfying (5.1) and (5.2). Then, there exists a solution u∞∈C(Ω) to (P∞) such that \|u(t, x) −u∞(x)\| −→ t→+∞0 locally uniformly in x ∈Ω. \|u(t, x) −u∞(x)\| −→ t→+∞0 locally uniformly in x ∈Ω. \|u(t, x) −u∞(x)\| −→ t→+∞0 locally uniformly in x ∈Ω. Remark 6.2. Since the convergence is local uniform, we also have \|u(t, x) −φ(x1 + ct)u∞(x)\| −→ t→+∞0 locally uniformly in x ∈Ω. (6.1) (6.1) \|u(t, x) −φ(x1 + ct)u∞(x)\| −→ t→+∞0 locally uniformly in x ∈Ω. (6.1) The proof of Proposition 6.1 relies on the following lemma: Lemma 6.3. Assume (1.4), (1.5), (1.6) and (1.9). Let u ∈C(Ω, [0, 1]) be a solution to the stationary equation Lu + f(u) = 0 in Ωsatisfying supΩu = 1. Then, Lemma 6.3. Assume (1.4), (1.5), (1.6) and (1.9). 6. Local behaviour after the encounter with K Of course, Jψ has no longer unit mass, but we still have that 0 < ´ RN Jψ ⩽1 which is enough to make the proof given in [19] work, including that of the sweeping-type principle (notice that all the other properties of Jrad are preserved). Arguing in this way, we may then remove the assumption that Jrad is square integrable. In the case when δ ∈Q(Ω) is not the Euclidean distance, this strategy still works: indeed, as it was already explained in [18, Remark 2.5], the proof requires only to work on convex regions far away from the obstacle K in which it trivially holds that δ(x, y) = \|x −y\|. □ We are now ready to prove Proposition 6.1. Proof of Proposition 6.1. By (5.1), one has that u(t, x) →u∞(x) ∈(0, 1] as t →∞for all x ∈Ω. Furthermore, using Lemma 4.1 (or (5.36)) one has that the convergence is (at least) locally uniform and that u∞is a continuous solution of Lu∞+f(u∞) = 0 in Ω(the continuity of u∞follows straightforwardly from (2.1) and the arguments in [19, Lemma 3.2]). Proof of Proposition 6.1. By (5.1), one has that u(t, x) →u∞(x) ∈(0, 1] as t →∞for all x ∈Ω. Furthermore, using Lemma 4.1 (or (5.36)) one has that the convergence is (at least) locally uniform and that u∞is a continuous solution of Lu∞+f(u∞) = 0 in Ω(the continuity of u∞follows straightforwardly from (2.1) and the arguments in [19, Lemma 3.2]). Proof of Proposition 6.1. By (5.1), one has that u(t, x) →u∞(x) ∈(0, 1] as t →∞for all x ∈Ω. Furthermore, using Lemma 4.1 (or (5.36)) one has that the convergence is (at least) locally uniform and that u∞is a continuous solution of Lu∞+f(u∞) = 0 in Ω(the continuity of u∞follows straightforwardly from (2.1) and the arguments in [19, Lemma 3.2]). g y ( ) g [ , ]) Let us now show that u∞(x) →1 as \|x\| →∞. By Proposition 5.7 we know that u(t, x) →1 as x1 →∞, for any ﬁxed (t, x) ∈R×RN−1. But since 0 < u(t, x) < 1 and since ∂tu(t, x) > 0 for all (t, x) ∈R × Ω, we have u(t, x) ⩽u∞(x) ⩽1 for all (t, x) ∈R × Ω. Hence letting x1 →∞, we deduce that u∞(x) →1 as x1 →∞. In particular, it holds that supΩu∞= 1. The conclusion now follows from Lemma 6.3. 6. Local behaviour after the encounter with K Let u ∈C(Ω, [0, 1]) be a solution to the stationary equation Lu + f(u) = 0 in Ωsatisfying supΩu = 1. Then, lim \|x\|→∞u(x) = 1. Proof. Let us ﬁrst consider the case when δ ∈Q(Ω) is the Euclidean distance. Then, Lemma 6.3 is exactly [19, Lemma 7.2] without the extra assumption that Jrad ∈L2(RN) (that was required in [19]). However, it turns out that the same arguments given there also yield Lemma 6.3 with only minor changes. As a matter of fact, the only place where the assumption that Jrad ∈L2(RN) comes into play is when showing the existence of a maximal solution w to ˆ BR(x0) Jrad(x −y)w(x)dy −w(x) + f(w(x)) = 0 for all x ∈BR(x0), (6.2) (6.2) and for any x0 ∈RN (provided that R ⩾d0 for some d0 = d0(f, J) > 0 large enough). This technical assumption is here only to ensure that the equation satisﬁes some compactness property which, in turn, is needed to establish the existence of nontrivial solutions. and for any x0 ∈RN (provided that R ⩾d0 for some d0 = d0(f, J) > 0 large enough). This technical assumption is here only to ensure that the equation satisﬁes some compactness property which, in turn, is needed to establish the existence of nontrivial solutions. The strategy of proof used in [19], consists in using this function w to construct a family of sub-solutions to (6.2) and to notice that any solution u to Lu + f(u) = 0 in Ωis a super-solution to (6.2) on balls BR(x0) that are suﬃciently far away from K. Then, using PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 39 the sweeping-type principle [19, Lemma 4.3], it can be shown that the so-constructed sub- solutions yield lower bounds on u which can be propagated in a way that yields that u(x) →1 as \|x\| →∞. This strategy still works if we replace Jrad by the truncation, Jψ, deﬁned by Jψ(z) = Jrad(z)ψ(z), where ψ ∈C∞ c (RN, [0, 1]) is a radial cut-oﬀfunction such that \|supp(Jψ)\| > 0 and Jψ ∈L2(RN). Indeed, since Jψ ∈L2(RN) there will then exist a solution wψ to (6.2) with Jψ instead of Jrad. Moreover, u is also a super-solution to (6.2) with Jψ instead of Jrad on balls BR(x0) that are suﬃciently far away from K (since Jψ ⩽Jrad). We may then simply work with the kernel Jψ instead of Jrad. 7. On the impact of the geometry So far, the geometry of K has not played any role in our analysis. The main purpose of this section is to understand how the geometry of K impacts the asymptotic behaviour of u(t, x) as t →∞. In a nutshell, we will show that the main information on the large time behaviour is contained in the properties of the solution, u∞, to the stationary problem (P∞). So far, the geometry of K has not played any role in our analysis. The main purpose of this section is to understand how the geometry of K impacts the asymptotic behaviour of u(t, x) as t →∞. In a nutshell, we will show that the main information on the large time behaviour is contained in the properties of the solution, u∞, to the stationary problem (P∞). We will ﬁrst discuss the validity of the Liouville-type property for (P∞) depending on the geometry of K (namely whether its only possible solution is u∞≡1). In particular, we extend some previous results of Hamel, Valdinoci and the authors to the case of a general δ ∈Q(Ω) and we prove that, when K is a convex set, then the Liouville-type property is satisﬁed (at least if J is non-increasing). Second, the prove that whether u(t, x) recovers the shape of the planar front φ(x1 + ct) as t →∞is equivalent to whether (P∞) satisﬁes the Liouville-type property. p p , , y p ( ) We will ﬁrst discuss the validity of the Liouville-type property for (P∞) depending on the geometry of K (namely whether its only possible solution is u∞≡1). In particular, we extend some previous results of Hamel, Valdinoci and the authors to the case of a general δ ∈Q(Ω) and we prove that, when K is a convex set, then the Liouville-type property is satisﬁed (at least if J is non-increasing). Second, the prove that whether u(t, x) recovers the shape of the planar front φ(x1 + ct) as t →∞is equivalent to whether (P∞) satisﬁes the Liouville-type property. 7.1. A Liouville type result. We establish a Liouville type result which extends the results obtained by Hamel, Valdinoci and the authors in [19] to arbitrary quasi-Euclidean distances. Proposition 7.1 (Liouville type result). Let K ⊂RN be a compact convex set and let δ ∈Q(Ω). Assume (1.5), (1.6), (1.7) and (2.1). If δ(x, y) ̸≡\|x −y\| suppose, in addition, that J is non-increasing. 6. Local behaviour after the encounter with K □ 6.2. Convergence near the horizon. Here, we shall prove that the encounter with K does not alter too much the entire solution u(t, x) to (P) with (5.1) and (5.2) in hyperplanes orthogonal to the x1-direction, in the sense that it remains close to the planar wave φ(x1+ct) locally uniformly in (t, x1) when \|x′\| →∞. Proposition 6.4. Assume (1.4), (1.5), (1.6), (1.7) and (2.1). Suppose that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1). Let u(t, x) be the unique entire solution to (P) satisfying (5.1) and (5.2). Then, for any sequence (x′ n)n⩾0 ⊂RN−1 such that \|x′ n\| →∞as n →∞, there holds \|u(t, x1, x′ + x′ n) −φ(x1 + ct)\| −→ n→∞0 locally uniformly in (t, x) = (t, x1, x′) ∈R × RN. Proof. The proof works essentially as in the local case, see [12, Proposition 4.1]. Let us, however, outline the main ingredients of the proof. For each n ⩾0, we set Ωn := Ω− (0, x′ n) and, for (t, x) ∈R × Ωn, we let un(t, x) := u(t, x1, x′ + x′ n). By Lemma 4.1 and the boundedness assumption on u, up to extraction of a subsequence, we have that un converges locally uniformly in (t, x) ∈R × RN to a solution V of Proof. The proof works essentially as in the local case, see [12, Proposition 4.1]. Let us, however, outline the main ingredients of the proof. For each n ⩾0, we set Ωn := Ω− (0, x′ n) and, for (t, x) ∈R × Ωn, we let un(t, x) := u(t, x1, x′ + x′ n). By Lemma 4.1 and the boundedness assumption on u, up to extraction of a subsequence, we have that un converges locally uniformly in (t, x) ∈R × RN to a solution V of ∂tV = Jrad ∗V −V + f(V ) in R × RN, 0 ⩽V ⩽1 in R × RN. 0 ⩽V ⩽1 in R × RN. JULIEN BRASSEUR AND J´ERˆOME COVILLE 40 By (5.2), V inherits from the limit behaviour of u as −∞, namely: By (5.2), V inherits from the limit behaviour of u as −∞, namely: lim t→−∞sup x∈RN \|V (t, x) −φ(x1 + ct)\| = 0. 6. Local behaviour after the encounter with K From here, we may reproduce the arguments in [12] using the trick of Fife and McLeod [39], to prove that V (t, x) ≡φ(x1+ct) which then completes the proof. Notice that the arguments in [12] adapt with no diﬃculty since the local structure of the operator ∆u does not come into play and can easily be replaced by Jrad ∗u −u. □ 7. On the impact of the geometry Let u∞: Ω→[0, 1] be a measurable function satisfying Lu∞+ f(u∞) = 0 a.e. in Ω, u∞(x) →1 as \|x\| →∞. (7.1) (7.1) Then, u∞≡1 a.e. in Ω. Then, u∞≡1 a.e. in Ω. Then, u∞≡1 a.e. in Ω. Proof. If δ is the Euclidean distance, then Proposition 7.1 is covered by [19, Theorem 2.2] together with [19, Lemma 3.2]. So it remains only to address the case when δ is not the Euclidean distance. It turns out that this case follows from the same arguments as in the case of the Euclidean distance, with only minor changes that we now explain in detail. First of all, as we already pointed out in [18, Remark 2.5], we note that the proof of [19, Lemma 3.2] can be adapted to prove that (2.1) still implies that u∞has a uniformly continuous representative in its class of equivalence. Hence, we may assume, without loss of generality, that u∞∈C(Ω). The strategy of proof used in [19] to show that u∞is necessarily identically 1 in the whole of Ω, consists in comparing a solution u∞to (7.1) to some planar function of the type φ(x · e −r0) with e ∈SN−1, r0 ∈R and where φ is as in (1.7). This is done using a sliding type method by letting r vary from +∞to −∞. To implement this method, two ingredients are needed: ﬁrst, we need to establish appro- priate comparison principles and, second, we need to be able to compare a given solution PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 41 Figure 6. The cone C (x) with boundary tangent to K. Figure 6. The cone C (x) with boundary tangent to K. Figure 6. The cone C (x) with boundary tangent to K. u∞to the planar function φ(x · e −r0) in half-spaces of the form He := x0 + {x ∈RN; x · e > 0} with He ⊂Ω. u∞to the planar function φ(x · e −r0) in half-spaces of the form H x + {x ∈RN x e > 0} ith H ⊂Ω u∞to the planar function φ(x · e −r0) in half-spaces of the form He := x0 + {x ∈RN; x · e > 0} with He ⊂Ω. It turns out that these two ingredients adapt to our generalized setting with no diﬃculty. 7. On the impact of the geometry Indeed, the proof of the comparison principles [19, Lemmata 4.1, 4.2] require only that (Ω, δ) has the J-covering property, that L maps continuous functions to continuous functions and that J δ ∈C(Ω). But all these requirements are guaranteed by assumption (1.6). ( ) ( ) On the other hand, to be able to compare u∞with φr0,e(x) := φ(x·e−r0) in He, it suﬃces to make sure that φr0,e is a sub-solution to Lw + f(w) = 0 in He. For it, we notice that Lφr0,e(x) = ˆ Ω J(\|x −y\|)(φr0,e(y) −φr0,e(x))dy + ˆ C (x)\K (J(δ(x, y)) −J(\|x −y\|))(φr0,e(y) −φr0,e(x))dy, where C (x) is the cone with vertex x tangent to ∂K (see Figure 6). Since φr0,e(y) ⩽φr0,e(x) for any x ∈He and any y ∈C (x) \ K (because φ′ > 0) and since J is non-increasing, it then holds that Lφr0,e(x) ⩾ ˆ Ω J(\|x −y\|)(φr0,e(y) −φr0,e(x))dy for all x ∈He. In other words, the problem reduces to the case δ(x, y) ≡\|x−y\|. At this stage, the arguments of [19] can be adapted without modiﬁcation. □ In other words, the problem reduces to the case δ(x, y) ≡\|x−y\|. At this stage, the arguments of [19] can be adapted without modiﬁcation. □ 7.2. The stationary solution encodes the geometry. In this section, we prove that the large time behaviour of u(t, x) is determined by the Liouville-type property of (P∞). ( ) ( ) In fact, we will prove a bit more than what we stated above: we will prove that the stationary solution u∞which arises in the large time limit is the minimal solution to (P∞). More precisely, we prove the following result: Proposition 7.2. Assume that (1.4), (1.5), (1.6), (1.9) and (2.1) hold. Suppose that J ∈ Bα 1,∞(Ω; δ) for some α ∈(0, 1). Let u(t, x) be the unique bounded entire solution to (P) satisfying (2.2). Let u∞∈C(Ω) be the solution to (P∞) such that (6.1) holds, i.e. such that \|u(t, x) −u∞(x)φ(x1 + ct)\| −→ t→+∞0 locally uniformly in x ∈Ω, \|u(t, x) −u∞(x)φ(x1 + ct)\| −→ t→+∞0 locally uniformly in x ∈Ω, and let eu∞∈C(Ω) be any solution to (P∞). Then, u∞⩽eu∞in Ω. and let eu∞∈C(Ω) be any solution to (P∞). Then, u∞⩽eu∞in Ω. JULIEN BRASSEUR AND J´ERˆOME COVILLE 42 Let us explain why proving Proposition 7.2 is indeed suﬃcient to establish Theorem 2.6. Proof of Theorem 2.6. Step 1. Preliminary observations Let eu∞∈C(Ω) be any solution to (P∞) and let s0, s1 > 0 be such that f ′ ⩽−s1 in [1 −s0, 1] (note that s0, s1 are well-deﬁned since f ′(1) < 0). Observe that, since eu∞is independent of t, it also satisﬁes ∂teu∞−Leu∞−f(eu∞) = 0 in R × Ω. (7.2) ∂teu∞−Leu∞−f(eu∞) = 0 in R × Ω. (7.2) Furthermore, since infΩeu∞> 0 (by the strong maximum principle [19, Lemma 4.2]) we may apply [19, Lemma 5.1] which yields the existence of a number r0 > 0 such that φ(\|x\| −r0) ⩽eu∞(x) for any x ∈Ω, (7.3) φ(\|x\| −r0) ⩽eu∞(x) for any x ∈Ω, (7.3) where φ is as in (1.7). (Note that the use of [19, Lemmata 4.2, 5.1] in the case of a general δ ∈Q(Ω) is licit, as can easily be seen by reasoning as in the proof of Proposition 7.1.) Lastly, we recall that, by construction of u(t, x), we have that ( ) Lastly, we recall that, by construction of u(t, x), we have that u(t, x) ⩽w+(t, x) for any (t, x) ∈(−∞, T1] × Ω, (7.4) (7.4) where w+ is given by (5.15) (remember (5.40)). 7. On the impact of the geometry If (P∞) satisﬁes the Liouville property, then, since the trivial solution is the only possible one, we clearly have that u∞≡1. On the other hand, if u∞≡1, then either (P∞) satisﬁes the Liouville property or it does not. Suppose, by contradiction, that (P∞) does not satisfy the Liouville property, namely that there exists a solution eu∞to (P∞) with 0 < eu∞< 1 a.e. in Ω. Because of assumption (2.1), by [19, Lemma 3.2], we know that every solution to (P∞) admits a representative in its class of equivalence that is uniformly continuous. Hence, we may always assume that eu∞∈C(Ω). Applying now Proposition 7.2, we ﬁnd that 1 ≡u∞⩽eu∞< 1 in Ω, a contradiction. □ ain why proving Proposition 7.2 is indeed suﬃcient to establish Theorem 2.6. Let us now prove Proposition 7.2. Proof of Proposition 7.2. For the convenience of the reader, the proof is split into three parts. After a preparatory step, where we collect some preliminary observations, we show that any solution to (P∞) bounds u(τ, x) from above, for some time τ ∈R in a neighborhood of −∞. Lastly, we show that this estimate holds for all t ∈(τ, ∞) using the comparison principle and we conclude using the convergence result obtained in Proposition 6.1. Step 2. A ﬁrst upper bound u(τ, ·) ⩽eu∞ in Ω\ H. Since, in addition, it holds that lim sup\|x\|→∞(u(τ, x) −eu∞(x)) ⩽0 and that eu∞⩾1 −s0 in H (remember the deﬁnition of RK), we may then apply the weak maximum principle [19, Lemma 4.1] (which we can do as pointed out in the proof of Proposition 7.1) to obtain that Since, in addition, it holds that lim sup\|x\|→∞(u(τ, x) −eu∞(x)) ⩽0 and that eu∞⩾1 −s0 in H (remember the deﬁnition of RK), we may then apply the weak maximum principle [19, Lemma 4.1] (which we can do as pointed out in the proof of Proposition 7.1) to obtain that u(τ, x) ⩽eu∞(x) for any x ∈Ω. (7.8) (7.8) It remains to show that this estimate holds for all t ∈(τ, ∞). It remains to show that this estimate holds for all t ∈(τ, ∞). Step 2. A ﬁrst upper bound Let RK > 0 be such that K ⊂BRK and eu∞⩾1 −s0 in RN \ BRK. Also, let τ ∈(−∞, T1] be suﬃciently negative so that cτ + ξ(τ) + r0 ⩽0 and Let RK > 0 be such that K ⊂BRK and eu∞⩾1 −s0 in RN \ BRK. Also, let τ ∈(−∞, T1] be suﬃciently negative so that cτ + ξ(τ) + r0 ⩽0 and max n 2φ(cτ +ξ(τ)) −φ(−r0), β0 + (cτ +ξ(τ)+r0) min{γ0, γ1e−µRK ax n 2φ(cτ +ξ(τ)) −φ(−r0), β0 + (cτ +ξ(τ)+r0) min{γ0, γ1e−µRK} o ⩽0, max n 2φ(cτ +ξ(τ)) −φ(−r0), β0 + (cτ +ξ(τ)+r0) min{γ0, γ1e−µRK} o ⩽0, where µ, β0, γ0, γ1 and ξ(t) are given by (5.6), (5.7), (5.8) and (5.12), respectively. (Note that τ is well-deﬁned since ξ(t) →0 as t →−∞, since φ(z) →0 as z →−∞, since φ′(z) > 0 for all z ∈R and since γ0, γ1 > 0.) where µ, β0, γ0, γ1 and ξ(t) are given by (5.6), (5.7), (5.8) and (5.12), respectively. (Note that τ is well-deﬁned since ξ(t) →0 as t →−∞, since φ(z) →0 as z →−∞, since φ′(z) > 0 for all z ∈R and since γ0, γ1 > 0.) γ0, γ ) Now, we notice that, if x1 < 0, then, by (7.3) and (7.4), we have γ γ ) Now, we notice that, if x1 < 0, then, by (7.3) and (7.4), we have u(τ, x) −eu∞(x) ⩽w+(τ, x) −φ(\|x\| −r0) ⩽2φ(cτ + ξ(τ)) −φ(−r0) ⩽0, u(τ, x) −eu∞(x) ⩽w+(τ, x) −φ(\|x\| −r0) ⩽2φ(cτ + ξ(τ)) −φ(−r0) ⩽0 where we have used that φ is increasing. In other words, we have that ( ) ⩽e ( ) f Ω ith 0 (7 5) where we have used that φ is increasing. In other words, we have that u(τ, x) ⩽eu∞(x) for any x ∈Ωwith x1 < 0. (7.5) where we have used that φ is increasing. In other words, we have that (7.5) u(τ, x) ⩽eu∞(x) for any x ∈Ωwith x1 < 0. (7.5) PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 43 Similarly, if x1 ⩾0, then \|x\| ⩾x1 and we have u(τ, x) −eu∞(x) ⩽u(τ, x) −φ(\|x\| −r0) ⩽φ(x1 + cτ + ξ(τ)) + φ(−x1 + cτ + ξ(τ)) −φ(x1 −r0) ⩽φ(cτ + ξ(τ)) + φ′(x1 + Θ)(cτ + ξ(τ) + r0) for some Θ ∈[cτ + ξ(τ), −r0]. Step 2. A ﬁrst upper bound u(τ, x) −eu∞(x) ⩽u(τ, x) −φ(\|x\| −r0) ⩽φ(x1 + cτ + ξ(τ)) + φ(−x1 + cτ + ξ(τ)) −φ(x1 −r0) ⩽φ(cτ + ξ(τ)) + φ′(x1 + Θ)(cτ + ξ(τ) + r0) for some Θ ∈[cτ + ξ(τ), −r0]. Let us now consider three subcases. First, if 0 ⩽x1 ⩽−Θ, then, by (5.7) and (5.8), we have u(τ, x) −eu∞(x) ⩽eλΘβ0 + γ0(cτ + ξ(t) + r0) ⩽0. ⩽φ(cτ + ξ(τ)) + φ′(x1 + Θ)(cτ + ξ(τ) + r0) for some Θ ∈[cτ + ξ(τ), −r0]. Let us now consider three subcases. First, if 0 ⩽x1 ⩽−Θ, then, by (5.7) and (5.8), we have u(τ, x) −eu∞(x) ⩽eλΘβ0 + γ0(cτ + ξ(t) + r0) ⩽0. ⩽φ(cτ + ξ(τ)) + φ′(x1 + Θ)(cτ + ξ(τ) + r0) for some Θ ∈[cτ + ξ(τ), −r0]. Let us now consider three subcases. First, if 0 ⩽x1 ⩽−Θ, then, by (5.7) and (5.8), we have u(τ, x) −eu∞(x) ⩽eλΘβ0 + γ0(cτ + ξ(t) + r0) ⩽0. Let us now consider three subcases. First, if 0 ⩽x1 ⩽−Θ, then, by (5.7) and (5.8), we have u(τ, x) −eu∞(x) ⩽eλΘβ0 + γ0(cτ + ξ(t) + r0) ⩽0. u(τ, x) −eu∞(x) ⩽eλΘβ0 + γ0(cτ + ξ(t) + r0) ⩽0 Therefore, we have that u(τ, x) ⩽eu∞(x) for any x ∈Ωwith 0 ⩽x1 ⩽−Θ. (7.6) Now, if −Θ < x1 ⩽RK −Θ, then φ′(x1 + Θ) ⩾γ1e−µ(x1+Θ) (by (5.7) and (5.8)). Hence, u(τ, x) −eu∞(x) ⩽β0eλ(cτ+ξ(τ)) + γ1(cτ + ξ(τ) + r0)e−µ(x1+Θ) ⩽β0 + γ1(cτ + ξ(τ) + r0)e−µRK ⩽0. (7.6) Thus, we have that Thus, we have that u(τ, x) ⩽eu∞(x) for any x ∈Ωwith −Θ < x1 ⩽RK −Θ. (7.7) Finally, let us consider the case x1 > RK −Θ. Let H be the half-space given by H := x ∈RN; x1 > RK −Θ ⊂RN \ BRK. u(τ, x) ⩽eu∞(x) for any x ∈Ωwith −Θ < x1 ⩽RK −Θ. (7.7) (7.7) Finally, let us consider the case x1 > RK −Θ. Let H be the half-space give H RN R Θ RN \ B H := x ∈RN; x1 > RK −Θ ⊂RN \ BRK. Since ∂tu > 0 in R × Ω, using (7.5), (7.6) and (7.7), we have      Leu∞+ f(eu∞) = 0 in H, Lu(τ, ·) + f(u(τ, ·)) ⩾0 in H, u(τ, ·) ⩽eu∞ in Ω\ H. Step 3. Conclusion Let T∗> τ be arbitrary. Then, using (7.8) and recalling (7.2), we arrive at Let T∗> τ be arbitrary. Then, using (7.8) and recalling (7.2), we arrive at ( ∂teu∞−Leu∞−f(eu∞) ⩾∂tu −Lu −f(u) in (τ, T∗] × Ω, eu∞(·) ⩾u(τ, ·) in Ω. Hence, by the comparison principle Lemma 3.1, we obtain that u(t, x) ⩽eu∞(x) for any (t, x) ∈[τ, T∗] × Ω. But since T∗> τ is arbitrary, we ﬁnd that Hence, by the comparison principle Lemma 3.1, we obtain that u(t, x) ⩽eu∞(x) for any (t, x) ∈[τ, T∗] × Ω. But since T∗> τ is arbitrary, we ﬁnd that u(t, x) ⩽eu∞(x) for any (t, x) ∈[τ, ∞) × Ω. □ Using Proposition 6.1, we obtain u∞⩽eu∞in Ω, which completes the proof. □ Using Proposition 6.1, we obtain u∞⩽eu∞in Ω, which completes the proof. □ 44 JULIEN BRASSEUR AND J´ERˆOME COVILLE JULIEN BRASSEUR AND J´ERˆOME COVILLE 44 8. Large time behaviour of super-level sets In this section, we characterise further the large time behaviour of the entire solution u(t, x). Precisely, we will prove that its super-level sets, namely the sets given by Eλ(t) := x ∈Ω; u(t, x) ⩾λ for λ ∈(0, 1) and t ∈R, are trapped between two moving frames which move at speed is c when t is large enough. In addition to the intrinsic interest of the results contained in this section, they will allow us to pave the way for the characterisation of the entire solution u(t, x) as a generalised transition front with global mean speed c. The main result of this section is the following: Proposition 8.1 (Large time behaviour of super-level sets). Assume (1.4), (1.5), (1.6), (1.9) and (2.1). Suppose that J ∈Bα 1,∞(Ω; δ) for some α ∈(0, 1). Let u(t, x) be the unique entire solution to (P) satisfying (5.1) and (5.2). Then, the following properties hold: (i) (Upper bound) For all λ ∈(0, 1), there exists a time t1 = t1(λ) ⩾0 and a constant Γ0 = Γ0(λ) ∈R such that, for all t ⩾t1, there holds Eλ(t) ⊂ x ∈Ω; x1 ⩾Γ0 −ct . (ii) (Lower bound) For all λ ∈(0, 1), there are some t2 = t2(λ) ⩾0, Γ1 = Γ1(λ) ∈R and Γ2 = Γ2(λ) ∈R such that, for all t ⩾t2, there holds (ii) (Lower bound) For all λ ∈(0, 1), there are some t2 = t2(λ) ⩾0, Γ1 = Γ1(λ) ∈R and Γ2 = Γ2(λ) ∈R such that, for all t ⩾t2, there holds Eλ(t) ⊃ x ∈Ω; Γ1 ⩾x1 ⩾Γ2 −ct . Let us make some comments on the strategy of the proof of Proposition 8.1. The heart of the proof consists in comparing u(t, x) with a sub- and a supersolution of the problem Let us make some comments on the strategy of the proof of Proposition 8.1. The heart of the proof consists in comparing u(t, x) with a sub- and a supersolution of the problem ∂tv = LRNv + f(v), (8.1) ∂tv = LRNv + f(v), (8.1) Now, let β0 > 0 be small enough so that −f ′(s) ⩾−max f ′(0), f ′(1) =: α0 for all s ∈[−2β0, 2β0] ∪[1 −2β0, 1 + 2β0]. Furthermore, let A > 0 be large enough so that −f ′(s) ⩾−max f ′(0), f ′(1) =: α0 for all s ∈[−2β0, 2β0] ∪[1 −2β0, 1 + 2β0]. Furthermore let A > 0 be large enough so that φ(z) ⩽β0 if z < −A, φ(z) ⩾1 −β0 if z > A. Note that A is well-deﬁned since limz→−∞φ(z) = 1 and limz→+∞φ(z) = 1. Now, let (t, x) ∈ [0, ∞) × RN be arbitrary. By a short computation, we obtain that Note that A is well-deﬁned since limz→−∞φ(z) = 1 and limz→+∞φ(z) = 1. Now, let (t, x) ∈ [0, ∞) × RN be arbitrary. By a short computation, we obtain that ∂tw+ ρ (t, x) = c + αβκe−αt φ′(x1 + ξ(t)) −αβ e−αt. Recalling (1.7), this rewrites ∂tw+ ρ (t, x) = LRNw+ ρ (t, x) + f w+ ρ (t, x) −β e−αt + αβκe−αtφ′(x1 + ξ(t)) −αβ e−αt. (8.3) Let us now distinguish between two diﬀerent cases. Let us now distinguish between two diﬀerent cases. Case 1: \|x1 + ξ(t)\| > A. Case 1: \|x1 + ξ(t)\| > A. Given the deﬁnition of w+ ρ (t, x) and A, we must have that either w+ ρ (t, x) −β e−αt ⩽β0 or w+ ρ (t, x) −β e−αt ⩾1 −β0. In both situations, we have Given the deﬁnition of w+ ρ (t, x) and A, we must have that either w+ ρ (t, x) −β e−αt ⩽β0 or w+ ρ (t, x) −β e−αt ⩾1 −β0. In both situations, we have f w+ ρ (t, x) −β e−αt −f(w+ ρ (t, x)) ⩾α0β e−αt, f w+ ρ (t, x) −β e−αt −f(w+ ρ (t, x)) ⩾α0β e−αt, for all 0 ⩽β ⩽β0 (recall that t ⩾0 so that β e−αt ⩽β). Since φ′ > 0, using (8.3), we get ∂tw+ ρ (t, x) ⩾LRNw+ ρ (t, x) + f(w+ ρ (t, x)) + β(α0 −α)e−αt. Thus, w+ ρ is a supersolution in the range \|x1+ξ(t)\| > A provided 0 ⩽α ⩽α0 and 0 ⩽β ⩽β0. Case 2: \|x1 + ξ(t)\| ⩽A. Thus, w+ ρ is a supersolution in the range \|x1+ξ(t)\| > A provided 0 ⩽α ⩽α0 and 0 ⩽β ⩽β0. (8.1) where LRNv := Jrad ∗v −v. The construction of the subsolution is slightly more involved than that of the supersolution due to the fact that the large time limit of u(t, x), namely u∞(x), may not be identically 1 in the whole of Ω. This forces us to consider subsolutions to (8.1) in some strips of RN which move at speed c′ < c. This will allow us to obtain a preliminary lower bound showing that the super-level sets move asymptotically at speed c. The desired lower bound on Eλ(t) will follow using a contradiction argument. For the convenience of the reader, the proof of Proposition 8.1 is divided into three subsections, each of which corresponds to a step of the proof of Proposition 8.1. 8.1. An upper bound: the super-level sets move at speed at most c. In this subsec- tion, we establish the upper bound on the super-level sets of u(t, x) stated at Proposition 8.1 (namely assertion (i)). To this end, we need to construct a supersolution to an auxiliary problem. This supersolution and the comparison principle will provide an upper bound on u(t, x), from which Proposition 8.1(i) will follow. So let us ﬁrst prove the following lemma: Lemma 8.2. There exists α0, β0 > 0 such that, for any 0 ⩽α ⩽α0 and any 0 ⩽β ⩽β0, there exists some κ0 = κ0(α) > 0 such that, for all κ ⩾κ0 and all ρ ∈R, the function w+ ρ (t, x) := φ x1 + ct + ρ + κβ (1 −e−αt) + β e−αt, (8.2) w+ ρ (t, x) := φ x1 + ct + ρ + κβ (1 −e−αt) + β e−αt, (8.2) is a supersolution to (8.1) in [0, ∞) × RN. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 45 Proof. Let ρ > 0 be arbitrary and set ξ(t) := ct + ρ + κβ (1 −e−αt). Let us extend f linearly outside [0, 1] by the function ef given by (3.21). For the sake of simplicity, let us still denote by f this extension. Now, let β0 > 0 be small enough so that Proof. Let ρ > 0 be arbitrary and set ξ(t) := ct + ρ + κβ (1 −e−αt). Let us extend f linearly outside [0, 1] by the function ef given by (3.21). For the sake of simplicity, let us still denote by f this extension. (8.1) (8.4) (8.4) On the other hand, by exploiting the asymptotic properties of φ (Lemma 5.2), we have On the other hand, by exploiting the asymptotic properties of φ (Lemma 5.2), we have w+ ρ (t, x) ⩾1 −γ1e−µ(x1+ct+ρ+κβ(1−e−αt)) + β e−αt = 1 + e−αt β −γ1eµ(2RJ−ρ)e−µκβ(1−e−αt)e−(µc−α)t , for all (t, x) ∈[0, ∞) × {x1 ⩾−2RJ}. Hence, choosing 0 ⩽α ⩽min {α0, µc} and ρ ⩾ 2RJ −µ−1 log(β/γ1), we have that for all (t, x) ∈[0, ∞) × {x1 ⩾−2RJ}. Hence, choosing 0 ⩽α ⩽min {α0, µc} and ρ ⩾ 2RJ −µ−1 log(β/γ1), we have that w+ ρ (t, x) ⩾1 > u(t, x) for all (t, x) ∈[0, ∞) × {x1 ⩾−2RJ}. (8.5) w+ ρ (t, x) ⩾1 > u(t, x) for all (t, x) ∈[0, ∞) × {x1 ⩾−2RJ}. (8.5) w+ ρ (t, x) ⩾1 > u(t, x) for all (t, x) ∈[0, ∞) × {x1 ⩾−2RJ}. (8.5) w+ ρ (t, x) ⩾1 > u(t, x) for all (t, x) ∈[0, ∞) × {x1 ⩾−2RJ}. (8.5) (8.5) Since K ⊂{x1 ⩾−RJ} it follows that u(t, x) is also a solution to (8.1) in (t, x) ∈[0, ∞) × {x1 ⩽−2RJ}. Hence, recalling (8.4) and (8.5), it follows from the comparison principle Lemma 3.1 (remember Remark 3.3) that Since K ⊂{x1 ⩾−RJ} it follows that u(t, x) is also a solution to (8.1) in (t, x) ∈[0, ∞) × {x1 ⩽−2RJ}. Hence, recalling (8.4) and (8.5), it follows from the comparison principle Lemma 3.1 (remember Remark 3.3) that u(t, x) ⩽w+ ρ (t, x) ⩽φ (x1 + ct + ρ + κβ) + β e−αt, (8.6) u(t, x) ⩽w+ ρ (t, x) ⩽φ (x1 + ct + ρ + κβ) + β e−αt, (8.6) (8.6) for all (t, x) ∈[0, ∞) × Ω. Now, let λ ∈(0, 1) and let t1(λ) := max{0, α−1 log(λ/(2β))}. Then, for all t ⩾t1(λ) and all x ∈Eλ(t), we have Γ0(λ) := φ−1(λ/2) −(ρ + κβ) ⩽x1 + ct. Therefore, the following inclusion holds Eλ(t) ⊂ x ∈Ω; x1 ⩾Γ0(λ) −ct , Eλ(t) ⊂ x ∈Ω; x1 ⩾Γ0(λ) −ct , □ which thereby proves Proposition 8.1(i). □ for all t ⩾t1(λ), which thereby proves Proposition 8.1(i). □ for all t ⩾t1(λ), which thereby proves Proposition 8.1(i). 8.2. A lower bound: the super-level sets move asymptotically at speed c. (8.1) Case 2: \|x1 + ξ(t)\| ⩽A. Case 2: \|x1 + ξ(t)\| ⩽A. et ζ := infz∈[−A,A] φ′(z) > 0. Since f ∈C0,1(R), it follows from (8.3) that ζ := infz∈[−A,A] φ′(z) > 0. Since f ∈C0,1(R), it follows from (8.3) that ∂tw+ ρ (t, x) ⩾LRNw+ ρ (t, x) + f(w+ ρ (t, x)) + β (ζακ −∥f ′∥∞−α)e−αt. Let ζ := infz∈[−A,A] φ′(z) > 0. Since f ∈C0,1(R), it follows from (8.3) that ∂tw+ ρ (t, x) ⩾LRNw+ ρ (t, x) + f(w+ ρ (t, x)) + β (ζακ −∥f ′∥∞−α)e−αt. ∂tw+ ρ (t, x) ⩾LRNw+ ρ (t, x) + f(w+ ρ (t, x)) + β (ζακ −∥f ′∥∞−α)e−αt. Thus, we, again, have that w+ ρ is a supersolution in the range \|x1 + ξ(t)\| ⩽A provided κ ⩾κ0(α), where we have set κ0(α) := (∥f ′∥∞+ α)/(ζα). Summing up, we have shown that w+ ρ is a supersolution to (8.1) for all (t, x) ∈[0, ∞)×RN provided that 0 ⩽α ⩽α0, that 0 ⩽β ⩽β0 and that κ ⩾κ0(α), as desired. □ Remark 8.3. Observe that α0 and β0 depend only on f ′. Remark 8.3. Observe that α0 and β0 depend only on f ′. We are now in position to prove Proposition 8.1(i). We are now in position to prove Proposition 8.1(i). Proof of Proposition 8.1(i). Since K is compact, up to immaterial translations, we may assume, without loss of generality, that K ⊂{x1 ⩾−RJ} where RJ > 0 is such that supp(J) ⊂[0, RJ]. By Lemma 8.2, we know that there exists α0 > 0 and β0 > 0 such that, for all 0 ⩽α ⩽α0 and all 0 ⩽β ⩽β0, there is some κ0 = κ0(α) > 0 such that, for all 0 ⩽κ ⩽κ0 and all ρ ∈R, the function w+ ρ given by (8.2), namely w+ ρ (t, x) := φ x1 + ct + ρ + κβ (1 −e−αt) + β e−αt, JULIEN BRASSEUR AND J´ERˆOME COVILLE 46 is a supersolution to (8.1) for all (t, x) ∈[0, ∞) × RN. Since u(0, x) →0 as x1 →−∞(by Proposition 5.7), since w(0, x) ⩾β for all x ∈Ωand since limz1→∞w+ ρ (0, z) = 1 + β > 1 > u(0, x) for all x ∈Ω, up to take ρ > 0 suﬃciently large, we may always assume that w+ ρ (0, x) ⩾u(0, x) for all x ∈Ω. (8.1) In this subsection, we establish a preliminary lower bound for the super-level sets of u(t, x) which will be useful to complete the proof of Proposition 8.1(ii) and, hence, of Proposition 8.1. This preliminary lower bound will allow us to obtain an asymptotic version of Proposition 8.1(ii). Namely, we will prove the following intermediary result: Lemma 8.4. For all λ ∈(0, 1), there is some n0 = n0(λ) ⩾1 such that, for all n ⩾n0, there exists Γ1,n ∈R, Γ2,n(λ) ∈R and t2,n(λ) ⩾0 such that, for all t ⩾t2,n(λ), there holds Eλ(t) ⊃ x ∈Ω; Γ1,n ⩾x1 ⩾Γ2,n(λ) −c 1 −1 n t . For the convenience of the reader, let us ﬁrst introduce a few notations which will be extensively used in this subsection. For any ﬁxed R0 ∈R, we let χR0 ∈C2(R, [0, 1]) be a cut-oﬀfunction such that χR0 ≡0 in {x1 ⩾R0 + 2RJ} and χR0 ≡1 in {x1 ⩽R0 + RJ}. Then, we have the following result: χR0 ≡0 in {x1 ⩾R0 + 2RJ} and χR0 ≡1 in {x1 ⩽R0 + RJ}. χR0 ≡0 in {x1 ⩾R0 + 2RJ} and χR0 ≡1 in {x1 ⩽R0 + RJ}. Then, we have the following result: Lemma 8.5. There exists α0, β0 > 0 such that, for any c′ < c, there exists ε0 > 0 with the property that, for all 0 < ε ⩽ε0, all 0 ⩽α ⩽α0 and all 0 ⩽β ⩽β0, there is some constant κ = κ(α) > 0 for which the function w− ρ (t, x) := (1 −ε)φ x1 + c′t −ρ −κβ(1 −e−αt) χR0(x1) −β e−αt, is a subsolution to (8.1) in [0, ∞) × {x1 ⩽R0} for all ρ ∈R and all R0 ∈R. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 47 Proof. Let ρ > 0 and c′ < c be arbitrary and set ξ(t) := c′t−ρ−κβ (1−e−αt). Let us extend f linearly outside [0, 1] by the function ef given by (3.21). For the sake of simplicity, let us still denote by f this extension. Now, let 0 < β0 < min{1 −θ, θ}/4 be small enough so that Proof. Let ρ > 0 and c′ < c be arbitrary and set ξ(t) := c′t−ρ−κβ (1−e−αt). Let us extend f linearly outside [0, 1] by the function ef given by (3.21). (8.1) For the sake of simplicity, let us still denote by f this extension. Now, let 0 < β0 < min{1 −θ, θ}/4 be small enough so that 5 4 f ′(0) ⩽f ′(s) ⩽3 4 f ′(0) for s ∈[−4β0, 4β0], f ′(s) ⩽1 2 f ′(1) for s ∈[1 −4β0, 1]. (8.7) (8.7) As in the proof of Lemma 8.2, we may ﬁnd some number A > 0 such that φ(z) ⩽β0/2 if z < −A, φ(z) ⩾1 −β0/2 if z > A. (8.8) (8.8) Now, let (t, x) ∈[0, ∞) × {x1 ⩽R0} be arbitrary. A short computation shows that ∂tw− ρ (t, x) = (1 −ε)(c′ −αβκe−αt)φ′(x1 + ξ(t)) + αβ e−αt. Now, let (t, x) ∈[0, ∞) × {x1 ⩽R0} be arbitrary. A short computation shows that ∂tw− ρ (t, x) = (1 −ε)(c′ −αβκe−αt)φ′(x1 + ξ(t)) + αβ e−αt. Now, let (t, x) ∈[0, ∞) × {x1 ⩽R0} be arbitrary. A short computation shows that ∂tw− ρ (t, x) = (1 −ε)(c′ −αβκe−αt)φ′(x1 + ξ(t)) + αβ e−αt. Recalling (1.7), this rewrites Recalling (1.7), this rewrites Recalling (1.7), this rewrites ∂tw− ρ (t, x) = (1 −ε)LRNφ(x1 + ξ(t)) + (1 −ε)f(φ(x1 + ξ(t))) + (1 −ε)(c′ −c −αβκe−αt)φ′(x1 + ξ(t)) + αβ e−αt. Since x1 ⩽R0, since supp(J) ⊂[0, RJ], since χR0 ≡1 in {x1 ⩽R0 + RJ} and since LRN(φ + v) = LRNφ for all functions v independent of x, we have (1 −ε)LRNφ(x1 + ξ(t)) = LRNw− ρ (t, x) for all x ∈{x1 ⩽R0}. Therefore, we have R (φ ) R φ p LRNw− ρ (t, x) for all x ∈{x1 ⩽R0}. Therefore, we have ∂tw− ρ (t, x) = LRNw− ρ (t, x) + f(w− ρ (t, x)) + I(t, x) for all x ∈{x1 ⩽R0} ∂tw− ρ (t, x) = LRNw− ρ (t, x) + f(w− ρ (t, x)) + I(t, x) for all x ∈{x1 ⩽R0}, where I(t, x) denotes the following expression where I(t, x) denotes the following expression I(t, x) := (1 −ε)(c′ −c −αβκe−αt)φ′(x1 + ξ(t)) + αβ e−αt + (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)). + (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)). (8.1) Hence, to prove that w− ρ (t, x) is a subsolution to (8.1) for all (t, x) ∈[0, ∞) × {x1 ⩽R0} it suﬃces to show that I(t, x) ⩽0 for all (t, x) ∈[0, ∞) × {x1 ⩽R0}. To this end, we distinguish between three diﬀerent cases. Hence, to prove that w− ρ (t, x) is a subsolution to (8.1) for all (t, x) ∈[0, ∞) × {x1 ⩽R0} it suﬃces to show that I(t, x) ⩽0 for all (t, x) ∈[0, ∞) × {x1 ⩽R0}. To this end, we distinguish between three diﬀerent cases. Case 1: x1 + ξ(t) < −A. Let us ﬁrst observe that (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) = (1 −ε) f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) −εf(w− ρ (t, x)). (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) = (1 −ε) f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) −εf(w− ρ (t, x)). Since x1 + ξ(t) < −A, we have φ(x1 + ξ(t)) ⩽β0/2 (by deﬁnition of A). Since, in addition, t ⩾0 and η ≡1 in {x1 ⩽R0}, we also have −3β0/2 ⩽w− ρ (t, x) ⩽β0/2. Hence, recalling the deﬁnition of w− ρ (t, x) and of β0 (remember (8.7)), we obtain that (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) = (1 −ε) f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) −εf(w− ρ (t, x)). Since x1 + ξ(t) < −A, we have φ(x1 + ξ(t)) ⩽β0/2 (by deﬁnition of A). Since, in addition, Since x1 + ξ(t) < −A, we have φ(x1 + ξ(t)) ⩽β0/2 (by deﬁnition of A). Since, in addition, t ⩾0 and η ≡1 in {x1 ⩽R0}, we also have −3β0/2 ⩽w− ρ (t, x) ⩽β0/2. Hence, recalling the deﬁnition of w− ρ (t, x) and of β0 (remember (8.7)), we obtain that f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) ⩽3 4f ′(0) εφ(x1 + ξ(t)) + β e−αt . (8.9) f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) ⩽3 4f ′(0) εφ(x1 + ξ(t)) + β e−αt . (8.9) Now, if w− ρ (t, x) = φ(x1 + ξ(t)) −β e−αt ⩽0, then, since f(s) = f ′(0)s ⩾0 for all construction), we have −εf(w− ρ (t, x)) ⩽0. (8.1) In this situation, since c > c′, φ > 0 an it follows that (8.9) f(φ(x1 + ξ(t))) f(wρ (t, x)) ⩽4f (0) εφ(x1 + ξ(t)) + β e . (8.9) Now, if w− ρ (t, x) = φ(x1 + ξ(t)) −β e−αt ⩽0, then, since f(s) = f ′(0)s ⩾0 for all s ⩽0 (by construction), we have −εf(w− ρ (t, x)) ⩽0. In this situation, since c > c′, φ > 0 and φ′ > 0, it follows that Now, if w− ρ (t, x) = φ(x1 + ξ(t)) −β e−αt ⩽0, then, since f(s) = f ′(0)s ⩾0 for all s ⩽0 (by construction), we have −εf(w− ρ (t, x)) ⩽0. In this situation, since c > c′, φ > 0 and φ′ > 0, it follows that I(t, x) ⩽αβ e−αt + 3 4f ′(0) εφ(x1 + ξ(t)) + β e−αt ⩽β e−αt α + 3 4f ′(0) . Th f I(t ) ⩽0 id d 0 ⩽ ⩽ 3f ′(0)/4 I(t, x) ⩽αβ e−αt + 3 4f ′(0) εφ(x1 + ξ(t)) + β e−αt ⩽β e−αt α + 3 4f ′(0) . I(t, x) ⩽αβ e−αt + 3 4f ′(0) εφ(x1 + ξ(t)) + β e−αt ⩽β e−αt α + 3 4f ′(0) . herefore, I(t, x) ⩽0 provided 0 ⩽α ⩽−3f ′(0)/4. Therefore, I(t, x) ⩽0 provided 0 ⩽α ⩽−3f ′(0)/4. Therefore, I(t, x) ⩽0 provided 0 ⩽α ⩽−3f ′(0)/4. JULIEN BRASSEUR AND J´ERˆOME COVILLE 48 On the other case, if w− ρ (t, x) = φ(x1 + ξ(t)) −β e−αt > 0, then, since φ(x1 + ξ(t)) ⩽β0/2, we also have w− ρ (t, x) ⩽β0/2 and, by (8.7), there holds On the other case, if w− ρ (t, x) = φ(x1 + ξ(t)) −β e−αt > 0, then, since φ(x1 + ξ(t)) ⩽β0/2, we also have w− ρ (t, x) ⩽β0/2 and, by (8.7), there holds −εf(w− ρ (t, x)) = ε f(0) −f(w− ρ (t, x)) ⩽−ε 5 4f ′(0)w− ρ (t, x). −εf(w− ρ (t, x)) = ε f(0) −f(w− ρ (t, x)) ⩽−ε 5 4f ′(0)w− ρ (t, x). (8.10) Since the right-hand side of (8.9) (resp. (8.10)) is nonpositive (resp. nonnegative), we have (8.10) Since the right-hand side of (8.9) (resp. (8.10)) is nonpositive (resp. (8.1) nonnegative), we have (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) ⩽f ′(0) 2 εφ(x1 + ξ(t)) + β e−αt −ε 3 2f ′(0)w− ρ (t, x) = −(1 −ε)εφ(x1 + ξ(t))f ′(0) + 2εf ′(0)β e−αt + ε2f ′(0) 2 φ(x1 + ξ(t)) + f ′(0) 2 β e−αt ⩽−(1 −ε)εφ(x1 + ξ(t))f ′(0) + f ′(0) 2 β e−αt (1 + 4ε) . Plugging this in the deﬁnition of I(t, x), we obtain I(t, x) ⩽β e−αt 1 + 4ε 2 f ′(0) + α −ακ(1 −ε)φ′(x1 + ξ(t)) + (1 −ε) (c′ −c)φ′(x1 + ξ(t)) −εφ(x1 + ξ(t))f ′(0) . Recalling (5.7), we know that there is a constant σ0 > 0 such that φ(z) ⩽σ0φ′(z) for all z < 0. Hence, using that c′ < c and that φ′ > 0, we obtain Recalling (5.7), we know that there is a constant σ0 > 0 such that φ(z) ⩽σ0φ′(z) for all z < 0. Hence, using that c′ < c and that φ′ > 0, we obtain I(t, x) ⩽β e−αt 1 + 4ε 2 f ′(0) + α + (1 −ε) c′ −c σ0 −εf ′(0) φ(x1 + ξ(t)). Therefore, I(t, x) ⩽0 provided 0 ⩽α ⩽−f ′(0)/2 and 0 < ε ⩽min{1/2, Therefore, I(t, x) ⩽0 provided 0 ⩽α ⩽−f ′(0)/2 and 0 < ε ⩽min{1/2, (c′ −c)/(σ0f ′(0))}. Case 2: x1 + ξ(t) > −A. Case 2: x1 + ξ(t) > −A. In this situation, we have φ(x1+ξ(t)) ⩾1−β0/2 (by construction of A). Since β0/2 < 1−θ (by construction of β0), we further have that f(φ(x1 + ξ(t))) > 0. Hence, using (8.7), we ﬁnd that, for all 0 < ε ⩽β0, there holds (1 −ε)f(φ(x1 + ξ(t))) −f((1 −ε)φ(x1 + ξ(t))) ⩽ε f ′(0) 2 φ(x1 + ξ(t)) ⩽0. (8.11) On the other hand, since 1 −β0/2 ⩽φ(x1 + ξ(t)) ⩽1, since 0 ⩽β ⩽β0 and since t ⩾0, for all 0 < ε < β0, we have (8.11) w− ρ (t, x) = φ(x1 + ξ(t)) −εφ(x1 + ξ(t)) −β e−αt ⩾1 −β0 2 −2β0 > 1 −4β0. w− ρ (t, x) = φ(x1 + ξ(t)) −εφ(x1 + ξ(t)) −β e−αt ⩾1 −β0 2 −2β0 > 1 −4β0. (8.1) Letting ζ := infz∈[−A,A] φ′(z) > 0 and recalling the deﬁnition of I(t, x), we get I(t, x) ⩽β e−αt α + [f]C0,1(R) −ζακ(1 −ε) +(1−ε) ζ(c′ −c) + ε[f]C0,1(R) +ε∥f∥L∞([0,1]) ζ := infz∈[−A,A] φ′(z) > 0 and recalling the deﬁnition of I(t, x), we get Letting ζ := infz∈[−A,A] φ′(z) > 0 and recalling the deﬁnition of I(t, x), we get I(t, x) ⩽β e−αt α + [f]C0,1(R) −ζακ(1 −ε) +(1−ε) ζ(c′ −c) + ε[f]C0,1(R) +ε∥f∥L∞([0,1]). Therefore, I(t, x) ⩽0 provided we choose ε and α such that I(t, x) ⩽β e−αt α + [f]C0,1(R) −ζακ(1 −ε) +(1−ε) ζ(c′ −c) + ε[f]C0,1(R) +ε∥f∥L∞([0,1]). Therefore, I(t, x) ⩽0 provided we choose ε and α such that 0 < ε ⩽ζ(c −c′)/(2∥f∥L∞([0,1]) + 2[f]C0,1(R)) and κ ⩾κ(α) := (α + [f]C0,1(R))/(ζα). Summing up, we have shown that w− ρ is a subsolution to (8.1) in [0, ∞) × {x1 ⩽R0} provided that 0 ⩽α ⩽α0 := −f ′(0)/2, that 0 ⩽β ⩽β0, that κ ⩾κ(α) and that 0 < ε ⩽ε0, where ε0 > 0 depends only on c, c′, f, β0 and φ, as desired. □ Remark 8.6. The constants α0 and β0 depend only on f ′ and the constant κ(α) depends only on f, φ and α. Moreover, by taking c′ := (1 −1/n)c for n ∈N \ {0}, it can easily be seen from the proof that there exists n0 ⩾1 such that ε0 reads ε0 = ϑc n where ϑ := min{σ−1 0 , ζ} 2([f]C0,1(R) + ∥f∥L∞([0,1])), for all n ⩾n0. Therefore, the function w− n,ρ(t, x) := 1 −ϑc n φ x1 + 1 −1 n ct −ρ −κ(α0)β0(1 −e−α0t) χR0(x1) −β0e−α0t, is a subsolution to (8.1) in [0, ∞) × {x1 ⩽R0} for all n ⩾n0, all ρ ∈R and all R0 ∈R. Remark 8.7. Notice that the same proof also works for the classical problem (1.2). is a subsolution to (8.1) in [0, ∞) × {x1 ⩽R0} for all n ⩾n0, all ρ ∈R and all R0 ∈R. Remark 8.7. Notice that the same proof also works for the classical problem ( We are now in position to prove Lemma 8.4. Proof of Lemma 8.4. Let λ ∈(0, 1) be arbitrary. Since K is compact, up to immaterial translations, we may assume, without loss of generality, that K ⊂{x1 ⩾−RJ}. (8.1) Otherwise said, we have Otherwise said, we have 1 −4β0 ⩽w− ρ (t, x) ⩽(1 −ε)φ(x1 + ξ(t)) ⩽1, which, together with (8.7), implies that, for all 0 < ε < β0, there holds which, together with (8.7), implies that, for all 0 < ε < β0, there holds which, together with (8.7), implies that, for all 0 < ε < β0, there holds f((1 −ε)φ(x1 + ξ(t))) −f(w− ρ (t, x)) ⩽β f ′(0) 2 e−αt. (8.12) (8.12) Plugging (8.11) and (8.12) in the deﬁnition of I(t, x), we obtain I(t, x) ⩽β α + f ′(0) 2 e−αt. I(t, x) ⩽β α + f ′(0) 2 e−αt. Therefore, I(t, x) ⩽0 provided 0 ⩽α ⩽−f ′(0)/2 and 0 < ε ⩽β0. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 49 Case 3: −A ⩽x1 + ξ(t) ⩽A. Case 3: −A ⩽x1 + ξ(t) ⩽A. Let us decompose the last two terms in the deﬁnition of I(t, x) as follows (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) = (1 −ε) f(φ(x1 + ξ(t))) −f((1 −ε)φ(x1 + ξ(t))) + f((1 −ε)φ(x1 + ξ(t))) −f(w− ρ (t, x)) −εf((1 −ε)φ(x1 + ξ(t))). Since f ∈C0,1(R) and since 0 < φ < 1, we then obtain the following estimate Since f ∈C0,1(R) and since 0 < φ < 1, we then obtain the following estimate Since f ∈C0,1(R) and since 0 < φ < 1, we then obtain the following estimate (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) ⩽[f]C0,1(R) ε(1 −ε) + β e−αt + ε∥f∥L∞([0,1]). Letting ζ := infz∈[−A,A] φ′(z) > 0 and recalling the deﬁnition of I(t, x), we get I(t, x) ⩽β e−αt α + [f]C0,1(R) −ζακ(1 −ε) +(1−ε) ζ(c′ −c) + ε[f]C0,1(R) +ε∥f∥L∞([0,1]). Therefore, I(t, x) ⩽0 provided we choose ε and α such that (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) ⩽[f]C0,1(R) ε(1 −ε) + β e−αt + ε∥f∥L∞([0,1]). (1 −ε)f(φ(x1 + ξ(t))) −f(w− ρ (t, x)) ⩽[f]C0,1(R) ε(1 −ε) + β e−αt + ε∥f∥L∞([0,1]). (8.1) u(t, x) ⩾w− n,ρ(t, x) for all (t, x) ∈[t∗ n, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}, u(t, x) ⩾w− n,ρ(t, x) for all (t, x) ∈[t∗ n, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}, u(t, x) ⩾w− n,ρ(t, x) for all (t, x) ∈[t∗ n, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}, where w− n,ρ(t, x) is as in Remark 8.6 with R0 := −Rn. But since w− n,ρ(t, x) ⩽0 ⩽u(t, x) for all x1 ⩾−Rn + 2RJ and all t ⩾0 (recall that χ--Rn ≡0 in {x1 ⩾−Rn + 2RJ}), we have (8.13) u(t, x) ⩾w− n,ρ(t, x) for all t ⩾t∗ n, all x1 ⩾−Rn and all ρ ∈R (8.13) u(t, x) ⩾w− n,ρ(t, x) for all t ⩾t∗ n, all x1 ⩾−Rn and all ρ ∈R. (8.13) u(t, x) ⩾wn,ρ(t, x) for all t ⩾tn, all x1 ⩾ Rn and al (8.13) Moreover, since limz→−∞φ(z) = 0, there is some ρn > 0 such that r, since limz→−∞φ(z) = 0, there is some ρn > 0 such that (1 −εn)φ −Rn + 1 −1 n ct∗ n −ρn −κ(α0)β0(1 −e−α0t∗ n) ⩽β0e−α0t∗ n. fore, w− n,ρn(t∗ n, x) ⩽0 for all x1 ⩽−Rn. But since u is positive, we obtain that u(t∗ n, x) ⩾w− n,ρn(t∗ n, x) for all x ∈Ω. (8.14) (8.14) Collecting (8.13), (8.14), recalling Lemma 8.5, Remark 8.6, that K ⊂{x1 ⩾−RJ} and that −Rn + RJ ⩽−RJ (because Rn ⩾2RJ, by construction of Rn), we have Collecting (8.13), (8.14), recalling Lemma 8.5, Remark 8.6, that K ⊂{x1 ⩾−RJ} and that −Rn + RJ ⩽−RJ (because Rn ⩾2RJ, by construction of Rn), we have          ∂tu ⩾LRNu + f(u) in [t∗ n, ∞) × {x1 ⩽−Rn}, ∂tw− n,ρn ⩽LRNw− n,ρn+ f(w− n,ρn) in [t∗ n, ∞) × {x1 ⩽−Rn}, u ⩾w− n,ρn in [t∗ n, ∞) × {x1 ⩾−Rn}, u(t∗ n, ·) ⩾w− n,ρn(t∗ n, ·) in Ω.          ∂tu ⩾LRNu + f(u) in [t∗ n, ∞) × {x1 ⩽−Rn}, ∂tw− n,ρn ⩽LRNw− n,ρn+ f(w− n,ρn) in [t∗ n, ∞) × {x1 ⩽−Rn}, u ⩾w− n,ρn in [t∗ n, ∞) × {x1 ⩾−Rn}, u(t∗ n, ·) ⩾w− n,ρn(t∗ n, ·) in Ω. (8.1) Let n ⩾n0 and εn := ϑc/n, where n0 ⩾1 and ϑ > 0 have the same meaning as in Remark 8.6. Let nλ ⩾n0 be such that 1 −εn > λ for all n ⩾nλ. Since u∞(x) →1 as \|x\| →∞, there exists then an increasing sequence (Rn)n⩾n0 ⊂[2RJ, ∞) such that u∞(x) ⩾1 −εn 2 for all x1 ⩽−Rn and all n ⩾n0. Let us now ﬁx some n ⩾nλ (⩾n0). Since limz→+∞φ(z) = 1, there is some tn ⩾0 such that φ(x1 + ct) ⩾1 −εn/2 for all (t, x) ∈[tn, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}. Since u(tn, x1, x′) →φ(x1 + ctn) for all −Rn ⩽x1 ⩽−Rn + 2RJ as \|x′\| →∞(by Proposi- tion 6.4), there exists R′ n > 0 such that u∞(x) ⩾1 −εn 2 for all x1 ⩽−Rn and all n ⩾n0. Let us now ﬁx some n ⩾nλ (⩾n0). Since limz→+∞φ(z) = 1, there is some tn ⩾0 such that φ(x1 + ct) ⩾1 −εn/2 for all (t, x) ∈[tn, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}. Since u(tn, x1, x′) →φ(x1 + ctn) for all −Rn ⩽x1 ⩽−Rn + 2RJ as \|x′\| →∞(by Proposi- tion 6.4), there exists R′ n > 0 such that u(tn, x) ⩾φ(x1 + ctn) −εn/2 > 1 −εn, JULIEN BRASSEUR AND J´ERˆOME COVILLE 50 for all −Rn ⩽x1 ⩽−Rn + 2RJ and all \|x′\| ⩾R′ n. Since ∂tu > 0, this implies that u(t, x) ⩾1 −εn, u(t, x) ⩾1 −εn, for all t ⩾tn, all \|x′\| ⩾R′ n and all −Rn ⩽x1 ⩽−Rn + 2RJ. Since, in addition, u(t, x) converges locally uniformly towards u∞(x) as t →∞, we may then ﬁnd some t′ n ⩾0 such that, for all \|x′\| ⩽R′ n and all −Rn ⩽x1 ⩽−Rn + 2RJ, there holds u(t′ n, x) ⩾u∞(x) −εn/2 ⩾1 −εn. Therefore, letting t∗ n := max{tn, t′ n}, we have Therefore, letting t∗ n := max{tn, t′ n}, we have Therefore, letting tn := max{tn, tn}, we have u(t, x) ⩾1 −εn for all (t, x) ∈[t∗ n, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}. In particular, for all ρ ∈R, we have u(t, x) ⩾1 −εn for all (t, x) ∈[t∗ n, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}. u(t, x) ⩾1 −εn for all (t, x) ∈[t∗ n, ∞) × {−Rn ⩽x1 ⩽−Rn + 2RJ}. (8.1)      ∂twn,ρn ⩽LRNwn,ρn+ f(wn,ρn) in [tn, ∞) × {x1 ⩽ Rn}, u ⩾w− n,ρn in [t∗ n, ∞) × {x1 ⩾−Rn}, u(t∗·) ⩾w− (t∗·) in Ω By the comparison principle Lemma 3.1 (whose application is licit as explained in Re- mark 3.3), we deduce that u(t, x) ⩾wn,ρn(t, x) for all (t, x) ∈[t∗ n, ∞) × Ω. By the comparison principle Lemma 3.1 (whose application is licit as explained in Re- mark 3.3), we deduce that u(t, x) ⩾wn,ρn(t, x) for all (t, x) ∈[t∗ n, ∞) × Ω. ) ( ) ,ρn( ) ( ) [ n ) Now, since n ⩾nλ, we may ﬁnd some τn ⩾t∗ n large enough so that λ + β0e−α0τn < 1 −εn (by construction of nλ). Since we also have κn(λ) := (λ+β0e−α0τn)/(1−εn) > 0, the number Θn(λ) := φ−1(κn(λ)) is well-deﬁned. Now, let Γn(λ) and Tn be given by Γn(λ) := ρn + β0κ(α0) + Θn(λ) and Tn := max τn, Γn(λ) + Rn c(1 −1/n) . th t t ∈[T ) d th t Γn(λ) := ρn + β0κ(α0) + Θn(λ) and Tn := max τn, Γn(λ) + Rn c(1 −1/n) . Suppose that t ∈[Tn, ∞) and that x ∈ x ∈Ω; −Rn ⩾x1 ⩾Γn(λ) −c 1 −1 n t . Γn(λ) := ρn + β0κ(α0) + Θn(λ) and Tn := max τn, Γn(λ) + Rn c(1 −1/n) . Suppose that t ∈[Tn, ∞) and that x ∈ x ∈Ω; −Rn ⩾x1 ⩾Γn(λ) −c 1 −1 n t . Then, by construction, we have that Then, by construction, we have that u(t, x) ⩾−β0e−α0t + (1 −εn)φ (Θn(λ)) ⩾λ + β0e−α0τn −β0e−α0t ⩾λ. u(t, x) ⩾−β0e−α0t + (1 −εn)φ (Θn(λ)) ⩾λ + β0e−α0τn −β0e−α0t ⩾λ. u(t, x) ⩾−β0e−α0t + (1 − □ □ Therefore, x ∈Eλ(t) for all t ⩾Tn, which thereby proves Lemma 8.4. □ Therefore, x ∈Eλ(t) for all t ⩾Tn, which thereby proves Lemma 8.4. □ PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 51 8.3. Conclusion: the super-level sets move exactly at speed c. This subsection is devoted to the proof of Proposition 8.1(ii). We will rely on some estimates obtained in the previous section together with a contradiction argument. So let us proceed. 8.3. Conclusion: the super-level sets move exactly at speed c. (8.1) This subsection is devoted to the proof of Proposition 8.1(ii). We will rely on some estimates obtained in the previous section together with a contradiction argument. So let us proceed. Proof of Proposition 8.1(ii). Let us extend f linearly outside [0, 1] by the function ef given by (3.21). For the sake of simplicity, let us still denote by f this extension. Now, let 0 < β0 < min{1 −θ, θ}/4 and A > 0 be as in the previous section, namely as in (8.7)-(8.8). Let us ﬁrst remark that the arguments in the proof of Lemma 8.4 also yield the existence of some ε∗, tε, ρε, Rε > 0 such that, for all 0 < ε ⩽ε∗, all t ⩾tε and all x ∈Ω, there holds u(t, x) ⩾w− ε (t, x) := 1 −ε 4 φ x1 + ct 2 −ρε −κ(α0)(1 −e−α0t) χ--Rε(x1) −β0e−α0t, χ--Rε has the same meaning as in the previous section. e where χ--Rε has the same meaning as in the previous section. e Now, since limz→+∞φ(z) = 1 and φ′ > 0, there exists eRε > 0 such that φ(z) ⩾1 −ε/4 for all z ⩾eRε. Moreover, if we let t∗:= max{tε, t′ ε}, where t′ ε ⩾0 is such that β0e−α0t′ ε ⩽ε2/16 and eRε −ct′ ε/4 + ρε + κ(α0) < −Rε, we have w− ε (t, x) > 1 −ε 4 φ eRε + c 4 (t −t∗) −β0e−α0t ⩾ 1 −ε 4 2 −ε2 16 = 1 −ε 2 t ⩾t∗and all x ∈{−Rε −ct/4 < x1 < −Rε + RJ}. Consequently, we have w− ε (t, x) > 1 −ε 4 φ eRε + c 4 (t −t∗) −β0e−α0t ⩾ 1 −ε 4 2 −ε2 16 = 1 −ε 2, for all t ⩾t∗and all x ∈{−Rε −ct/4 < x1 < −Rε + RJ}. Consequently, we have u(t, x) > 1 −ε for all (t, x) ∈[t∗, ∞) × −Rε −ct < x1 < −Rε + RJ (8.15) u(t, x) > 1 −ε 2 for all (t, x) ∈[t∗, ∞) × −Rε −ct 4 < x1 < −Rε + RJ . (8.1) Deﬁne M(J) := ˆ R J1(z1)\|z1\|e\|z1\|dz1. (8.16) (8.16) Moreover, set , α := σc 4 and κ∗:= 4ζ−1 c(1 −ε0) M(J) + c 2 + 3[f]C0,1(R) σ + ∥f∥L∞([0,1]) σ , where ζ := infz∈[−A,A] φ′(z) (remember that A is given by (8.8)) and σ is giv σ := min ( 1, \|f ′(0)\| 2M(J) + c, \|f ′(1)\| c , \|f ′(1)\|(1 −β0 2 ) 4M(J) ) , Recall that RJ is an arbitrary constant such that supp(J) ⊂[0, RJ], hence we have the freedom to chose RJ arbitrarily large. So even if it means increasing RJ (which is always possible and which does not impact the value of the constants involved in the deﬁnition of σ and ε0), we may always assume that Recall that RJ is an arbitrary constant such that supp(J) ⊂[0, RJ], hence we have the freedom to chose RJ arbitrarily large. So even if it means increasing RJ (which is always possible and which does not impact the value of the constants involved in the deﬁnition of σ and ε0), we may always assume that RJ ⩾1 σ log 1 ε0 . (8.17) RJ ⩾1 σ log 1 ε0 . (8.17) Furthermore, since K is compact, up to immaterial translations, we may assume, without loss of generality, that K ⊂{x1 ⩾−RJ}. Even if it means increasing Rε0 (and, therefore, increasing t∗), we may further assume that K ⊂{x1 ⩾−Rε0 + 2RJ} (notice that this does not aﬀect the validity of any of the previous estimates). Now, since u(t∗, x) > 0 and since limx1→∞u(t∗, x) = 1 (by Proposition 5.7), thanks to following asymptotic behaviour of φ, namely lim x1→−∞φ(x1 + ct∗−ρ) −2ε0e−αt∗= −2ε0e−αt∗< 0, lim x1→−∞φ(x1 + ct∗−ρ) −2ε0e−αt∗= −2ε0e−αt∗< 0, lim x1→+∞φ(x1 + ct∗−ρ) −2ε0e−αt∗= 1 −2ε0e−αt∗< 1, we may ﬁnd some ρσ > 0 such that u(t∗, x) ⩾φ(x1 + ct∗−ρσ) −2ε0e−αt∗> ewσ,ρσ(t∗, x) for all x ∈Ω. u(t∗, x) ⩾φ(x1 + ct∗−ρσ) −2ε0e−αt∗> ewσ,ρσ(t∗, x) for all x ∈Ω. u(t∗, x) ⩾φ(x1 + ct∗−ρσ) −2ε0e−αt∗> ewσ,ρσ(t∗, x) for all x ∈Ω. The goal is to show that this inequality remains true for all t ⩾t∗. Thanks to the uniform continuity of u and φ, there exists t0 > t∗such that The goal is to show that this inequality remains true for all t ⩾t∗. (8.1) (8.15) (t, x) := 1 −ε0eσ(x1+Rε0) φ x1 + ct −ρ −2ε0κ 1 −e−σct 4 −2ε0e−σct 4 , ewσ,ρ(t, x) := 1 −ε0eσ(x1+Rε0) φ x1 + ct −ρ −2ε0κ 1 −e−σct 4 −2ε0e−σct 4 , where Rε0 has the same meaning as Rε with ε0 instead of ε. We claim that Claim 8.8. There exists κ∗> 0, σ∗> 0 and ρσ∗> 0 such that u(t, x) ⩾ewσ∗,ρσ∗(t, x) for all (t, x) ∈[t∗, ∞) × Ω. Note that by proving Claim 8.8, we end the proof of Proposition 8.1(ii). To see this, ﬁx some λ ∈(0, 1). Set ϱ := (1 −λ)/2 and Rλ := φ−1(λ + ϱ). Also, let us set Γ1 := −Rε0 −1 σ∗ log ϱ 2ε0(λ + ϱ) and Γ2 := Rλ + ρσ∗+ 2ε0κ∗. Let t ⩾max{t∗, (Γ2 −Γ1)/c} and let x ∈ x ∈Ω; Γ1 ⩾x1 ⩾Γ2 −ct . Then, we have ewσ∗,ρσ∗(t, x) ⩾2λ + ϱ 2(λ + ϱ) φ(Rλ) −2ε0e−σ∗ct 4 = λ + ϱ 2 −2ε0e−σ∗ct 4 . If also t ⩾tλ := max{t∗, (Γ2 −Γ1)/c, 4(σ∗c)−1 log(4ε0/ϱ)}, then, by Claim 8.8, we have u(t, x) ⩾ewσ∗,ρσ∗(t, x) ⩾λ + ϱ 2 −2ε0e−σ∗ct 4 ⩾λ. Γ1 := −Rε0 −1 σ∗ log ϱ 2ε0(λ + ϱ) and Γ2 := Rλ + ρσ∗+ 2ε0κ∗. ( ϱ) If also t ⩾tλ := max{t∗, (Γ2 −Γ1)/c, 4(σ∗c)−1 log(4ε0/ϱ)}, then, by Claim 8.8, we have u(t, x) ⩾ewσ∗ρσ (t, x) ⩾λ + ϱ 2 −2ε0e−σ∗ct 4 ⩾λ. If also t ⩾tλ := max{t∗, (Γ2 −Γ1)/c, 4(σ∗c)−1 log(4ε0/ϱ)}, then, by Claim 8.8, we have ϱ σ ct If also t ⩾tλ := max{t∗, (Γ2 −Γ1)/c, 4(σ∗c)−1 log(4ε0/ϱ)}, then, by Claim 8.8, we have If also t ⩾tλ := max{t∗, (Γ2 −Γ1)/c, 4(σ∗c)−1 log(4ε0/ϱ)}, then, by Claim 8.8, we have u(t, x) ⩾ewσ∗,ρσ∗(t, x) ⩾λ + ϱ 2 −2ε0e−σ∗ct 4 ⩾λ. Therefore, we obtain that x ∈Ω; Γ1 ⩾x1 ⩾Γ2 −ct ⊂Eλ(t) for all t ⩾t Therefore, we obtain that x ∈Ω; Γ1 ⩾x1 ⩾Γ2 −ct ⊂Eλ(t) for all t ⩾tλ. Therefore, we obtain that x ∈Ω; Γ1 ⩾x1 ⩾Γ2 −ct ⊂Eλ(t) for all t ⩾tλ. □ □ To complete the proof of Proposition 8.1(ii), it remains to establish Claim 8.8. To complete the proof of Proposition 8.1(ii), it remains to establish Claim 8.8. JULIEN BRASSEUR AND J´ERˆOME COVILLE 52 Proof of Claim 8.8. (8.1) Thanks to the uniform continuity of u and φ, there exists t0 > t∗such that u(t, x) > ewσ,ρσ(t, x) for all (t, x) ∈[t∗, t0] × Ω. Observe also that u(t, x) > 0 ⩾ewσ,ρσ(t, x) for all (t, x) ∈[t∗, ∞) × x1 ⩾−Rε0 + 1 σ log 1 ε0 . (8.18) (8.18) Now, let us deﬁne the following set Now, let us deﬁne the following set E := n t ⩾t∗such that u(t, x) < ewσ,ρσ(t, x) for some x ∈Ω o . E := n t ⩾t∗such that u(t, x) < ewσ,ρσ(t, x) for some x ∈Ω o . If we can prove that E = ∅, then Claim 8.8 will automatically follow. So let us assume, by contradiction, that E ̸= ∅and set t := inf E < +∞. Readily, we observe that t ⩾t0 > t∗. Next, we claim that If we can prove that E = ∅, then Claim 8.8 will automatically follow. So let us assume, by contradiction, that E ̸= ∅and set t := inf E < +∞. Readily, we observe that t ⩾t0 > t∗. l h If we can prove that E = ∅, then Claim 8.8 will automatically follow. So let us assume, by contradiction, that E ̸= ∅and set t := inf E < +∞. Readily, we observe that t ⩾t0 > t∗. Next, we claim that Claim 8.9. There exists a point x ∈Ωsuch that u(t, x) = ewσ,ρσ(t, x). Claim 8.9. There exists a point x ∈Ωsuch that u(t, x) = ewσ,ρσ(t, x). PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 53 Proof. Let (tn)n∈N ⊂E be such that tn →t as n →∞. Then, for all n ∈N, there is some point xn ∈Ωsuch that u(tn, xn) ⩽ewσ,ρn(tn, xn). We claim that the sequence (xn)n∈N stay in a compact set. If not, then \|xn\| →∞as n →∞. Since lim x1→−∞ewσ,ρσ(t, x) = −2ε0e−αt < 0, and since (by (8.18)) we have and since (by (8.18)) we have u(t, x) > ewσ,ρσ(t, x) for all (t, x) ∈[t∗, ∞) × x1 ⩾−Rε0 + 1 σ log 1 ε0 , we must have that −R′ ⩽x1,n < −Rε0 + σ−1 log(1/ε0) for some R′ > 0. Hence, we must have \|x′ n\| →∞. (8.1) However, by Proposition 6.4, we have \|u(tn, x1,n, x′ n) −φ(x1,n + ctn)\| →0 as b t φ(x1,n + ctn) > 2ε0e−αtn + ewσ,ρσ(tn, xn), and so we have lim sup n→∞ u(tn, xn) ⩾2ε0e−αt + lim sup n→∞ ewσ,ρσ(tn, xn) > lim sup n→∞ ewσ,ρσ(tn, xn), ontradiction. Therefore, (xn)n∈N cannot be unbounded and stays in a compac As a consequence, we may ﬁnd a subsequence, still denoted by xn, which converges towards some x ∈Ω. By (8.18), we deduce that x1 < −Rε0 + σ−1 log(1/ε0) ⩽−Rε0 + RJ. In addition, we have u(t, x) ⩽ewσ,ρσ(t, x) and u(t, x) > ewσ,ρσ(t, x) for all t < t and all x ∈Ω(since otherwise this would contradict the deﬁnition of t). In fact, the latter implies that u(t, x) = ewσ,ρσ(t, x) (because u and ewσ,ρσ are both continuous). □ By deﬁnition of t and x, the function z(t, x) := u(t, x) −wσ,ρσ(t, x) ⩾0 achieves its global minimum at x. Hence, LRNz(t, x) > 0, z(t, x) = 0 and ∂tz(t, x) ⩽0. (Remember that K ⊂{x1 ⩾−Rε0 +2RJ} and that x1 < −Rε0 +RJ.) Thus, we deduce that ∂t ewσ,ρσ(t, x) −LRN ewσ,ρσ(t, x) −f( ewσ,ρσ(t, x)) > ∂tu(t, x) −LRNu(t, x) −f(u(t, x)) = 0. The whole game is now to obtain a contradiction with this by showing that I(t, x) := ∂t ewσ,ρσ(t, x) −LRN ewσ,ρσ(t, x) −f( ewσ,ρσ(t, x)) < 0. (8.19) Set η(x1) := 1 −ε0eσ(x1+Rε0) and ξ(t) := ct −ρ −2ε0κ∗(1 −e−σct 4 ). A short computation (using the equation satisﬁed by φ) shows that (8.19) I(t, x) = ˆ R J1(x1 −y1)φ(y1 + ξ(t))(η(x1) −η(y1))dy1 + 2ε0αe−αt + η(x1)f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt −2ε0ακ∗e−αtφ′(x1 + ξ(t))η(x1). Using the deﬁnition of η, we have Using the deﬁnition of η, we have sing the deﬁnition of η, we have sing the deﬁnition of η, we have ˆ R J1(x1 −y1)φ(y1 + ξ(t))(η(x1) −η(y1))dy1 ˆ ˆ R J1(x1 −y1)φ(y1 + ξ(t))(η(x1) −η(y1))dy1 ⩽ε0eσ(x1+Rε0) ˆ R J1(z1)φ(x1 + z1 + ξ(t))(eσ\|z1\| −1)dz1. )(η( 1) η(y1)) y1 ⩽ε0eσ(x1+Rε0) ˆ R J1(z1)φ(x1 + z1 + ξ(t))(eσ\|z1\| −1)dz1. JULIEN BRASSEUR AND J´ERˆOME COVILLE 54 Using that eX −1 ⩽XeX for all X ⩾0, together with σ ⩽1 and 0 < φ < 1 we obtain ˆ R J1(x1 −y1)φ(y1 + ξ(t))(η(x1) −η(y1))dy1 ⩽σε0M(J)eσ(x1+Rε0), where MJ > 0 is as in (8.16). (8.1) Thus, we have that where MJ > 0 is as in (8.16). Thus, we have that I(t, x) ⩽σε0M(J)eσ(x1+Rε0) + 2ε0αe−αt −2ε0ακ∗e−αtφ′(x1 + ξ(t))η(x1) + η(x1)f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt . (8.20) I(t, x) ⩽σε0M(J)eσ(x1+Rε0) + 2ε0αe−αt −2ε0ακ∗e−αtφ′(x1 + ξ ( , ) ⩽ ε0 (J) + ε0 ε0 ∗ φ ( 1 + ξ( ) + η(x1)f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−α (8.20) (8.20) We now distinguish between three diﬀerent cases. We now distinguish between three diﬀerent cases. We now distinguish between three diﬀerent cases. Case 1: x1 + ξ(t) < −A. Case 1: x1 + ξ(t) < −A. By deﬁnition of A, β0 and ε0, we have φ(x1 + ξ(t)) < φ(−A) ⩽β0 2 < 1 −β0 2 < 1 −ε0. Therefore, we have that ewσ,ρσ(t, x) < 1 −ε0. Thanks to (8.15), (8.17), (8.18) and the deﬁnition of σ, we have x1 ⩽−Rε0 −ct/4. This, in turn, implies that Therefore, we have that ewσ,ρσ(t, x) < 1 −ε0. Thanks to (8.15), (8.17), (8.18) and the deﬁnition of σ, we have x1 ⩽−Rε0 −ct/4. This, in turn, implies that eσ(x1+Rε0) ⩽e−σct/4 = e−αt. (8.21) eσ(x1+Rε0) ⩽e−σct/4 = e−αt. (8.21) (8.21) Combining (8.21) with (8.20), using that η(x1) > 0, that α = σc/4 and that Combining (8.21) with (8.20), using that η(x1) > 0, that α = σc/4 and that φ′ > 0, we get I(t, x) ⩽σε0 M(J) + c 2 e−αt + η(x1)f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt . (8.22) I(t, x) ⩽σε0 M(J) + c 2 e−αt + η(x1)f(φ(x1 + ξ(t))) I(t, x) ⩽σε0 M(J) + c 2 e−αt + η(x1)f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt . −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt . (8.22) (8.22) Let us rewrite the last two terms in the right-hand side as follows η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt = f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt + (η(x1) −1)f(φ(x1 + ξ(t))). Since φ(x1 + ξ(t)) ⩽β0/2 and since x1 ⩽−Rε0 −ct/4, we have f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽ε0 3f ′(0) 4 eσ(x1+Rε0)φ(x1 + ξ(t)) + 2e−αt , η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt = f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt + (η(x1) −1)f(φ(x1 + ξ(t))). (8.1) Therefore, E = ∅and so ewσ,ρσ(t, x) ⩽u(t, x) for all (t, x) ∈[t∗, ∞) × Ω, as desired. The proof is thereby complete. □ (8.1) = f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt + (η(x1) −1)f(φ(x1 + ξ(t))). Since φ(x1 + ξ(t)) ⩽β0/2 and since x1 ⩽−Rε0 −ct/4, we have f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽ε0 3f ′(0) 4 eσ(x1+Rε0)φ(x1 + ξ(t) Since φ(x1 + ξ(t)) ⩽β0/2 and since x1 ⩽−Rε0 −ct/4, we have f(φ(x + ξ(t))) f η(x )φ(x + ξ(t)) 2ε e−αt Since φ(x1 + ξ(t)) ⩽β0/2 and since x1 ⩽−Rε0 −ct/4, we have ⩽ε0 3f ′(0) 4 eσ(x1+Rε0)φ(x1 + ξ(t)) + 2e−αt , ⩽ε0 3f ′(0) 4 eσ(x1+Rε0)φ(x1 + ξ(t)) + 2e−αt , In addition, by (8.7), we have In addition, by (8.7), we have (η(x1)−1)f(φ(x1+ξ(t))) = −ε0eσ(x1+Rε0)f(φ(x1+ξ(t))) ⩽−ε0 5f ′(0) 4 eσ(x1+Rε0)φ(x1+ξ(t)). (η(x1)−1)f(φ(x1+ξ(t))) = −ε0eσ(x1+Rε0)f(φ(x1+ξ(t))) ⩽−ε0 5f ′(0) 4 eσ(x1+Rε0)φ(x1+ξ(t)). Hence, recalling (8.21) and using that φ ⩽1, we ﬁnd that η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0 Hence, recalling (8.21) and using that φ ⩽1, we ﬁnd that Hence, recalling (8.21) and using that φ ⩽1, we ﬁnd that η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽ε0f ′(0)e−αt. nce, recalling (8.21) and using that φ ⩽1, we ﬁnd that η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽ε0f ′(0) Therefore, recalling (8.22) and the deﬁnition of σ, we have Therefore, recalling (8.22) and the deﬁnition of σ, we have I(t, x) ⩽ε0 h σ M(J) + c 2 + f ′(0) i e−αt < 0. (8.23) (8.23) Case 2: −A ⩽x1 + ξ(t) ⩽A. PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 55 In this case, we have φ(x1 + ξ(t)) < 1 −β0/2 < 1 −ε0. Hence, the estimate (8.21) remains true. Moreover, φ′(x1+ξ(t)) ⩾infz∈[−A,A] φ′(z) =: ζ > 0 and, since x1 < −Rε0−ct/4 < −Rε0, we also have η(x1) ⩾1 −ε0. Hence, using (8.20), we obtain that In this case, we have φ(x1 + ξ(t)) < 1 −β0/2 < 1 −ε0. Hence, the estimate (8.21) remains true. Moreover, φ′(x1+ξ(t)) ⩾infz∈[−A,A] φ′(z) =: ζ > 0 and, since x1 < −Rε0−ct/4 < −Rε0, we also have η(x1) ⩾1 −ε0. Hence, using (8.20), we obtain that I(t, x) ⩽σε0 M(J) + c 2 −c 2(1 −ε0)ζ κ∗ e−αt + η(x1)f(φ(x1 + ξ(t))) −f η(x1)φ(x1 I(t, x) ⩽σε0 M(J) + c 2 −c 2(1 −ε0)ζ κ∗ e−αt + η(x1)f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt . (8.1) Using that f ∈C0,1(R), we further have Using that f ∈C , (R), we further have η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽ε0[f]C0,1(R) 2e−αt + eσ(x1+Rε0)φ(x1 + ξ(t)) −ε0e−σ(x1+Rε0)f(φ(x1 + ξ(t))) ⩽ε0e−αt 3[f]C0,1(R) + ∥f∥L∞([0,1]) . η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽ε0[f]C0,1(R) 2e−αt + eσ(x1+Rε0)φ(x1 + ξ(t)) −ε0e−σ(x1+Rε0)f(φ(x1 + ξ(t))) ⩽ε0e−αt 3[f]C0,1(R) + ∥f∥L∞([0,1]) . Therefore, recalling the deﬁnition of κ∗, we have Therefore, recalling the deﬁnition of κ∗, we have I(t, x) ⩽σε0 M(J) + c 2 −c 2(1 −ε0)ζ κ∗+ 3[f]C0,1(R) σ + ∥f∥L∞([0,1]) σ e−αt < 0. (8.24) Case 3: x1 + ξ(t) > A. ξ( ) In this case, we have φ(x1 +ξ(t)) ⩾1−β0/2 > θ, so that f(φ(x1 +ξ(t))) ⩾0. Thus, using the fact that η(x1) ⩽1 and the deﬁnition of ε0, we ﬁnd that In this case, we have φ(x1 +ξ(t)) ⩾1−β0/2 > θ, so that f(φ(x1 +ξ(t))) ⩾0. Thus, using the fact that η(x1) ⩽1 and the deﬁnition of ε0, we ﬁnd that η(x1)f(φ(x1 + ξ(t)))−f η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽f(φ(x1 + ξ(t))) −f η(x1)φ(x1 + ξ(t)) −2ε0e−αt ⩽ε0 f ′(1) 2 e−σ(x1+Rε0) 1 −β0 2 + 2e−αt . Plugging this in (8.20), using that φ′ > 0 and recalling the deﬁnition of σ, we obtain that f ′(1) β f ′(1) Plugging this in (8.20), using that φ′ > 0 and recalling the deﬁnition of σ, we obtain that Plugging this in (8.20), using that φ > 0 and recalling the deﬁnition of σ, we obtain that I(t, x) ⩽ε0e−σ(x1+Rε0) σM(J) + f ′(1) 2 1 −β0 2 + 2ε0e−αt σc 4 + f ′(1) 2 < 0. (8.25) I(t, x) ⩽ε0e−σ(x1+Rε0) σM(J) + f ′(1) 2 1 −β0 2 + 2ε0e−αt σc 4 + (8.25) I(t, x) ⩽ε0e−σ(x1+Rε0) σM(J) + f ′(1) 2 1 −β0 2 + 2ε0e−αt σc 4 + f ′(1) 2 < 0. (8.25) Collecting (8.23), (8.24) and (8.25) we obtain that (8.19) holds, which is the desired contradiction. Therefore, E = ∅and so ewσ,ρσ(t, x) ⩽u(t, x) for all (t, x) ∈[t∗, ∞) × Ω, as desired. The proof is thereby complete. □ Collecting (8.23), (8.24) and (8.25) we obtain that (8.19) holds, which is the desired contradiction. 9. The entire solution u(t, x) is a generalised transition wave This last section is devoted to the proof of Theorem 2.10. Namely, we will prove that u(t, x) is a generalised transition almost-planar invasion front between 0 and u∞with global mean speed c. That is, we will prove that sup (t,x)∈R×Ω, x1+ct⩾A \|u(t, x) −u∞(x)\| −→ A→∞0, (9.1) and that sup (t,x)∈R×Ω, x1+ct⩽−A u(t, x) −→ A→∞0. JULIEN BRASSEUR AND J´ERˆOME COVILLE 56 We will adapt to our situation the arguments developed in [12] using the characterisation of the large time behaviour of the super-level sets of u(t, x) derived at the previous section. For the convenience of the reader, we prove separately (9.1) and (9.2). We will adapt to our situation the arguments developed in [12] using the characterisation of the large time behaviour of the super-level sets of u(t, x) derived at the previous section. For the convenience of the reader, we prove separately (9.1) and (9.2). Proof of (9.1). Assume, by contradiction, that (9.1) does not hold. Then, there exists ε > 0 and a sequence (tn, xn)n∈N such that x1,n + ctn →∞as n →∞and u∞(xn) −u(tn, xn) > ε for all n ∈N. Up to extraction of a subsequence, two cases may occur: either \|xn\| →∞or xn →x. In the latter case, we must have that tn →∞and, since u(t, x) →u∞(x) locally uniformly in Ωas t →∞, we deduce that Up to extraction of a subsequence, two cases may occur: either \|xn\| →∞or xn →x. In the latter case, we must have that tn →∞and, since u(t, x) →u∞(x) locally uniformly in Ωas t →∞, we deduce that 0 < ε ⩽lim n→∞(u∞(xn) −u(tn, xn)) = 0, a contradiction. In the former case, we have \|xn\| →∞and so u∞(xn) →1 as n →∞. Therefore, up to extraction of a subsequence, we have a contradiction. In the former case, we have \|xn\| →∞and so u∞(xn) →1 as n →∞. Therefore, up to extraction of a subsequence, we have a contradiction. In the former case, we have \|xn\| →∞ Therefore, up to extraction of a subsequence, we have 1 −u(tn, xn) > ε for all n ∈N. (9.3) 1 −u(tn, xn) > ε for all n ∈N. At this stage, up to extraction of a subsequence, three diﬀerent subcases may occur. At this stage, up to extraction of a subsequence, three diﬀerent subcases may occur. Subcase 1: tn →−∞. Subcase 1: tn →−∞. 9. The entire solution u(t, x) is a generalised transition wave Since \|u(t, x)−φ(x1+ct)\| →0 uniformly in x ∈Ωas t →−∞, we have u(tn, xn)−φ(x1,n+ ctn) →0 as n →∞. But since x1,n + ctn →∞, we must have that φ(x1,n + ctn) →1 which, in turn, implies that u(tn, xn) →1 as n →∞, contradicting (9.3). Subcase 2: tn →t. Subcase 2: tn →t. Since x1,n + ctn →∞, we must have x1,n →∞. Since \|u(t, x) −φ(x1 + ct)\| →0 uniformly in x ∈Ωas t →−∞, we may then ﬁnd some T ≪−1 such that Since x1,n + ctn →∞, we must have x1,n →∞. Since \|u(t, x) −φ(x1 + ct)\| →0 uniformly in x ∈Ωas t →−∞, we may then ﬁnd some T ≪−1 such that Since x1,n + ctn →∞, we must have x1,n →∞. Since \|u(t, x) −φ(x1 + ct)\| →0 uniformly in x ∈Ωas t →−∞, we may then ﬁnd some T ≪−1 such that sup x∈Ω \|u(T, x) −φ(x1 + cT)\| ⩽ε 2. sup ∈Ω \|u(T, x) −φ(x1 + cT)\| ⩽ε 2. Otherwise said, we have that u(T, x) ⩾φ(x1 + cT) −ε/2 for all x ∈Ω. Up to decrease further T, we may assume that T < t. Now, using that ∂tu > 0, we have u(t, x) ⩾u(T, x) ⩾ φ(x1 + cT) −ε/2 for all t ⩾T and all x ∈Ω. Thus, recalling (9.3), we obtain that 1 −ε > lim sup n→∞ u(tn, xn) ⩾lim sup n→∞ φ(x1,n + cT) −ε 2 = 1 −ε 2, 1 −ε > lim sup n→∞ u(tn, xn) ⩾lim sup n→∞ φ(x1,n + cT) −ε 2 = 1 −ε 2, a contradiction. Subcase 3: tn →∞. Subcase 1: tn →−∞. Subcase 1: tn →−∞. Since \|u(t, x)−φ(x1+ct)\| →0 uniformly in x ∈Ωas t →−∞, we have \|u(tn, xn)−φ(x1,n+ ctn)\| →0 as n →∞. But since x1,n + ctn →−∞as n →∞, we have φ(x1,n + ctn) →0 as n →∞which, in turn, implies that u(tn, xn) →0 as n →∞, contradicting (9.4). Subcase 2: tn →t. Subcase 2: tn →t. Subcase 2: tn →t. Since x1,n + ctn →−∞as n →∞, we must have x1,n →−∞. By Proposition 5.7, we further have that limx1→−∞u(t + 1, x) = 0. Hence, there exists R0 > 0 such that u(t + 1, x) ⩽ε 2 for all x ∈{x1 ⩽R0}. u(t + 1, x) ⩽ε 2 for all x ∈{x1 ⩽R0}. But since ∂tu > 0 and since x1,n →−∞, we infer that ε ⩽lim sup n→∞ u(tn, xn) ⩽lim n→∞u(t + 1, xn) ⩽ε 2, a contradiction. Subcase 3: tn →∞. Subcase 3: tn →∞. By (8.6), there exists α, β, ρ > 0 such that By (8.6), there exists α, β, ρ > 0 such that u(t, x) ⩽φ(x1 + ct + ρ) + β e−αt for all (t, x) ∈[0, ∞) × Ω. Since x1,n + ctn →−∞and since tn →∞, we obtain that ε ⩽lim sup n→∞ u(tn, xn) ⩽lim n→∞ φ(x1,n + ctn + ρ) + β e−αtn ⩽ε 2, u(t, x) ⩽φ(x1 + ct + ρ) + β e−αt for all (t, x) ∈[0, ∞) Since x1,n + ctn →−∞and since tn →∞, we obtain that Since x1,n + ctn →−∞and since tn →∞, we obtain that ε ⩽lim sup n→∞ u(tn, xn) ⩽lim n→∞ φ(x1,n + ctn + ρ) + β e−αtn ⩽ε 2, a contradiction. Hence, this case is ruled out too. The proof of (9.2) is thereby complete. □ a contradiction. Hence, this case is ruled out too. The proof of (9.2) is thereby complete. □ Subcase 3: tn →∞. Up to extraction of a subsequence, three situations may occur: either x1,n →−∞, or x1,n →∞or x1,n →x. If x1,n →∞, we readily get a contradiction (by repeating the arguments of Subcase 2), so this situation is ruled out. Next, if x1,n →x, then, since \|xn\| →∞, we must have that \|x′ n\| →∞. Hence, by Proposition 6.4, we have lim inf n→∞u(T, xn) = φ(x1 + cT) for all T ∈R. lim inf n→∞u(T, xn) = φ(x1 + cT) for all T ∈R. Choose T > 0 large enough so that φ(x1 + cT) ⩾1 −ε/2. Since tn ⩾T for n large enough and since ∂tu > 0, we then have Choose T > 0 large enough so that φ(x1 + cT) ⩾1 −ε/2. Since tn ⩾T for n large enough and since ∂tu > 0, we then have ε ⩽lim sup n→∞ (1 −u(tn, xn)) ⩽lim sup n→∞ (1 −u(T, xn)) ⩽1 −φ(x1 + cT) ⩽ε 2, ε ⩽lim sup n→∞ (1 −u(tn, xn)) ⩽lim sup n→∞ (1 −u(T, xn)) ⩽1 −φ(x1 + cT) ⩽ε 2, ε ⩽lim sup n→∞ (1 −u(tn, xn)) ⩽lim sup n→∞ (1 −u(T, xn)) ⩽1 −φ(x1 + cT) ⩽ε 2, 57 PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION which, again, yields a contradiction. Lastly, let us consider the case when x1,n →−∞. Since tn →∞, x1,n + ctn →∞and x1,n →−∞as n →∞, it follows immediately from Claim 8.8 and (9.3) that, for n large enough, there holds which, again, yields a contradiction. Lastly, let us consider the case when x1,n →−∞. Since tn →∞, x1,n + ctn →∞and x1,n →−∞as n →∞, it follows immediately from Claim 8.8 and (9.3) that, for n large enough, there holds 1 −ε > u(tn, xn) ⩾1 −ε 2, 1 −ε > u(tn, xn) ⩾1 −ε 2, 1 −ε > u(tn, xn) ⩾1 −ε 2, a contradiction. Hence, this case is ruled out too. The proof of (9.1) is thereby complete. □ Finally, let us now prove (9.2). a contradiction. Hence, this case is ruled out too. The proof of (9.1) is thereby complete. □ Finally, let us now prove (9.2). Finally, let us now prove (9.2). Proof of (9.2). Subcase 3: tn →∞. As above, we argue by contradiction and we assume that there exist some ε > 0 and a sequence (tn, xn)n∈N such that x1,n + ctn →−∞as n →∞and (t ) > f ll ∈N (9 4) (9.4) u(tn, xn) > ε for all n ∈N. u(tn, xn) > ε for all n ∈N. 9.4) u(tn, xn) > ε for all n ∈N. u(tn, xn) > ε for all n ∈N. Up to extraction, three situations may occur. Up to extraction, three situations may occur. Up to extraction, three situations may occur. Up to extraction, three situations may occur. Up to extraction, three situations may occur. Subcase 1: tn →−∞. JULIEN BRASSEUR AND J´ERˆOME COVILLE JULIEN BRASSEUR AND J´ERˆOME COVILLE 58 ANR DEFI project NONLOCAL (ANR-14-CE25-0013) and the ANR JCJC project MOD- EVOL (ANR-13-JS01-0009). Acknowledgments The authors warmly thank Guillaume Legendre (CEREMADE, Universit´e Paris-Dauphine) for its great help in the implementation of the numerics involving the geodesic distance. This work has been carried out in the framework of the Archim`ede Labex (ANR-11-LABX- 0033) and of the A*MIDEX project (ANR-11-IDEX-0001-02), funded by the “Investisse- ments d’Avenir” French Government program managed by the French National Research Agency (ANR). The research leading to these results has also received funding from the Appendix A. The J-covering property Appendix A. The J-covering property In this Appendix, we list some additional results regarding the properties of quasi-Euclidean distances. Precisely, we prove the assertions made in Remark 1.7. Incidentally, this will jus- tify that the ﬁrst assumption in (1.6) is satisﬁed in a wide range of situations (and is, therefore, not an empty assumption). Firstly, we show that if δ is the Euclidean distance, then the J-covering property always holds. Proposition A.1. Let E ⊂RN be a connected set and let δ ∈Q(E) be the Euclidean distance. Let J : [0, ∞) →[0, ∞) be a measurable function with \|supp(J)\| > 0. Then, (E, δ) has the J-covering property. Proof. Let x0 ∈E. By deﬁnition of Π2(J, x0), we have Proof. Let x0 ∈E. By deﬁnition of Π2(J, x0), we have Π2(J, x0) = (x0 + supp(Jrad) + supp(Jrad)) ∩E. Let R > 0 be such that Λ := supp(Jrad) ∩BR has positive Lebesgue measure. Since the function G : RN →[0, ∞) given by G(x) := 1Λ ∗1Λ(x) is continuous and since, on the other hand, G(0) = \|Λ\| > 0, we deduce that there is some τ > 0 such that Bτ ⊂supp(G) ⊂Λ + Λ ⊂supp(Jrad) + supp(Jrad). Bτ ⊂supp(G) ⊂Λ + Λ ⊂supp(Jrad) + supp(Jrad). Then, we make some preliminary geometric observations and, ﬁnally, we complete the proof by estimating the sets Πj(J, ·). Step 1. Some preparatory deﬁnitions Prior to proving Proposition A.2, we will need to introduce a few deﬁnitions and notations. For any x ∈Ω, we deﬁne eΠ1(x, r1, r2) := {x} and, for j ⩾0, we set ﬁne eΠ1(x, r1, r2) := {x} and, for j ⩾0, we set eΠj+1(x, r1, r2) := [ z∈eΠj(x,r1,r2) supp 1[r1,r2](δ(·, z)) . Clearly, eΠj(x, r1, r2) ⊂Πj(J, x) for all j ⩾1. Also, for all x ∈Ω, we set star(x) := y ∈Ωs.t. [x, y] ⊂Ω . Clearly, eΠj(x, r1, r2) ⊂Πj(J, x) for all j ⩾1. Also, for all x ∈Ω, we set star(x) := y ∈Ωs.t. [x, y] ⊂Ω . Roughly speaking, star(x) is the set of all points which are reachable from x without “jump- ing” through K. By deﬁnition, it is the largest subset of Ωwhich is starshaped with respect to x. In addition, for any x ∈Ω, we let C (x) be the closed cone with vertex x whose bound- ary ∂(C (x)) is tangent to ∂K. Notice that, since K is a compact convex set, C (x) is always well-deﬁned and we have K ⊂C (x) for any x ∈Ω. For later purposes, it will be useful to denote by C +(x) := C (x) ∩star(x) the upper part of the cone C (x). Step 2. Preliminary geometric observations First of all, we notice that, since [r1, r2] ⊂supp(J), we also have [r1, er2] ⊂supp(J) for any er2 ∈(r1, r2). Hence, up to replace r2 by some er2 ∈(r1, r2) arbitrarily close to r1, we have the freedom to choose κ := r1 −r2 arbitrarily small (A.1) Bτ ⊂supp(G) ⊂Λ + Λ ⊂supp(Jrad) + supp(Jrad). Bτ ⊂supp(G) ⊂Λ + Λ ⊂supp(Jrad) + supp(Jrad). Hence, Bτ(x0) ∩E ⊂Π2(J, x0). Since x0 ∈E was chosen arbitrarily, we may apply the same reasoning to any boundary point z0 of Bτ(x0) ∩E and we have Bτ(z0) ∩E ⊂Π2(J, z0). But since z0 ∈Π2(J, x0), we have Π2(J, z0) ⊂Π4(J, z0) and so Bδ(z0) ∩E ⊂Π4(J, x0). This being true for any boundary point of Bτ(x0) ∩E, we then obtain that B2τ(x0) ∩E ⊂ Π2(J, x0) ∪Π4(J, x0). By induction, we ﬁnd that Bτk(x0) ∩E ⊂ k[ j=1 Π2j(J, x0) for all k ∈N∗. Bτk(x0) ∩E ⊂ k[ j=1 Π2j(J, x0) for all k ∈N∗. In turn, this implies that the following chain of inclusions hold: In turn, this implies that the following chain of inclusions hold E = [ k⩾0 Bτk(x0) ∩E ⊂ [ k⩾0 Π2k(J, x0) ⊂ [ j⩾0 Πj(J, x0) ⊂E. □ Therefore, (Ω, δ) has the J-covering property. Lastly, we prove that (Ω, δ) has the J-covering property for all δ ∈Q(Ω), whenever Ω is the complement of a compact convex set with C2 boundary and J satisﬁes some mild additional assumptions. Proposition A.2. Let K ⊂RN be a compact convex set with nonempty interior and C2 boundary, let Ω:= RN \ K and let δ ∈Q(Ω). Suppose that J : [0, ∞) →[0, ∞) is such that [r1, r2] ⊂supp(J) for some 0 ⩽r1 < r2. Then, (Ω, δ) has the J-covering property. Proposition A.2. Let K ⊂RN be a compact convex set with nonempty interior and C2 boundary, let Ω:= RN \ K and let δ ∈Q(Ω). Suppose that J : [0, ∞) →[0, ∞) is such that [r1, r2] ⊂supp(J) for some 0 ⩽r1 < r2. Then, (Ω, δ) has the J-covering property. Proof. The proof follows roughly the same structure as the one of Proposition A.1. However, it is slightly more involved due to the presence of an arbitrary quasi-Euclidean distance, which forces us to “secure” starshaped regions in which it behaves like the Euclidean distance. To keep the proof as clear as possible, we split it into three main steps. First, we introduce some PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 59 useful notations and terminology. Then, we make some preliminary geometric observations and, ﬁnally, we complete the proof by estimating the sets Πj(J, ·). useful notations and terminology. (A.1) we have the freedom to choose κ := r1 −r2 arbitrarily small. (A.1) Let m ∈∂K be arbitrary and let Rmin := (max∂K γ)−1 where γ is the maximum principal curvature of ∂K. Since, by deﬁnition, Rmin is the minimum of the radii of curvature of ∂K, there is then an osculating open ball B with radius Rmin such that ∂B ∩∂K = {m} and that B ⊂int(K). Although this is classical, we recall that max∂K γ > 0 (since K is a compact convex set) and that max∂K γ < ∞(since the Weingarten map is bounded, as follows from the fact that K has C2 boundary), so that Rmin and B are well-deﬁned. Now, let p := m + κν(m), where ν(m) is the outward unit normal to ∂K at m. Then, the ball Bκ(p) is tangent to ∂K at p, satisﬁes Bκ(p) ∩K = {m} and Bκ(p) ⊂Ω(remember that K is convex). Let q := m + r1ν(m) and let C +(q) be the upper part of C (q). Also, let CB(q) be the closed cone with vertex q and tangent to B and let C + B (q) := CB(q) ∩star(q) be its upper part. Clearly, CB(q) ⊂C (q) and C + B (q) ⊂C +(q). ( ) ( ) B ( ) ( ) Now, by Thales’ theorem, up to choose κ small (remember (A.1)), say if 0 < κ < min r1 3 , r1Rmin 2Rmin + r1 , we may assume that Bκ(p) ⊂C + B (q) (regardless of the choice of m). Since p = m + κν(m) and since the orthogonal cross section of the cone CB(q) is increasing in the direction −ν(m) (in the sense of the inclusion), we also have we may assume that Bκ(p) ⊂C + B (q) (regardless of the choice of m). Since p = m + κν(m) and since the orthogonal cross section of the cone CB(q) is increasing in the direction −ν(m) (in the sense of the inclusion), we also have Bκ(m + ℓν(m)) ∩Ω⊂C + B (q) ⊂C +(q) for all ℓ∈[0, κ], (A.2) (A.2) see Figure 7 for a visual evidence. Moreover, since ℓ+ r1 ⩾r1 for all ℓ∈[0, κ], we have the see Figure 7 for a visual evidence. Moreover, since ℓ+ r1 ⩾r1 for all ℓ∈[0, κ], we have the JULIEN BRASSEUR AND J´ERˆOME COVILLE 60 60 Figure 7. (A.1) Illustration of the balls B and Bκ(p) and the cones C (q) and CB(q), when K is an ellipse. The upper cone C +(q) (resp. C + B (q)) correspond to the region of the cone C (q) (resp. CB(q)) which lie above K. The translates of the ball Bκ(p) appearing in (A.2) are represented in thin dashed lines. Figure 7. Illustration of the balls B and Bκ(p) and the cones C (q) and CB(q), when K is an ellipse. The upper cone C +(q) (resp. C + B (q)) correspond to the region of the cone C (q) (resp. CB(q)) which lie above K. The translates of the ball Bκ(p) appearing in (A.2) are represented in thin dashed lines. straightforward inclusion straightforward inclusion C +(q) = C +(m + r1ν(m)) ⊂C +(m + (ℓ+ r1)ν(m)). Recalling (A.2), we obtain that Recalling (A.2), we obtain that Bκ(m + ℓν(m)) ∩Ω⊂C +(m + (ℓ+ r1)ν(m)) for all ℓ∈[0, κ]. Since C +(m + (ℓ+ r1)ν(m)) ⊂star(m + (ℓ+ r1)ν(m)) (by deﬁnition), it follows that Bκ(m + ℓν(m)) ∩Ω⊂star(m + (ℓ+ r1)ν(m)) for all ℓ∈[0, κ], (A.3) (A.3) and all m ∈∂K. Now that we have (A.3), we are in position to complete th and all m ∈∂K. Now that we have (A.3), we are in position to complete the proof. Step 3. Estimates for eΠj(·, r1, r2) and conclusion Now, let us ﬁx an arbitrary point x0 ∈Ω. Since δ ∈Q(Ω), we have that δ(x0, y) = \|x0 −y\| for every y ∈star(x0). In particular, star(x0) ∩A(x0, r1, r2) ⊂eΠ1(x0, r1, r2). (A.4) (A.4) Since RN \ C (x0) is starshaped with respect to x0 and since (RN \ C (x0)) ∩K = ∅, we have RN \ C (x0) ⊂star(x0). (A.5) Since RN \ C (x0) is starshaped with respect to x0 and since (RN \ C (x0)) ∩K = ∅, we have RN \ C (x0) ⊂star(x0). (A.5) RN \ C (x0) ⊂star(x0). (A.5) Now, let S(x0) be the set of all e ∈SN−1 such that x0 + et ∈RN \ C (x0) for all t ⩾0 (note that S(x0) is well-deﬁned because RN \ C (x0) is also a cone). Since K is convex, it follows that C (x0) has a maximum opening angle less than π. In particular, the cone RN \ C (x0) has a minimum opening angle greater than π. Hence, S(x0) contains a half-sphere. (A.1) Since δ(x⊥ 0 , y) = \|x⊥ 0 −y\| for all y ∈star(x⊥ 0 ), this then implies that Bκ(x0) ∩Ω\ star(x0) ⊂eΠ1(x⊥ 0 , r1, r2) ⊂eΠ2(x0, r1, r2), Since δ(x⊥ 0 , y) = \|x⊥ 0 −y\| for all y ∈star(x⊥ 0 ), this then implies that e e ce δ(x⊥ 0 , y) = \|x⊥ 0 −y\| for all y ∈star(x⊥ 0 ), this then implies that where, in the last inclusion, we have used that x⊥ 0 ∈eΠ1(x0, r1, r2). Together with (A.6), this yields that Bκ(x0) \ Ω⊂eΠ2(x0, r1, r2). At this stage, we may conclude exactly as in the proof of Proposition A.1 (remember that eΠj(x, r1, r2) ⊂Πj(J, x) for all x ∈Ω) and we therefore obtain that (Ω, δ) has the J-covering property, as desired. □ where, in the last inclusion, we have used that x⊥ 0 ∈eΠ1(x0, r1, r2). Together with (A.6), this yields that Bκ(x0) \ Ω⊂eΠ2(x0, r1, r2). At this stage, we may conclude exactly as in the proof of Proposition A.1 (remember that eΠj(x, r1, r2) ⊂Πj(J, x) for all x ∈Ω) and we therefore obtain that (Ω, δ) has the J-covering property, as desired. □ where, in the last inclusion, we have used that x⊥ 0 ∈eΠ1(x0, r1, r2). Together with (A.6), this yields that Bκ(x0) \ Ω⊂eΠ2(x0, r1, r2). At this stage, we may conclude exactly as in the proof of Proposition A.1 (remember that eΠj(x, r1, r2) ⊂Πj(J, x) for all x ∈Ω) and we therefore obtain that (Ω, δ) has the J-covering property, as desired. □ (A.1) Now, let S(x0) be the set of all e ∈SN−1 such that x0 + et ∈RN \ C (x0) for all t ⩾0 (note that S(x0) is well-deﬁned because RN \ C (x0) is also a cone). Since K is convex, it follows that C (x0) has a maximum opening angle less than π. In particular, the cone RN \ C (x0) has a minimum opening angle greater than π. Hence, S(x0) contains a half-sphere. ( ) Let e ∈S(x0) and let q ∈[x0 −κe, x0 + κe] ∩star(x0) be arbitrary. Then, there exist t, τ ∈[r1, r2] such that q = x0 + (t −τ)e. Hence, letting p := x0 + et, we have p ∈A(x0, r1, r2) \ C (x0), p −τ e = x0 + (t −τ)e = q and \|p −q\| ∈[r1, r2 PROPAGATION PHENOMENA WITH NONLOCAL DIFFUSION 61 Recalling (A.4) and (A.5), we have that p ∈eΠ1(x0, r1, r2). Moreover, by construction, we further have δ(p, q) = \|p −q\| ∈[r1, r2]. Therefore, for all e ∈S(x0) and all q ∈[x0 −κe, x0 + κe] ∩star(x0), there exists p ∈eΠ1(x0, r1, r2) such that r1 ⩽δ(p, q) ⩽r2. Consequently, Recalling (A.4) and (A.5), we have that p ∈eΠ1(x0, r1, r2). Moreover, by construction, we further have δ(p, q) = \|p −q\| ∈[r1, r2]. Therefore, for all e ∈S(x0) and all q ∈[x0 −κe, x0 + κe] ∩star(x0), there exists p ∈eΠ1(x0, r1, r2) such that r1 ⩽δ(p, q) ⩽r2. Consequently, Recalling (A.4) and (A.5), we have that p ∈eΠ1(x0, r1, r2). Moreover, by construction, we further have δ(p, q) = \|p −q\| ∈[r1, r2]. Therefore, for all e ∈S(x0) and all q ∈[x0 −κe, x0 + κe] ∩star(x0), there exists p ∈eΠ1(x0, r1, r2) such that r1 ⩽δ(p, q) ⩽r2. Consequently, [ e∈S(x0) [x0 −κe, x0 + κe] ∩star(x0) ⊂eΠ2(x0, r1, r2). But since S(x0) contains a half-sphere, the left-hand side in the above equation is nothing but Bκ(x0) ∩star(x0). Hence, we have that But since S(x0) contains a half-sphere, the left-hand side in the above equation is nothing but Bκ(x0) ∩star(x0). Hence, we have that But since S(x0) contains a half-sphere, the left-hand side in the above equation is nothing but Bκ(x0) ∩star(x0). Hence, we have that Bκ(x0) ∩star(x0) ⊂eΠ2(x0, r1, r2). (A.6) Bκ(x0) ∩star(x0) ⊂eΠ2(x0, r1, r2). (A.6) Let us now prove that Bκ(x0) ∩Ω\ star(x0) ⊂eΠ2(x0, r1, r2). (A.1) We may suppose, without loss of generality, that Bκ(x0) ∩Ω\star(x0) ̸= ∅, since otherwise there is nothing to prove. So, we have, in particular, that Bκ(x0) ∩K ̸= ∅. Let m ∈∂K be the orthogonal projection of x0 to ∂K. Then, by construction, we have x0 = m+\|x0−m\|ν(m), where ν(m) denotes the outward unit normal to ∂K at m. Set x⊥ 0 := x0 + r1ν(m). Notice that x⊥ 0 ∈A(x0, r1, r2) \ C (x0) (by construction of x⊥ 0 ), so that x⊥ 0 ∈eΠ1(x0, r1, r2) (remember (A.4) and (A.5)). Moreover, we have Bκ(x0) ∩Ω\ star(x0) ⊂RN \ Br1(x⊥ 0 ) and Bκ(x0) ⊂Br2(x⊥ 0 ). Therefore, we have Let us now prove that Bκ(x0) ∩Ω\ star(x0) ⊂eΠ2(x0, r1, r2). We may suppose, without loss of generality, that Bκ(x0) ∩Ω\star(x0) ̸= ∅, since otherwise there is nothing to prove. So, we have, in particular, that Bκ(x0) ∩K ̸= ∅. Let m ∈∂K be the orthogonal projection of x0 to ∂K. Then, by construction, we have x0 = m+\|x0−m\|ν(m), where ν(m) denotes the outward unit normal to ∂K at m. Set x⊥ 0 := x0 + r1ν(m). Notice that x⊥ 0 ∈A(x0, r1, r2) \ C (x0) (by construction of x⊥ 0 ), so that x⊥ 0 ∈eΠ1(x0, r1, r2) (remember (A.4) and (A.5)). Moreover, we have Bκ(x0) ∩Ω\ star(x0) ⊂RN \ Br1(x⊥ 0 ) and Bκ(x0) ⊂Br2(x⊥ 0 ). 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https://openalex.org/W2791726380	https://scindeks-clanci.ceon.rs/data/pdf/0547-3330/2017/0547-33301703423V.pdf	Serbian	null	The role and importance of consulting children in education	Nastava i vaspitanje	2,017	cc-by-sa	6,811	UDK-37.064.2 371.8 DOI:10.5937/nasvas1703423V Прегледни рад НВ год. LXVI 3/2017 UDK-37.064.2 371.8 DOI:10.5937/nasvas1703423V Прегледни рад НВ год. LXVI 3/2017 Јелена Д. Врањешевић1 Филозофски факултет Универзитета у Београду УЛОГА И ЗНАЧАЈ КОНСУЛТАЦИЈА СА ДЕЦОМ У ОБРАЗОВАЊУ У рау се искуује значај који консулаивни роцес са ецом у школи има у креирању конекса који ће осицаи њихов развој и учење, обезбеии ошовање рава ееа и оношење олука које су у њиховом најбољем инересу. Пресављене су и најчешће ререке које оежавају роцес консулација с ученицима, како на нивоу ојеинаца (насавника) и њихове иеје о рирои ееа и улози арициаивних искусава у развоју и учењу ако и на нивоу образовно сисема који је ораслоценричан и неовољно инклузиван каа је о реч о слушању и уважавању ерсекиве ученика. Пререке за консулаивни роцес су искуоване у свелу различиих арициаивних ројекаа и исраживања која указују на важнос консулација ученика. Дае су реоруке за усосављање раксе емокраско ијалоа у школама, које реосављају како ресрукурацију, ј. ромену савова ораслих акера образовно роцеса, ако и рекулурацију, ј. ромену начина на који школски сисем функционише. Асрак консулаивни роцес, арициација, школа, насавници, ученици. Кључне речи: 1 E-маил: jelena.vranjesevic@f.bg.ac.rs Консултативни процес у школи: Због чега треба да слушамо децу? Перспектива деце и одраслих може бити веома различита, што се углавном не узима у обзир, већ се одлуке доносе на основу онога што одрасли (као врховни ау- торитети када је реч о деци и њиховим потребама) мисле да је у најбољем интересу деце. Консултовање деце о питањима која су значајна за њих одраслима помаже да се еценрирају и да сагледају проблем са тачке гледишта деце, да разумеју шта је деци важно и које приоритете она постављају у одређеним областима. Истраживање које се бавило разумевањем начина на који деца учествују у доношењу одлука које их се тичу, како би се подстакла њихова партиципација, показало је велики несклад између перспективе деце и перспективе одраслих. Деца су сматрала да су им свакодневне, краткорочне одлуке које могу да доносе (шта ће да раде, с ким ће да се играју или да уче и сл.) једнако важне као и дугорочне одлуке које се тичу будућности. Одрасли, с друге стране, дугорочне одлуке сматрају далеко важнијим и занемарују значај свако- дневних, „малих“ одлука (O’Kane, 2008). Истраживања у школском контексту показала су да деца помињу као значајне и неке проблеме које одрасли не сматрају толико важним и не баве се њима, као што су нпр. проблем избора, количине и квалитета школских оброка, редови у кафетерији због којих деца немају времена да поједу свој оброк, распоред часова после паузе за ручак и сл. (Bennathan, 2012). Перспектива деце и одраслих често се разликује и када је у питању начин на који тумаче одређене појаве и процесе. У истраживању које је спроведено у једној од средњих школа у Кембриџу, у оквиру пројекта Ефикасно учење (Flutter & Rudduck, 2004), наставници су желели да сазнају шта ученици мисле о критеријумима за оцењивање (Шта чини један задатак успешно урађеним?). Консултативни процес с ученицима показао је да су они прилично збуњени када је реч о нивоу постигнућа које се тражи за сваку од оцена (иако су наставници мислили да су то довољно јасно образложили), као и када је реч о информативности оцене у смислу повратне информације о томе шта могу да ураде да унапреде квалитет свог рада (већина наставника је мислила да је то при- лично јасно дефинисано самом оценом). У том истом истраживању, наставници су сазнали и да, за разлику од увреженог мишљења да је критика најбољи мотиватор, ученици сматрају да честе (негативне) критике делују негативно на њихово самопо- уздање и смањују мотивацију за учење. Увод „Они који се баве образовањем су некако заборавили важну везу између на- ставника и ученика. Слушамо спољне експерте који нам говоре шта да радимо и због тога заборављамо благо у сопственом дворишту – ученике“ (SooHoo, 1993: 389). Овим речима Сузан Су Ху покушава да укаже на значај уважавања дечје перс- пективе у образовном процесу, као и на важност партнерства са децом у процесу из- градње квалитетног система образовања, које обезбеђује развој потенцијала сваког детета до његових крајњих могућности. Процес консултовања деце и укључивање њихове перспективе у различите области друштвеног живота представља први ва- жан корак у остваривању права деце да учествују у одлукама које их се тичу, што има значај не само са становишта њихових права и образовних политика, већ и са становишта подстицања и подржавања њиховог развоја и учења и улоге одраслих (наставника) у том процесу. Овај рад се бави значајем који консултативни процес са 423 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. децом у школи има у креирању контекста који ће подстицати њихов развој и учење, најчешћим препрекама (индивидуалним и системским) које отежавају процес кон- султација са ученицима, као и препорукама за успостављање праксе демократског дијалога у школама. Консултативни процес у школи: Због чега треба да слушамо децу? Консултације с децом помажу одраслима и да стекну бољи уви у роблеме са којима се деца суочавају. У једном истраживању вршњачког насиља у школама ис- траживачи су схватили да је ученицима важније где се насиље одвија, тј. која места у школи су најмање безбедна, него ко су насилници и најчешће жртве (Rowe, 1999). У другим истраживањима која су се бавила условима успешног учења, слушајући ученике наставници су разумели значај вршњачке групе за учење и успех ученика, а сазнали су и да ученицима велике проблеме представља недостатак времена за 424 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. одређене активности и немогућност да учествују у управљању временом у школи (Flutter & Rudduck, 2004). Када је реч о политикама усмереним на остваривање најбоље инереса ее- а, слушање деце и разумевање њихове перспективе (која се често разликује од перспективе одраслих) повећава вероватноћу да ће одлуке које се о деци доносе за- иста бити у њиховом најбољем интересу. Као пример могу да послуже истраживања перспективе деце и њиховог искуства вршњачког насиља. Разговори с ученицима о томе ко су најчешћи насилници, како се насиље испољава и на којим местима се дешава, као и њихови предлози за спречавање насиља помогли су наставницима у неким школама да креирају програм заштите деце од насиља (Charlton, 2012). Ис- траживање дечје перспективе често резултира и акцијом коју сама деца предлажу и спроводе, што представља сложенији облик партиципације. Тако је у једној школи оформљен тим вршњачких савеника који су давали подршку деци која су жртве на- сиља (Sharp et al., 1994). У другим истраживањима која су рађена у области образо- вања, консултовање деце и уважавање њихове перспективе водило је до промене различитих аспеката који чине квалитет и доступност образовања: повећана безбед- ност деце (Bennathan, 2012), промена односа између курикуларних и екстракурику- ларних активности и веће учешће деце у избору како курикуларних (избор садржаја/ тема и метода учења) тако и екстракурикуларних активности (Flutter & Rudduck, 2004), повећана партиципација деце која имају специјалне образовне потребе у избору на- чина процене знања (завршних тестова) и процена адекватности стручне подршке (Armstrong & Galloway, 2012), као и боља прилагођеност школе маргинализованим групама – деци која имају тешкоће у учењу, деци из различитих етничких група и сл. (Davie & Galloway, 2012). Слушање и уважавање мишљења деце у образовном процесу може да има и значајан развојни потенцијал. Консултативни процес у школи: Због чега треба да слушамо децу? Нека истраживања показују да консултације с уче- ницима о процесу учења (Како најбоље уче? Шта им помаже да уче боље?) могу да утичу на њихова постигнућа (Watkins, 2001), мотивацију за учење и позитиван став према школи и наставницима (Flutter & Rudduck, 2004), самопоуздање и позитивну слику о себи, као и на жељу да се активно укључују у различите аспекте живота шко- ле (Jelly et al., 2000). Консултативни процес у школи такође доприноси и развоју ме- такогниције ученика, тј. ученици јасније разумеју начин на који уче и ефекте учења (Flutter & Rudduck, 2004; Black et al., 2002). Развојни потенцијал консултативног про- цеса огледа се и у стицању компетенција неопходних за живот у грађанском демо- кратском друштву. Консултативни процес представља прекид уобичајене праксе неслушања, дискриминације деце и (зло)употребе моћи одраслих. Тиме што консул- тују децу, одрасли поручују да их уважавају, да њихово мишљење узимају озбиљно у разматрање и да се њихово мишљење поштује. Истраживања показују да овај на- чин учешћа деце утиче на развој просоцијалног понашања и низа компетенција које су важне за одрастање у демократским друштвима (Wооdhead, 1998). Деца уче кроз процес консултација: уче се да јасно изражавају своја искуства и да слушају искуства других; уче се да функционишу у групи, да се договарају и преговарају са другима, да трагају за конструктивним решењима проблема; уче се да критички мисле, да буду самостална и одговорна, као и да уважавају друге (пошто су и сама уважена). 425 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. Дијалог с ученицима и уважавање њихове перспективе има импликације и за саме наставнике и квалитет њихове праксе. Слушајући ученике, наставници упознају начин на који деца уче, факторе који им олакшавају/отежавају процес учења, проб- леме са којима се суочавају у образовном процесу и на тај начин прилагођавају свој рад контексту у којем се учење одвија, као и компетенцијама и потребама ученика. За разлику од традиционално успостављених односа моћи и хијерархијског ауторитета (у којем су наставници на врху, а ученици на дну хијерархије), дијалог с ученицима и слушање онога што имају да кажу претпоставља један другачији модел односа који је заснован на узајамности, договарању и сарадњи и у којем ученици имају свест о томе да су уважени партнери у комуникацији. Консултативни процес у школи: Због чега треба да слушамо децу? То доводи и до промене етоса шко- ле која постаје заједница која учи, у којој су искуснији (наставници) и мање искусни партнери (ученици) активни у процесу размене и деле одговорност за процес учења (Rogoﬀ, 1994). Консултативни процес у школи: Због чега не слушамо децу? Упркос бројним примерима који показују трансформативни и развојни потен- цијал консултовања с децом у образовању, ученици су и даље прилично невидљиви и њихов глас се ретко чује када се доносе одлуке које су непосредно значајне за њих. Постоје бројне препреке како на нивоу појединаца (наставника) и њихове идеје о природи детета и улози партиципације у развоју и учењу, тако и на нивоу образовног система који је одраслоцентричан и недовољно инклузиван када је о реч о партици- пацији ученика. Најчешће заблуде које доводе до маргинализације деце (ученика) у образовном процесу су следеће: ■ Орасли су (боље о саме еце) у сању а роцене њихове оребе и нај- бољи инерес. ■ Орасли су (боље о саме еце) у сању а роцене њихове оребе и нај- бољи инерес. Цртање (Angelides & Michaelidou, 2009; Tay-Lim & Lim, 2013), вајање и мо- деловање, фото и видео записи (Wang & Burris, 1997), као и различите драмске улоге попут игара улога, пантомиме, лутака које деца сама праве или које су претходно из- рађене – персона лутке (Etienne et al., 2008), форум театар / интерактивно позориште (Veale, 2005) само су неке од метода/техника које се могу користити самостално или у комбинацији са другим техникама у процесу разумевања сложеног искуства деце. Узраст и компетенције деце не могу да представљају препреку за слушање деце, а посебно деце у периоду средњег детињства и адолесценције, будући да се тада раз- вијају компетенције које су важне и за сложеније форме партиципације (Lansdown, 2005; Vranješević, 2004). Примери консултација са децом у основним и средњим шко- лама показују да је већина ученика (без обзира на узраст) изузетно компетентна да разговара о процесу учења, иако (посебно млађи ученици) нису увек у стању да своје идеје и увиде артикулишу језиком формалног образовања (Flutter & Rudduck, 2004). Оно што најчешће представља баријеру за консултовање деце је несигурност одрас- лих у погледу својих компетенција за разговор са децом, као и недостатак комуника- цијских вештина и способности прилагођавања стила и начина комуникације сваком појединачном детету (Alderson, 2000). Од одраслих се зато захтева да истраже нове могућности комуникације са децом (који превазилазе уобичајени образац у којем једна особа прича, а друга слуша), да науче нове „језике“ у раду са њима, као и нове начине слушања њиховог искуства (Moss, 2006). ■ Орасли су (боље о саме еце) у сању а роцене њихове оребе и нај- бољи инерес. Цртање (Angelides & Michaelidou, 2009; Tay-Lim & Lim, 2013), вајање и мо- деловање, фото и видео записи (Wang & Burris, 1997), као и различите драмске улоге попут игара улога, пантомиме, лутака које деца сама праве или које су претходно из- рађене – персона лутке (Etienne et al., 2008), форум театар / интерактивно позориште (Veale, 2005) само су неке од метода/техника које се могу користити самостално или у комбинацији са другим техникама у процесу разумевања сложеног искуства деце. Узраст и компетенције деце не могу да представљају препреку за слушање деце, а посебно деце у периоду средњег детињства и адолесценције, будући да се тада раз- вијају компетенције које су важне и за сложеније форме партиципације (Lansdown, 2005; Vranješević, 2004). Примери консултација са децом у основним и средњим шко- лама показују да је већина ученика (без обзира на узраст) изузетно компетентна да разговара о процесу учења, иако (посебно млађи ученици) нису увек у стању да своје идеје и увиде артикулишу језиком формалног образовања (Flutter & Rudduck, 2004). Оно што најчешће представља баријеру за консултовање деце је несигурност одрас- лих у погледу својих компетенција за разговор са децом, као и недостатак комуника- цијских вештина и способности прилагођавања стила и начина комуникације сваком појединачном детету (Alderson, 2000). Од одраслих се зато захтева да истраже нове могућности комуникације са децом (који превазилазе уобичајени образац у којем једна особа прича, а друга слуша), да науче нове „језике“ у раду са њима, као и нове начине слушања њиховог искуства (Moss, 2006). асиметричној интеракцији (Виготски). Деца нису пасивни примаоци утицаја околине, већ активно сазнају и осмишљавају свет око себе, у чему им одрасли свесрдно пома- жу. Кључно питање које одрасли треба да поставе приликом консултовања деце није Да ли она разумеју, или не?, већ Ша реба а ураимо а би нас она разумела?, бу- дући да вештина слушања перспективе и потреба других не зависи од компетенција онога ко прича, већ од компетенција онога ко слуша (Alderson, 2000: 16–17). Теорија о соину језика еце (Malaguzzi, 1993) представља метафору која говори о бројности различитих начина на које деца могу да изразе своје мишљење и идеје које имају. Поред најчешће коришћених техника којима се може истраживати искуство деце као што су интервју, фокус-групни интервју, партиципативно посматрање и сл., постоје и друге визуелне индивидуалне и групне технике истраживања искуства чак и сасвим мале деце. ■ Орасли су (боље о саме еце) у сању а роцене њихове оребе и нај- бољи инерес. Корен ове заблуде је у слици о детету која је у друштву доминантна и која ум- ногоме утиче на сагледавање развоја, његових циљева и начина на који се развој подстиче. Та слика нуди прихваћене моделе односа деце и одраслих, указује на по- жељне облике образовања и васпитања деце и представља важан део режима ис- ине (Foucault, 1995), тј. система друштвених претпоставки које дефинишу шта ће се у једном друштву сматрати истином, нормалношћу, чињеницом која се не доводи у питање, као и ко то може да процени и на који начин. Као део режима истине, слика о детету постаје основ (и оправдање) за различите манифестације опресивних пракси над децом, које су подржане јасним дискриминаторским ставовима. Једна од експли- цитних дискриминаторских пракси је неслушање и неуважавање мишљења детета, будући да је дете незрело, некомпетентно и самим тим непоуздан извор података о себи. Одрасли су зато једини у стању да процене најбољи интерес деце и њихове потребе. Овај аргумент је тешко одржив са становишта (социо)конструктивистичког приступа развоју у оквиру којег се на дете гледа као на научника, активног у констру- исању сазнања о свету око себе (Пијаже), креатора значења (Брунер) и партнера у 426 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. асиметричној интеракцији (Виготски). Деца нису пасивни примаоци утицаја околине, већ активно сазнају и осмишљавају свет око себе, у чему им одрасли свесрдно пома- жу. Кључно питање које одрасли треба да поставе приликом консултовања деце није Да ли она разумеју, или не?, већ Ша реба а ураимо а би нас она разумела?, бу- дући да вештина слушања перспективе и потреба других не зависи од компетенција онога ко прича, већ од компетенција онога ко слуша (Alderson, 2000: 16–17). Теорија о соину језика еце (Malaguzzi, 1993) представља метафору која говори о бројности различитих начина на које деца могу да изразе своје мишљење и идеје које имају. Поред најчешће коришћених техника којима се може истраживати искуство деце као што су интервју, фокус-групни интервју, партиципативно посматрање и сл., постоје и друге визуелне индивидуалне и групне технике истраживања искуства чак и сасвим мале деце. ■ Не можемо а излазимо у сусре свим ечјим оребама ученика – о би овело о анархије у школи. Наставници често потпуно занемарују могућност да консултују ученике о различитим аспектима живота школе, зато што процес консултација повезују с ис- пуњавањем свих потреба и захтева деце (Vranješević, 2012). Овде је реч о неразуме- вању суштине консултативног процеса, тј. мешању консултација с децом и учешћа деце у процесу одлучивања. Консултација с ученицима подразумева да се њихово мишљење озбиљно узима у обзир приликом одлучивања, да се оно третира равноп- равно као мишљење наставника и да, уколико се донесе одлука која није у складу са ставовима и потребама ученика, та одлука буде јасно образложена. Она може да води до сложенијег учешћа у процесу одлучивања, али чак и тада не значи да се пот- ребама деце мора изаћи у сусрет, нити да је мишљење наставника мишљење другог реда. Консултативни процес помаже наставницима да се децентрирају, да пробле- ме сагледавају из угла ученика и да одлуке које доносе буду у складу са потреба- ма деце и њиховим најбољим интересом. То не значи губитак ауторитета (како неки наставници мисле), већ његово редефинисање у правцу међусобног уважавања и сарадње, што доводи до бољег односа између наставника и ученика, подстицања мотивације за учење и партиципацију у школи, о чему је већ било речи. ■ Не можемо консуловаи ецу у мноим сварима зао шо о није у њиховом најбољем инересу, ми орасли смо у а их шиимо. ■ Не можемо консуловаи ецу у мноим сварима зао шо о није у њиховом најбољем инересу, ми орасли смо у а их шиимо. За разлику од претходног експлицитно дискриминаторског става о некомпе- тентности детета, у овом случају реч је о мање видљивој, суптилнијој форми дискри- минације која је исказана као заштита деце и покушај да се обезбеди њихов најбољи интерес. У једном истраживању у којем су наставници питани да наведу разлоге због којих о неким областима школског живота не би консултовали децу, као најчешће разлоге за неуважавање мишљења деце, они наводе безбедност и заштиту најбољег интереса деце (Vranješević, 2012). У овом случају дискриминација је заснована на уве- рењу наставника да могу сами (без консултације с ученицима) да дефинишу у чему се огледа безбедност деце и шта представља њихов најбољи интерес. У истраживању 427 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. су се издвојиле две групе наставника које су се разликовале у начину на који виде најбољи интерес деце. Они наставници који најбољи интерес дефинишу као заштиту физичког и психичког интегритета детета (да се дете не повреди, да се заштити од различитих облика угрожавања, заштита здравља детета и сл.) само у тим случајеви- ма преузимају на себе процес одлучивања без консултације са децом. По мишљењу тих наставника, постоје неке ситуације у којима треба реаговати брзо и спречити да дете настрада (туче у школском дворишту, излазак ван дворишта и сл.) и да одлуку како да поступе у тим ситуацијама доносе они сами, тј. користе тзв. протективну упо- требу силе. За разлику од њих, они наставници који најбољи интерес дефинишу као заштиту деце и у другим областима које нису нужно угрожавајуће, као што су орга- низација боравка у учионици (правила седења, организација часа, начин рада, ева- луација рада на часу), па чак и ваннаставне активности (излети, посете, такмичења) сматрају да деца веома често не знају шта је за њих добро и због тога наставници већину одлука преузимају на себе. ■ Ученике не реба консуловаи о сварима које их се неосрено не ичу. ■ Ученике не реба консуловаи о сварима које их се неосрено не ичу. Овај став, као и претходни, произилази из уверења наставника да су они не- прикосновени ауторитети који осим најбољег интереса и потреба деце, могу да де- финишу и области од њиховог (не)посредног значаја. Међутим, перспектива деце је другачија. Млади истраживачи (узраста од 8 до 18 година) из групе Children’s Express поручују одраслима да могу да „траже да чују мишљење деце о свему, јер она су део друштва. Она су директно укључена у разне области, као нпр. образовање, али ни- када о њима нису питана“ („Kids these days...“, 1999: 136). Истраживања показују да је консултовање деце чешће на млађим него на старијим узрастима (Vranješević, 2012; 428 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. Pešić, 1999) и да учитељи децу консултују у вези са већим бројем области у школском животу него наставници. С обзиром на то да као један од разлога за неконсултовање деце наставници наводе недовољно развијене компетенције, очекивало би се да ређе консултују млађу децу „која немају шта да кажу“, него старију која су зрелија и компетентнија, што очигледно није случај. Консултовање деце и учитељи и наставни- ци најчешће практикују у процесу наставе, и то углавном као део наставног процеса (одговори на питања која се тичу садржаја наставе и провере наученог). Међутим, учитељи консултују децу и када је реч о начину рада на часу (индивидуално, у ма- лим групама, у великој групи), о самој структури и току часа (подела задужења, ко ће шта да ради и на који начин), као и у процесу евалуације сопственог знања и знања других ученика. С друге стране, и учитељи и наставници се слажу у томе да теме које ће се обрађивати, циљеви часа и уџбеници који се користе нису од непосредног интереса за децу будући да о томе не би тражили дечје мишљење. Такође, ретко се тражи и мишљење деце када су у питању области које излазе из оквира конкретне учионице: кућни ред школе – правила понашања у школи, рад школских служби, мо- гућност евалуације наставника и њиховог рада, евалуација програма који се у школи спроводе, сарадња с родитељима и могући облици те сарадње, професионално уса- вршавање наставника и сл. ■ Ученике не реба консуловаи о сварима које их се неосрено не ичу. (Vranješević, 2012) Друга истраживања показују да постоји широк дијапазон области школског жи- вота у којима је до сада коришћено консултовање деце (укључујући и млађе основ- ношколске узрасте) као облик њиховог учешћа у школи. Посебна пажња посвећена је процесу учења и факторима од којих зависи успешно учење (Flutter & Rudduck, 2004; Frost & Roberts, 2011; MacBeath et al., 2006). У истраживању које се бавило стратегијама учења, ученици су заједно са наставницима причали о сопственим (успешним и неус- пешним) стратегијама учења, факторима који олакшавају и отежавају процес учења, врсти подршке која им треба и та дискусија је представљала почетну основу за пра- вљење акционог плана којим ће се те тешкоће превазићи (Bullock & Wickeley, 2001). Деца су у неким школама активно учествовала у процесу школске самоевалуације (Flutter & Rudduck, 2004), као и у различитим аспектима реформе школе, као што су промена курикулума: садржај наставних и ненаставних активности, начини подуча- вања, партиципација ученика у процесу одлучивања, начини провере знања, односи наставник–ученик, облици сарадње са родитељима и сл. (Bennathan, 2012; Flutter & Rudduck, 2004; Mitra, 2001). Ученици су били консултовани и у истраживањима која су се бавила специфичним проблемима које одређене групе ученика или појединци имају и на које треба обратити пажњу и решавати их (напуштање школе деце из мар- гинализованих група, слабе оцене ученика одређених разреда из појединих предме- та, и слично). Постоје и истраживања у којима је испитиван ефекат награде и казне (које врсте казни и награда су по мишљењу деце ефикасне, а које нису и због чега), као и мишљење деце о проблемима у вези са дисциплином и школским правилима (Charlton, 2012). У неким школама су консултације с ученицима биле предуслов за успостављање демократичног система у школи и примену грађанског образовања у пракси, кроз организовање школских парламената у млађим разредима основне школе (Holden, 1998). 429 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. Број истраживања у којима су деца консултована, њихова разноврсност (дија- пазон области у школском животу на које се односе), као и ефекти које имају у проце- су унапређења квалитета образовно-васпитног рада, захтева од наставника да раз- мишљају шире о областима које су у непосредном интересу ученика, тј. да прихвате да постоје бројне области у образовном процесу о којима ученици могу и треба да кажу своје мишљење. ■ Обезбеие нам време, а ћемо чешће а консулујемо ученике! ■ Ученике не реба консуловаи о сварима које их се неосрено не ичу. Ова се препрека само делимично односи на уверења наставника, а већим де- лом је условљена структуром образовног система и начином његовог функциони- сања. Наставници себе виде разапете између обавезних планова и програма и онога што сматрају ваннаставним активностима које нису обавезне, али су пожељне. Ар- гумент да деца често не могу да буду консултована пошто за то нема времена, тј. да би процес консултовања деце „појео време“ које у школи треба да буде посвећено подучавању и учењу, показује колико је идеја партиципације ученика кроз консулта- тивни процес неелаборисана и неуклопљена у формални курикулум школе. Разлог због којег учитељи виде консултативни процес као терет само делимично може да се објасни идејом коју они имају о његовој суштини и циљевима, а већим делом је усло- вљен системом вредности, норми и правила на којем је школа заснована: Колико је у школи чест дијалог у ком је заиста важно шта деца мисле? На који начин је пракса консултовања деце повезана са консултовањем одраслих (родитеља и наставника) у школи? На који начин је консултативни процес уткан у организациону културу и етос школе? У којој мери културне норме и вредности школе укључују слушање деце у образовном процесу и схватање школе као „заједнице која учи“? Све су то централна питања која указују на место које консултативни процес има у школској пракси и кул- тури школе (Vranješević, 2012). Трансформаивни оенцијал консулаивно роцеса Консултовање деце није само правно и развојно-психолошко питање, већ је и важно етичко питање (Dahlberg & Мoss, 2005) будући да слушање деце умногоме мери зависи од ставова и вредности које постоје у једном демократском друштву. Речи данског истраживача Олеа Лангстеда указују на везу између консултативног процеса и ставова према деци: „Важније од техника и процедура јесте клима у друштву која обликује идеје које одрасли у том друштву имају о деци. Жеља да се деца саслушају и укључе потиче из те друштвене климе. Наравно, постоје средства и про- цедуре које су важне – међутим, питање које треба да поставимо је: Да ли као култура, друштво, желимо да слушамо једни друге? Да ли желимо да градимо демократске односе?“ (према: Moss, 2006: 21) 430 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. Консултовање деце подразумева културу слушања деце која је израз демократ- ских пракса у друштву: способност да се препознају, прихвате и уважавају различите перспективе, да се о њима дискутује и да се кроз дијалог са децом креирају нова значења. Да би се пракса демократског дијалога у школама успоставила, потребна је рекултурација, тј. промена ставова одраслих који су у контакту са децом, али и реструктурација, тј. промена начина на који школа као систем функционише (Fullan, 2007). Као полазна основа у промишљању могућих праваца промене на нивоу поје- динаца и на нивоу система, може да послужи оквир за анализу консултативног про- цеса у школи, који се заснива на три питања: Ко рича? Ко слуша? и Ге се ијало овија? (Fielding, 2001). Питање ко рича односи се на то да ли се у школи чује глас свих ученика, да ли су видљива деца из различитих мањинских и маргинализованих група (деца са тешкоћама у развоју / деца са посебним образовним потребама, деца која живе у институцијама, сиромашна деца, деца из разних етничких група и сл.), и ако нису, који су разлози за то. Подједнако је важно и питање о чему ученици причају, тј. да ли могу да причају и о стварима које одрасли сматрају неважним или непри- хватљивим и којим „језиком“, односно да ли се подржавају различити начини изра- жавања мишљења који су прилагођени ученицима, или се инсистира на формалном образовном језику који је прилагођен одраслима. Трансформаивни оенцијал консулаивно роцеса Питања ко слуша, због чега и на који начин односе се на ставове и компетен- ције наставника и других одраслих који су укључени у образовни процес и умногоме одређују ток процеса рекултурације. Уколико се консултовање деце користи некри- тички и у манипулативне сврхе, уместо да ојача онога ко прича (децу), може ојачати онога ко слуша (одрасле), тако да се онда лакше манипулише мишљењем деце, или се оно користи у декоративне сврхе (Моss, 2006). Питање разлога због којих одрасли у школи слушају децу такође је веома важно, будући да показује слику о ученику коју наставници имају: да ли ученике виде као извор корисних информација које ће им помоћи да их боље контролишу, или као експерте за сопствено искуство од којих могу много да науче (Fielding, 2001). Компетенције наставника важне за консултатив- ни процес са децом представљају још једну важну област на коју се односи питање како наставници слушају. Култура слушања деце подразумева аутентичну заинтере- сованост наставника за перспективу ученика, симетричност у односима тако што се поштује и уважава дечје мишљење и стављање у заграду сопствених предубеђења. Етичку основу слушања, чини тзв. етика сусрета (Dahlberg & Моss, 2005), која под- разумева однос са другом особом током којег смо са њом повезани тако да поштује- мо њене границе и не поистовећујемо је са нама самима: не тумачимо другу особу на основу сопственог референтног оквира (сопствених појмова, теорија и искуства), не покушавамо да је асимилујемо у наш систем разумевања и не минимизирамо и/ или не поричемо различитости које постоје. Етика сусрета указује на важност препо- знавања и уважавања различитости (између одраслог и детета и између деце), као и уздржавање од поистовећивања других са нама самима (уздржавање од одрасло- центричне перспективе). Консултовање ученика представља веома сложен процес који захтева од на- ставника комуникационе компетенције и знања у области развоја детета и вештине 431 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. које се односе на различите начине слушања деце. Тај процес захтева од наставника да прилагођава начин на који пита ученика његовим развојним могућностима. На- ставник мора стално да проверава начин на који је ученик разумео оно што се од њега тражи, као и да даје повратну информацију која служи да ученик разуме смисао онога што се од њега тражи. Трансформаивни оенцијал консулаивно роцеса Због тога је најадекватнији подстицај развоју и учењу пажљиво праћење, слушање и уважавање дечјег погледа на свет, као и стално преис- питивање своје улоге да би се избегла центрација и неосетљивост за дететову тачку гледишта. Важно питање је и на који начин наставници стичу компетенције које су важне у процесу слушања и уважавања мишљења деце. Да ли је предвиђена могућ- ност њиховог стицања у току иницијалног образовања наставника? У којој мери се предвиђа њихово усавршавање током професионалног развоја наставника? На који начин су те компетенције укључене у процес (само)евалуације школе? Вештина слу- шања и уважавања мишљења ученика подразумева разумевање учења као заједнич- ког процеса у којем се одвија сарадња, размена и коконструкција значења и предста- вљају важан корак ка успостављању етоса школе као заједнице која учи. Међутим, промене на нивоу ставова и компетенција наставника нису довољне уколико се не мења и образовни систем који ће те промене подржати. Због тога кон- екс у којем се консултације одвијају има од кључну важност. Да ли се у школској пракси подржава видљивост ученика и њихова перспектива? Да ли су консултовање ученика и сарадња с њима вредности које се у школи негују? Да ли постоји могућ- ност партиципације и других актера у образовном процесу, нпр. наставника и роди- теља? У којој мери је школа отворена за различите перспективе? То су важна питања будући да указују на спремност школе да учини видљивим и уважи различите перс- пективе, не само ученика. Видљивост перспективе наставника је по неким ауторима нужан предуслов за видљивост перспективе ученика будући да „наставници неће подржати партиципацију ученика ако она учини видљивом њихову немогућност да партиципирају“ (Fielding, 2001). Култура слушања треба да укључи и механизме који ће деци омогућити да буду видљива и да се њихов глас у чује (Lensdaun, 2004). То значи да школа треба да осигу- ра да ученици имају приступ одговарајућим информацијама које су примерене њи- ховом узрасту и развојним могућностима, да би на основу њих могла да формирају ставове и искажу своје информисано мишљење. Школа такође треба да обезбеди простор за дијалог о важним питањима квалитета образовања у којем ће (поред дру- гих заинтересованих страна) важну улогу имати и ученици. То значи да се одрасли уче да говоре у сарањи са ученицима, а не у њихово име и да ученике третирају као равноправне учеснике у дијалогу који су компетентни да учествују и у доношењу од- лука у областима које их се тичу. Трансформаивни оенцијал консулаивно роцеса Да би консултативни процес могао да подржи и подстакне развој ученика и да допринесе трансформацији образовног контекста, он треба да постане саставни део школске праксе, а не изоловани случај који зависи од добре воље поједине школе или појединаца у њој. 432 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. London & New York: Routledge. Bennathan, M. (2012). Listening to Children in Schools: An Empirical Study. In R. Davie & D. Galloway (Eds.), Listening to Children in Education (pp. 90–109). London & New York: Routledge. Black, P., Harrison, C., Lee, C., Marshall, B. & William, D. (2002). Working Inside the Black Box. 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Прихваћено: 20. 01. 2018. 434 Врањешевић, Ј. • Улога и значај консултација са децом... • НВ год. LXVI бр. 3/2017, стр. 423–435. THE ROLE AND IMPORTANCE OF CONSULTING CHILDREN IN EDUCATION This paper discusses the importance of the consultation process with children in schools for creating a context that will stimulate their development and learning and ensure that children’s rights are respected and that decisions are made in their best interest. The paper also looks at the most common obstacles that impede the process of consultation with students, both at the level of the individual (the teacher) and their ideas about the nature of children and the role of participatory experiences in development and learning, and at the level of the education system which is adult-centric and insuﬃciently inclusive with regard to listening to and acknowledging the students’ perspective. Obstacles to the consultation process are discussed with reference to various participatory projects and studies that highlight the importance of consulting with students. Recommendations are given for establishing the practice of democratic dialogue in schools, which requires both restructuring, i.e. a change in the attitude of adult actors in the education process, and reculturation, i.e. a change in the way in which the school system functions. Abstract consultation process, participation, school, teachers, students. Keywords: РОЛЬ И ЗНАЧЕНИЕ КОНСУЛЬТАЦИЙ С ДЕТЬМИ В ОБРАЗОВАНИИ В статье обсуждается важность процесса консультативной работы с детьми в школе для создания условий, способствующих их развитию и обучению, обеспечивая при этом уважение прав детей и принятие решений в их интересе. Приводятся наиболее распространенные препятствия, которые затрудняют процесс консультаций с учениками как на уровне отдельных лиц (учителей), их представлений о природе ребенка и роли совместного участия в процессе развития и обучения, а также на уровне системы образования, которая является взрослоцентричной и недостаточно инклюзивной, когда речь идет о понимании и уважении мнения и точки зрения ученика. Препятствия на пути консультативной работе обсуждаются в свете различных совместных проектов и исследований, которые указывают на важность консультаций с учениками. Приводятся рекомендации по установлению практики демократического диалога в школах, которые предполагают реструктуризацию, т. е. изменение отношения взрослых субъектов образовательного процесса, а также рекультивацию, т. е. изменение способа функционирования школьной системы. Резюме процесс консультации, партиципация, школа, учитель, ученик. Ключевые слова: 435
https://openalex.org/W3129402593	https://www.jmir.org/2021/3/e25726/PDF	English	null	Adoption of COVID-19 Contact Tracing Apps: A Balance Between Privacy and Effectiveness	JMIR. Journal of medical internet research/Journal of medical internet research	2,021	cc-by	5,481	KEYWORDS mobile apps; COVID-19; contact tracing; exposure notification; privacy; effectiveness; app; surveillance; tracing; transmission; security; digital health Corresponding Author: Corresponding Author: Emily Seto, PhD, PEng Institute of Health Policy, Management and Evaluation Dalla Lana School of Public Health University of Toronto 155 College Street, Suite 425 Toronto, ON, M5T 3M6 Canada Phone: 1 416 669 9295 Email: emily.seto@utoronto.ca Phone: 1 416 669 9295 Email: emily.seto@utoronto.ca JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Seto et al Abstract With the relative ubiquity of smartphones, contact tracing and exposure notification apps have been looked to as novel methods to help reduce the transmission of COVID-19. Many countries have created apps that lie across a spectrum from privacy-first approaches to those that have very few privacy measures. The level of privacy incorporated into an app is largely based on the societal norms and values of a particular country. Digital health technologies can be highly effective and preserve privacy at the same time, but in the case of contact tracing and exposure notification apps, there is a trade-off between increased privacy measures and the effectiveness of the app. In this article, examples from various countries are used to highlight how characteristics of contract tracing and exposure notification apps contribute to the perceived levels of privacy awarded to citizens and how this impacts an app’s effectiveness. We conclude that finding the right balance between privacy and effectiveness, while critical, is challenging because it is highly context-specific. J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 1 (page number not for citation purposes) Adoption of COVID-19 Contact Tracing Apps: A Balance Between Privacy and Effectiveness Emily Seto1,2,3, PhD, PEng; Priyanka Challa1, MHI; Patrick Ware2, MPH, PhD Emily Seto , PhD, PEng; Priyanka Challa , MHI; Patrick Ware , MPH, PhD 1Institute of Health Policy, Management and Evaluation, Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada 2Centre for Global eHealth Innovation, University Health Network, Toronto, ON, Canada 3Techna Institute, University Health Network, Toronto, ON, Canada Introduction Many countries around the world have released contact tracing and exposure notification apps in an attempt to help combat the spread of COVID-19 [1,2]. However, the technologies used, adoption rates, and potential impact of the apps have been extremely varied across countries. Moreover, each country has developed contact tracing apps that meet the level of privacy required for their citizens. Often, increased privacy has been deemed a fair trade-off for a decrease in the potential effectiveness of the app. Although privacy laws provide a foundation that can inform the design and implementation of exposure notification and true contact tracing apps [3], it is the types of technologies used (eg, quick response [QR] codes, GPS, Bluetooth Low Energy) and the way they are applied within those legal frameworks that determine the level of privacy afforded to citizens. The important distinction between contact tracing apps and exposure notification apps is that the former collects tracking data so that public health authorities can determine who individuals have been in contact with, as well as the location and time of the contact. On the other hand, exposure notification apps collect only the data required to determine if an individual may have https://www.jmir.org/2021/3/e25726 J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 1 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX Seto et al JOURNAL OF MEDICAL INTERNET RESEARCH COVID-19 apps in various countries. The examples have been selected to demonstrate a spectrum of privacy-related features in countries where information about their apps is publicly available, as well as to demonstrate that the optimal balance between privacy and effectiveness may be culturally dependent. been in close contact with someone who has been identified as being positive for COVID-19, which provides significantly more privacy. Table 1 expands upon research by Liu and Guo [4], which was published in the initial months of the pandemic. Specifically, it presents privacy-related characteristics of been in close contact with someone who has been identified as being positive for COVID-19, which provides significantly more privacy. Table 1 expands upon research by Liu and Guo [4], which was published in the initial months of the pandemic. Specifically, it presents privacy-related characteristics of Table 1. Privacy characteristics of COVID-19 contact tracing and exposure notification apps. Introduction Approximate adoption rate, % (month of report- ing) Centralized or decen- tralized data storage Consent for data sharing required App data bound by pri- vacy laws Technology Voluntary or mandatory App name (month of launch) Country 21.6 (July 2020) [6] Centralized Yes Yes Bluetooth [5] Voluntary COVIDSafe (April 2020) [5] Australia ~64 (April 2020)a [8] Information not found No Yes May use GPS or records of individ- ual’s location Voluntary (re- quired to move around cities) HealthCode (February 2020) [7] China ~15 (December 2020)a,b [10] Decentralized Yes Yes Bluetooth Voluntary COVID Alert (July 2020) [9] Canada 1.4 (July 2020) [6] Centralized (pseudonymized con- tact identifiers) Yes Yes Bluetooth Voluntary Corona-Warn-App (June 2020) [11] Germany Information not found Decentralized Yes Yes Bluetooth and ge- ofencing technol- ogy using wrist- bands Mandatory for 14-day home quarantine StayHomeSafe (April 2020) [12] Hong Kong Information not found Decentralized Yes Yes QRc codes Voluntary LeaveHomeSafe (November 2020) [12] Hong Kong 10.7 (July 2020) [6] Decentralized Yes Yes Bluetooth, QR codes, location alert through push notifications Voluntary NZ COVID Tracer App (May 2020) [13] New Zealand Information not found Centralized Yes Yes App seeks con- sent to access Bluetooth, GPS, and camera Mandatory for individuals with COVID- 19; voluntary for others Social Monitoring (April 2020) [14] Russia 70 (December 2020) [16] Centralized Not for in- fected per- sons Yes Bluetooth Voluntary (re- quired to move around the city) TraceTogether (March 2020) [15] Singa- pore Information not found Centralized Yes Yes Bluetooth, GPS, credit card trans- actions, surveil- lance cameras, and others Required for new arrivals for 2 weeks (telephone calls are alter- native option) Self-Quarantine Safety Protection (March 2020) [17] South Ko- rea ~2 (end of Febru- ary 2020, 3 weeks after roll- out)a [18] Decentralized No Yes GPS, uses data from public gov- ernment sources Voluntary Corona 100m (February 2020) [18] South Ko- rea 4.9 (July 2020) [6] Centralized Information not found Information not found GPS Voluntary Tawakkalna (May 2020) [19] Saudi Arabia 40 (October 2020) [21] Centralized (noniden- tifiable information) Yes Yes Bluetooth 4.0 or higher Voluntary NHS COVID-19 (September 2020) [20] United Kingdom aEstimated adoption rate based on number of downloads divided by the country’s entire population. bIn Canada, the app was rolled out in Ontario first; as of December 2020, only 9 of the 13 provinces and territories had adopted the app. cQR: quick response Table 1. Privacy Measures Related to Adoption Beyond the inherent value of privacy as a human right and as a protection against bias and stigma, the intent of increasing an app’s privacy measures is to broaden its adoption. This has been confirmed by studies conducted since the start of the pandemic, which have demonstrated the important role of trust and perceived privacy in influencing the adoption and use of COVID-19 apps [22-24]. The perception of privacy is not only determined by a user’s interpretation of existing safeguards (eg, the underlying technology, whether the app is voluntary, and the degree of data centralization), but also requires those details be effectively communicated to citizens, which is not always the case. For example, an analysis of COVID-19 app privacy policies concluded that improvements to the readability of privacy policies could lead to increased usage [25]. Modelling by a research group at Oxford has indicated that the pandemic can be stopped if about 60% of the population uses the app. Lower adoption rates of 15% would still reduce infection and deaths by about 8% and 6%, respectively [26]. At the time of writing, Singapore and China have been able to reach the 60% threshold, while many other countries have reported adoption rates greater than 15%, including Ireland, Canada (ie, Ontario, where the app was first launched), Germany, and Iceland (the first country in Europe to launch their app) [27]. Third, data governance in terms of privacy laws related to the app data, user consent for data sharing, and centralization of data storage are also important factors that can impact citizens’ comfort level with using the apps. Countries with data governance laws and policies that protect privacy, such as the United Kingdom and Canada, appear to have relatively good adoption rates. On the other hand, a lower adoption rate was reported in Saudi Arabia, where it is not clear if the apps are bound by privacy laws and whether consent for data sharing is required. Some countries have centralized the storage of data into a database controlled by a public health authority, while others have decentralized data storage (ie, data is stored only on an individual’s smartphone). While a centralized data storage system could provide added value through the ability to analyze the data for trends and adoption information, decentralized systems may invoke more trust in the app, which could drive up adoption. Introduction Privacy characteristics of COVID-19 contact tracing and exposure notification apps. aEstimated adoption rate based on number of downloads divided by the country’s entire population. bIn Canada, the app was rolled out in Ontario first; as of December 2020, only 9 of the 13 provinces and territories had adopted the app. cQR: quick response https://www.jmir.org/2021/3/e25726 XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Seto et al information has been made available on the details of how the algorithms work [28]. As another example, in South Korea, all people coming into the country are required to quarantine for two weeks and to download the “Self-Quarantine Safety Protection” app, which tracks a person’s movement via GPS to monitor compliance with isolation procedures (telephone calls are an option if someone does not have a smartphone) [30]. After the two-week quarantine period, the app tells the user that they are able to delete the app from their phones. In addition, Corona 100m is a voluntary app in South Korea that was built by a private developer after the government made certain data about patients with COVID-19 freely available [31]. The Corona 100m app shows the location of people infected with the virus, the date the infection was confirmed, and the nationality, sex, and age of the infected person. Alerts are sent to users when they are within 100 meters of the latest tracked location visited by someone positive for COVID-19. Data used by the app comes from smartphone location logs, credit and debit card transactions, and an extensive network of surveillance cameras [32]. https://www.jmir.org/2021/3/e25726 Privacy Measures Related to Adoption Germany’s exposure notification app was initially developed to support a centralized approach, but was met with much criticism, leading to a change to a decentralized model [33]. While adoption rates are affected by numerous nuanced factors, they are likely highly influenced by three broad categories of privacy-related factors highlighted in Table 1. First, apps that are mandatory would presumably result in higher adoption rates than voluntary apps. For example, although use of the apps in Singapore and China is technically voluntary, the apps are required for citizens to move around freely in cities, which may have resulted in the high adoption rates of those apps. Specifically, citizens in China are unable to move freely within cities and enter establishments without showing their color-coded individual QR codes at checkpoints. A color code (green, yellow, or red) is assigned depending on the user’s travel history and health status, with green allowing unrestricted movement, and yellow and red indicating different quarantine requirements [28]. J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 3 (page number not for citation purposes) Conclusion With increased privacy, there are inherent trade-offs in the effectiveness of COVID-19 contact tracing and exposure notification apps. The effectiveness of the apps might be impossible to evaluate fully due to the lack of collected data, especially for apps with privacy-first approaches, as well as confounding factors like community lockdowns. However, given the assumption that higher adoption translates into increased effectiveness, broadening adoption of voluntary apps is a goal of many countries, which can be achieved through several techniques. These include investing in a promotional campaign that may involve hiring a professional marketing firm, partnering with high-profile personalities to endorse the app, and increasing ease of app download via smartphone alerts by the government that link to the app. While the level of privacy required for a COVID-19 contact tracing and exposure notification app will depend on factors including whether it is voluntary, the underlying technology, and degree of data centralization, translation of those important safeguards into a user’s perception of privacy will occur within the context of the norms and values of their country. Therefore, striking the right balance between privacy and effectiveness requires careful consideration, especially as the urgency to reduce transmission of the virus evolves based on fluctuating case numbers and vaccination efforts. The Privacy Versus Effectiveness Second, the technology employed in the apps will largely dictate how intrusive the apps are to an individual’s privacy. Public transparency about the technology used may also increase confidence in the use of the app. To increase adoption, some countries—including Canada, Germany, and the United Kingdom—have opted to use the well-documented and highly vetted Google and Apple Exposure Notification application programming interface (API), which enables the swapping of anonymous identifier beacons (ie, random strings of numbers that are frequently changed) between phones in close proximity via Bluetooth Low Energy [29]. This provides a very high level of privacy because no identifiable data is transmitted. Other countries, such as China, have opted for technology that tracks the location of individuals (eg, GPS), which may be a deterrent to their use. Local Chinese governments have developed their own apps with algorithms that assign the color code, but little The trade-off between privacy and effectiveness is apparent at both the individual and system level. For example, apps leveraging the Google and Apple Exposure Notification API, which was designed with a privacy-first approach, do not provide users with the identity of the person with COVID-19 that they were in close proximity to, or information about the location or time of the potential exposure beyond the fact that it was within the past 14 days. Therefore, the user is provided with little context to determine the actual risk (eg, whether personal protective equipment was used) and how long they should be exercising extra precaution to reduce the risk of transmission of the virus. The limited collection of data and decentralized systems that are used to protect privacy also hinder the ability of governments https://www.jmir.org/2021/3/e25726 J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 3 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX Seto et al JOURNAL OF MEDICAL INTERNET RESEARCH TraceTogether token, which works by swapping identifier beacons via Bluetooth, similar to the app [36]. to analyze aggregate data, including demographics, time stamps, and geolocalization, which could inform the design and implementation of more targeted public health strategies. Specifically, COVID-19 apps that prioritize effectiveness over privacy could aggregate reliable demographic information about potentially exposed users that is grounded in a specific place and time. The Privacy Versus Effectiveness In its absence, governments and public health officials must largely rely on information gathered when patients positive for COVID-19 interact with the health system (which is almost always temporally dissociated from the time of infection) or human-driven contact tracing, which relies on citizens’ recall and willingness to report accurate information. While useful, these data may be less reliable and comprehensive compared to data collected via a COVID-19 app, thus limiting the data’s ability to inform the types of targeted interventions that could simultaneously decrease the spread of COVID-19 and avoid the negative societal consequences of more generalized lockdowns. There are other methods that have been used to try to increase adoption of voluntary exposure notification apps. One is to incorporate features in the app to increase its perceived value [37]. For example, some exposure notification apps can provide COVID-19 test results. In the United Kingdom, the NHS COVID-19 app has features such as ordering COVID-19 tests, receiving test results, regional risk score alerts, symptom recorders, and a self-isolation countdown and advice [38]. A second way to increase adoption of these apps is through social influence and media campaigns. In Canada, influential brands and high-profile individuals like athletes have partnered to promote the use of the COVID Alert app [39]. New Zealand has used humor and creativity in their efforts to inform citizens about the NZ COVID Tracer app, and the pandemic more generally, through comedic skits with well-known television personalities and a call for filmmakers to submit short videos [40]. Opportunities to communicate the privacy safeguards as well as personal and societal benefits should also be explored in these campaigns. Furthermore, the lack of detailed and centralized data limits evaluations of the effectiveness of these apps. For instance, many exposure notification systems will only be able to determine the number of downloads and the number of users who have chosen to identify themselves as positive for COVID-19 through the app. It is not known whether the user has since deleted the app or has chosen to turn off Bluetooth. Other unknowns include how many people have been notified of a potential exposure, how many people chose to be tested because of an alert, and how many people tested positive for COVID-19 earlier than they would have otherwise due to an alert. The Privacy Versus Effectiveness In these situations, there is mainly anecdotal effectiveness evidence of users getting alerted about an exposure, getting tested, and then modifying their behavior to reduce the transmission of the virus. Evidence of the effectiveness of the app can help drive adoption of the app, as well as inform future implementations of and improvements to the app. Furthermore, the perception that the app is effective may incentivize individuals to download and use the app, which in itself would presumably increase its effectiveness. Finally, reducing user effort by making the app easier to download and use may also increase adoption. For example, during the launch of the COVID Alert app in Ontario, Canada, a government alert was sent to smartphones regarding the app, with information on how to download the app, which runs in the background after setup without further user interaction. J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 4 (page number not for citation purposes) Other Factors Impacting the Adoption of COVID-19 Apps In addition to privacy concerns, a barrier to adoption is the inability for some to download the app. While the proportion of people who own smartphones is high and increasing (upwards of 80% in some countries), there is still a considerable number of people who do not own smartphones [34]. In addition, a criticism of using the Google and Apple Exposure Notification API is that it works only on phones that were released in the past five years or so, which could have the effect of excluding lower-income communities that may have particularly high rates of COVID-19 transmission [35]. Singapore’s innovative solution to help reach citizens that are unable to download the TraceTogether app was to distribute a device called the Conflicts of Interest None declared. Conflicts of Interest Conflicts of Interest None declared. None declared. 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URL: https://english.alarabiya.net/en/coronavirus/2020/05/26/ Coronavirus-How-to-use-Saudi-Arabia-s-Tawakkalna-app-to-get-movement-permits [accessed 2020-12-19] pp g p 20. White L, Cwalina C, Evans M, Flockhart F, Gamvros A. References Contact tracing apps in the UK. Norton Rose Fulbright. 2020 Oct 05. URL: https://www.nortonrosefulbright.com/-/media/files/nrf/nrfweb/contact-tracing/uk-contact-tracing. pdf?la=en-us&revision= [accessed 2020-12-20] 20. White L, Cwalina C, Evans M, Flockhart F, Gamvros A. Contact tracing apps in the UK. Norton Rose Fulbright. 2020 Oct 05. URL: https://www.nortonrosefulbright.com/-/media/files/nrf/nrfweb/contact-tracing/uk-contact-tracing. pdf?la=en-us&revision= [accessed 2020-12-20] p [ ] 21. Kent C. A comedy of errors: the UK’s contact-tracing apps. Medical Device Network. 2020 Oct 06. URL: https://www. medicaldevice-network.com/features/uk-contact-tracing-app-problems/ [accessed 2020-12-21] p 21. Kent C. A comedy of errors: the UK’s contact-tracing apps. Medical Device Network. 2020 Oct 06. URL: https://www. medicaldevice-network.com/features/uk-contact-tracing-app-problems/ [accessed 2020-12-21] J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 5 (page number not for citation purposes) https://www.jmir.org/2021/3/e25726 XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Seto et al 22. Kaspar K. Motivations for Social Distancing and App Use as Complementary Measures to Combat the COVID-19 Pandemic: Quantitative Survey Study. J Med Internet Res 2020 Aug 27;22(8):e21613 [FREE Full text] [doi: 10.2196/21613] [Medline: 32759100] 22. Kaspar K. Motivations for Social Distancing and App Use as Complementary Measures to Combat the COVID-19 Pandemic: Quantitative Survey Study. J Med Internet Res 2020 Aug 27;22(8):e21613 [FREE Full text] [doi: 10.2196/21613] [Medline: 32759100] ] 23. Walrave M, Waeterloos C, Ponnet K. Adoption of a Contact Tracing App for Containing COVID-19: A Health Belief Model Approach. JMIR Public Health Surveill 2020 Sep 01;6(3):e20572 [FREE Full text] [doi: 10.2196/20572] [Medline: 32755882] 23. Walrave M, Waeterloos C, Ponnet K. Adoption of a Contact Tracing App for Containing COVID-19: A Health Belief Model Approach. JMIR Public Health Surveill 2020 Sep 01;6(3):e20572 [FREE Full text] [doi: 10.2196/20572] [Medline: 32755882] ] 24. Abuhammad S, Khabour O, Alzoubi K. COVID-19 Contact-Tracing Technology: Acceptability and Ethical Issues of Use. PPA 2020 Sep 18;Volume 14:1639-1647. [doi: 10.2147/ppa.s276183] ] 24. Abuhammad S, Khabour O, Alzoubi K. COVID-19 Contact-Tracing Technology: Acceptability and Ethical Issues of Use. PPA 2020 Sep 18;Volume 14:1639-1647. [doi: 10.2147/ppa.s276183] 25. Zhang M, Chow A, Smith H. COVID-19 Contact-Tracing Apps: Analysis of the Readability of Privacy Policies. J Med Internet Res 2020 Dec 03;22(12):e21572 [FREE Full text] [doi: 10.2196/21572] [Medline: 33170798] 25. Zhang M, Chow A, Smith H. COVID-19 Contact-Tracing Apps: Analysis of the Readability of Privacy Policies. J Med Internet Res 2020 Dec 03;22(12):e21572 [FREE Full text] [doi: 10.2196/21572] [Medline: 33170798] 26. Abueg M, Hinch R, Wu N, Liu L, Probert W, Wu A, et al. References Modeling the combined effect of digital exposure notification and non-pharmaceutical interventions on the COVID-19 epidemic in Washington state. medRxiv. Preprint published online on September 2, 2020. [doi: 10.1101/2020.08.29.20184135] 26. Abueg M, Hinch R, Wu N, Liu L, Probert W, Wu A, et al. Modeling the combined effect of digital exposure notification and non-pharmaceutical interventions on the COVID-19 epidemic in Washington state. medRxiv. Preprint published online on September 2, 2020. [doi: 10.1101/2020.08.29.20184135] p , [ ] 27. Farronato C, Iansiti M, Bartosiak M, Denicolai S, Ferretti L, Fontana R. How to get people to actually use contact-tracing apps. Harvard Business Review. 2020 Jul 15. URL: https://hbr.org/2020/07/ how-to-get-people-to-actually-use-contact-tracing-apps [accessed 2020-12-21] 27. Farronato C, Iansiti M, Bartosiak M, Denicolai S, Ferretti L, Fontana R. How to get people to actually use contact-tracing apps. Harvard Business Review. 2020 Jul 15. URL: https://hbr.org/2020/07/ how-to-get-people-to-actually-use-contact-tracing-apps [accessed 2020-12-21] 28. Utzerath J, Bird R, Cheng G, Ohara J. Contact tracing apps in China, Hong Kong, Singapore, Japan, and South Korea. Freshfields Bruckhaus Deringer. 2020 Apr 24. URL: https://digital.freshfields.com/post/102g5my/ contact-tracing-apps-in-china-hong-kong-singapore-japan-and-south-korea [accessed 2020-12-19] 28. Utzerath J, Bird R, Cheng G, Ohara J. Contact tracing apps in China, Hong Kong, Singapore, Japan, and South Korea. Freshfields Bruckhaus Deringer. 2020 Apr 24. URL: https://digital.freshfields.com/post/102g5my/ contact-tracing-apps-in-china-hong-kong-singapore-japan-and-south-korea [accessed 2020-12-19] 29. Exposure Notifications: Using technology to help public health authorities fight COVID-19. Goog google.com/covid19/exposurenotifications/ [accessed 2020-12-12] g g p 30. Smith J, Hyonhee S, Cha S. Ahead of the curve: South Korea's evolving strategy to prevent a coronavirus resurgence. Reuters. 2020 Apr 15. URL: https://www.reuters.com/article/us-health-coronavirus-southkorea-respons-idUSKCN21X0MO [accessed 2020-12-16] 30. Smith J, Hyonhee S, Cha S. Ahead of the curve: South Korea's evolving strategy to prevent a coronavirus resurgence. Reuters. 2020 Apr 15. URL: https://www.reuters.com/article/us-health-coronavirus-southkorea-respons-idUSKCN21X0MO [accessed 2020-12-16] 31. Klatt K. Corona apps: South Korea and the dark side of digital tracking. The Brussels Times. 2020 Apr 30. 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J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 6 (page number not for citation purposes) References Germany flips to Apple-Google approach on smartphone contact tracing. Reuters. 2020 Apr 26. URL: https://www.reuters.com/article/us-health-coronavirus-europe-tech-idUSKCN22807J [accessed 2020-12-21] 33. Busvine D, Rinke A. Germany flips to Apple-Google approach on smartphone contact tracing. Reuters. 2020 Apr 26. URL: https://www.reuters.com/article/us-health-coronavirus-europe-tech-idUSKCN22807J [accessed 2020-12-21] 34. Taylor K, Silver L. Smartphone ownership is growing rapidly around the world, but not always equally. Pew Research Center. 2019 Feb 05. URL: https://www.pewresearch.org/global/2019/02/05/smartphone-ownership-is-growing- rapidly-around-the-world-but-not-always-equally/ [accessed 2020-12-19] 34. Taylor K, Silver L. Smartphone ownership is growing rapidly around the world, but not always equally. Pew Research Center. 2019 Feb 05. 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JMIR Public Health Surveill 2020 Sep 01;6(3):e20572 [FREE Full text] [doi: 10.2196/20572] [Medline: 32755882] 37. Walrave M, Waeterloos C, Ponnet K. Adoption of a Contact Tracing App for Containing COVID-19: A Health Belief Model Approach. JMIR Public Health Surveill 2020 Sep 01;6(3):e20572 [FREE Full text] [doi: 10.2196/20572] [Medline: 32755882] 38. NHS COVID-19: the new contact-tracing app from the NHS. National Cyber Security Centre. 2020 May 04. URL: https:/ /www.ncsc.gov.uk/information/nhs-covid-19-app-explainer [accessed 2020-12-18] 38. NHS COVID-19: the new contact-tracing app from the NHS. National Cyber Security Centre. 2020 May 04. URL: https:/ /www.ncsc.gov.uk/information/nhs-covid-19-app-explainer [accessed 2020-12-18] g pp p 39. Millions Across Canada Now Using Made-in-Ontario COVID Alert App. Ontario Government. 2020 Oct 15. URL: https:/ /news.ontario.ca/en/release/58831/millions-across-canada-now-using-made-in-ontario-covid-alert-app [accessed 2020-12-19] 40. Bernacki E. Covid19 as wicked problem-solving: did your country change behaviours and make people laugh? LinkedIn. 2020 Jul 08. URL: https://www.linkedin.com/pulse/covid19-wicked-problem-solving-did-your-country-change-ed-bernacki/ [accessed 2020-12-19] 40. Bernacki E. Covid19 as wicked problem-solving: did your country change behaviours and make people laugh? LinkedIn. 2020 Jul 08. URL: https://www.linkedin.com/pulse/covid19-wicked-problem-solving-did-your-country-change-ed-bernacki/ [accessed 2020-12-19] J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 7 (page number not for citation purposes) Abbreviations API: application programming interface QR: quick response J Med Internet Res 2021 \| vol. 23 \| iss. 3 \| e25726 \| p. 6 (page number not for citation purposes) https://www.jmir.org/2021/3/e25726 XSL•FO RenderX Seto et al JOURNAL OF MEDICAL INTERNET RESEARCH Edited by G Eysenbach, C Basch; submitted 12.11.20; peer-reviewed by J Li, J Ropero; comments to author 04.12.20; revised version received 24.12.20; accepted 19.02.21; published 04.03.21 Please cite as: Seto E, Challa P, Ware P Adoption of COVID-19 Contact Tracing Apps: A Balance Between Privacy and Effectiveness J Med Internet Res 2021;23(3):e25726 URL: https://www.jmir.org/2021/3/e25726 doi: 10.2196/25726 PMID: 33617459 Edited by G Eysenbach, C Basch; submitted 12.11.20; peer-reviewed by J Li, J Ropero; comments to author 04.12.2 received 24.12.20; accepted 19.02.21; published 04.03.21 Please cite as: Seto E, Challa P, Ware P Adoption of COVID-19 Contact Tracing Apps: A Balance Between Privacy and Effectiveness J Med Internet Res 2021;23(3):e25726 URL: https://www.jmir.org/2021/3/e25726 doi: 10.2196/25726 PMID: 33617459 ©Emily Seto, Priyanka Challa, Patrick Ware. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 04.03.2021. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in the Journal of Medical Internet Research, is properly cited. The complete bibliographic information, a link to the original publication on http://www.jmir.org/, as well as this copyright and license information must be included. https://www.jmir.org/2021/3/e25726 XSL•FO RenderX
https://openalex.org/W4391904573	http://catalog.liha-pres.eu/index.php/liha-pres/catalog/download/251/6946/15613-1	Ukrainian	null	Industry 4.0 and the management of a higher education institution	ГУМАНІТАРНИЙ І ІННОВАЦІЙНИЙ РАКУРС ПРОФЕСІЙНОЇ МАЙСТЕРНОСТІ: ПОШУКИ МОЛОДИХ ВЧЕНИХ	2,023	cc-by	1,111	DOI https://doi.org/10.36059/978-966-397-357-9-49 DOI https://doi.org/10.36059/978-966-397-357-9-49 INDUSTRY 4.0 AND THE MANAGEMENT OF A HIGHER EDUCATION INSTITUTION Lebediev М. K. Postgraduate Student at the Department of Smart Economics Kyiv National University of Technologies and Design Kyiv, Ukraine The constantly changing business environment is hyperdynamic, volatile, and ambiguous. Alongside this, the socio-economic landscape is highly unpredictable, and the national economy is undergoing structural shifts also due to increasing involvement and the level of participation in globalization processes. In this context, digitalization is considered as an objective and integral factor in a rapidly evolving society. The expansion of this phenomenon is widespread at different levels and in different directions. These conditions present a challenge for employers seeking workers who meet their professional requirements. One of the key links in the formation of the human potential of the society is the higher education institution. The world is changing too fast, and higher education institutions are faced with the urgent need not only to adapt, but to change dramatically. In the modern educational scenario, educators must prepare students to use technologies that do not yet exist [1, с. 65]. Society is undergoing the fourth industrial revolution, Industry 4.0 [2], which integrates advanced technologies including the Internet, cloud technology, cyber-physical systems, artificial intelligence, big data analytics, and the Internet of Things. The growth of a service society and a communications-based economy necessitates strong communication skills and competencies such as critical and creative thinking, emotional intelligence, resilience, problem-solving, cognitive flexibility, digital fluency, growth mindset, cross-cultural literacy, rapid decision-making under uncertainty, and risk management ability. Computer-mediated communication is transforming the structure of the workflow in enterprises, in some cases dramatically decreasing the necessity for physical attendance at the workplace. In turn, one crucial criterion encompasses performing professional tasks within a digital smart environment. This paradigm highlights the need for a fresh outlook regarding education processes in higher education institutions, which remain pivotal in cultivating adept professionals. 177 The university is a part of formal education. In this context, the key challenge is the capacity to deviate from standardized uniform learning, by introducing changes that align with the trend toward personalized learning. Additionally, it’s essential to create a strong value proposition that focuses on meeting individual preferences, customizing educational paths, and increasing opportunities for self-education. Interactive learning environments and EdTech tools, such as modern learning management system (LMS), like Moodle, offer great potential for planning, implementing, and assessing specific learning processes either alongside or in place of in-person learning. DOI https://doi.org/10.36059/978-966-397-357-9-50 TQM ЯК ЕФЕКТИВНИЙ ІНСТРУМЕНТ УПРАВЛІННЯ ЯКІСТЮ У ФАРМАЦЕВТИЧНОМУ БІЗНЕСІ Ляшенко-Щербакова В. В. здобувачка IV курсу навчання за спеціальністю 073 – Менеджмент Міжнародний гуманітарний університет м. Одеса, Україна Якість надаваємих послуг є важливим чинником конкурентоспроможності будь-якого аптечного закладу. Задля досягнення необхідного рівня якості товарів і послуг, а також економічної ефективності діяльності аптечних закладів в умовах високого рівня конкуренції на фармацевтичному ринку України, найбільш перспективним й оптимальним напрямком розвитку цих закладів є розробка і впровадження системи менеджменту якості. Загалом система менеджменту якості охоплює усі бізнес– і технологічні процеси в організації задля підвищення якості продукції або послуг. Її DOI https://doi.org/10.36059/978-966-397-357-9-50 INDUSTRY 4.0 AND THE MANAGEMENT OF A HIGHER EDUCATION INSTITUTION On the other hand, today the education sector constitutes a relevant sector of the economy. «The global smart education and learning market was valued at $260 billion in 2022, and is projected to reach $1.4 trillion by 2032, growing at a CAGR of 18.4% from 2023 to 2032» [3]. A balanced organizational and economic mechanism (OEM) for managing higher education institution is essential to achieving significant progress in its development. Such coordination promotes the implementation of a qualitative leap that propels the institution forward. This mechanism ensures the synergy and synchronization of internal processes (including academic, research, and business operations) and external interactions (with university students, stakeholders, the state, and international partners). Operations management [4, p. 5] is a fundamental component of the management mechanism of an institution that is concerned with the management of resources that facilitate the creation and delivery of products and services. The primary objective of an OEM for managing higher education institution is to ensure the relevant scientific-intellectual, financial, material- technical and organizational-administrative aspects necessary for such institution to function as a center of innovative knowledge creation and startup cluster. That includes also formation of academic and scientific-innovative center, integrated into the national production sector and European ecosystem of higher education, which also is manifested through the position in global rankings. It is preferable for OEM for managing higher education institution to implement a strategy that emphasizes not only current but also strategic flexibility, since innovation changes constantly accompany the activities of the institution. Adapting an institution’s value proposition requires constant market testing. Analysis and forecasting should take into account the feedback from university students and stakeholders. Such an approach ensures relevance, effectiveness and readiness to review and adjust the value proposition as needed. A holistic approach to OEM for managing higher education institution also includes guaranteeing the safety of their internal and external communication 178 interactions. In a highly digitized and globalized society, cyber threats can pose significant risks [5]. interactions. In a highly digitized and globalized society, cyber threats can pose significant risks [5]. Bibliography: 1. Mete R., Riegel C., Kozen A., Polka W. What Every Contemporary Teacher Educator Needs to Know About Technology... But, Didn’t Know Who to Ask! Critical Issues in Teacher Education. 2017. Vol. XXIV. Р. 65–79. 1. Mete R., Riegel C., Kozen A., Polka W. What Every Contemporary Teacher Educator Needs to Know About Technology... But, Didn’t Know Who to Ask! Critical Issues in Teacher Education. 2017. Vol. XXIV. Р. 65–79. 2. Матюшенко І. Ю. Технологічна конкурентоспроможність України в умовах нової промислової революції і розвитку конвергентних технологій. Проблеми економіки. 2016. № 1. С. 108–120. 3. Smart Education and Learning Market Research, 2032. URL: https://www.alliedmarketresearch.com/smart-education-learning-market. (дата звернення: 10.12.2023). ( р ) 4. Slack N., Brand-Jones A., Johnston R. Operations Management, 8th ed. Harlow: Pearson, 2016. ( р ) 4. Slack N., Brand-Jones A., Johnston R. Operations Management, 8th ed. Harlow: Pearson, 2016. 5. European Union Agency for Cybersecurity: ENISA Threat Landscape 2023. URL: https://www.enisa.europa.eu/publications/enisa-threat- landscape-2023. (дата звернення: 10.12.2023). TQM ЯК ЕФЕКТИВНИЙ ІНСТРУМЕНТ УПРАВЛІННЯ ЯКІСТЮ У ФАРМАЦЕВТИЧНОМУ БІЗНЕСІ Ляшенко-Щербакова В. В. здобувачка IV курсу навчання за спеціальністю 073 – Менеджмент Міжнародний гуманітарний університет м. Одеса, Україна Якість надаваємих послуг є важливим чинником конкурентоспроможності будь-якого аптечного закладу. Задля досягнення необхідного рівня якості товарів і послуг, а також економічної ефективності діяльності аптечних закладів в умовах високого рівня конкуренції на фармацевтичному ринку України, найбільш перспективним й оптимальним напрямком розвитку цих закладів є розробка і впровадження системи менеджменту якості. Загалом система менеджменту якості охоплює усі бізнес– і технологічні процеси в організації задля підвищення якості продукції або послуг. Її 179
https://openalex.org/W2169473616	https://bmcwomenshealth.biomedcentral.com/counter/pdf/10.1186/1472-6874-10-35	English	null	Awareness and knowledge of HPV, cervical cancer, and vaccines in young women after first delivery in São Paulo, Brazil - a cross-sectional study	BMC women's health	2,010	cc-by	5,695	© 2010 Rama 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. Cristina H Rama1, Luisa L Villa2, Sonia Pagliusi3, Maria A Andreoli2, Maria C Costa2, Aline L Aoki4, Adhemar Longatto-Filho5,6, José Eluf-Neto7 Cristina H Rama1, Luisa L Villa2, Sonia Pagliusi3, Maria A Andreoli2, Maria C Costa2, Aline L Aoki4, Adhemar Longatto-Filho5,6, José Eluf-Neto7 Abstract Background: The success of HPV vaccination programs will require awareness regarding HPV associated diseases and the benefits of HPV vaccination for the general population. The aim of this study was to assess the level of awareness and knowledge of human papillomavirus (HPV) infection, cervical cancer prevention, vaccines, and factors associated with HPV awareness among young women after birth of the first child. Methods: This analysis is part of a cross-sectional study carried out at Hospital Maternidade Leonor Mendes de Barros, a large public maternity hospital in Sao Paulo. Primiparous women (15-24 years) who gave birth in that maternity hospital were included. A questionnaire that included questions concerning knowledge of HPV, cervical cancer, and vaccines was applied. To estimate the association of HPV awareness with selected factors, prevalence ratios (PR) were estimated using a generalized linear model (GLM). Results: Three hundred and one primiparous women were included; 37% of them reported that they “had ever heard about HPV”, but only 19% and 7%, respectively, knew that HPV is a sexually transmitted infection (STI) and that it can cause cervical cancer. Seventy-four percent of interviewees mentioned the preventive character of vaccines and all participants affirmed that they would accept HPV vaccination after delivery. In the multivariate analysis, only increasing age (P for trend = 0.021) and previous STI (P < 0.001) were factors independently associated with HPV awareness ("had ever heard about HPV”). Conclusions: This survey indicated that knowledge about the association between HPV and cervical cancer among primiparous young women is low. Therefore, these young low-income primiparous women could benefit greatly from educational interventions to encourage primary and secondary cervical cancer prevention programs. * Correspondence: crisrama@usp.br 1Hospital Maternidade Leonor Mendes de Barros, Sao Paulo, Brazil Av. Celso Garcia, 2477; 03015-000; Belenzinho Full list of author information is available at the end of the article Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Open Access Background or shortly thereafter to achieve optimal vaccine effective- ness. The US Advisory Committee on Immunization Practices (ACIP) recommended routine HPV vaccina- tion of girls at age 9-12 years and catch-up vaccination for females aged 13-26 years who have not been pre- viously vaccinated or who have not completed the full immunization series [4]. Genital infection by oncogenic human papillomavirus (HPV) is a necessary factor in the development of cancer of the cervix [1]. Although the HPV family of viruses includes more than 100 different viral genotypes, HPV 16 and 18 were identified in about 70% of cervical cancer cases [2], while HPV 6 and 11 can cause genital warts [3]. Two prophylactic vaccines to prevent infections by high risk HPV viral genotypes 16 and 18 were available; ideally they should be administered before sexual debut However, providing a vaccine “against cancer” to ado- lescent girls raises several challenges because many young unmarried girls and women face significant chal- lenges in accessing the health care necessary to meet their sexual and reproductive health needs [5]. For this reason, in some countries school vaccination programs have been established as a strategy to vaccinate girls * Correspondence: crisrama@usp.br 1Hospital Maternidade Leonor Mendes de Barros, Sao Paulo, Brazil Av. Celso Garcia, 2477; 03015-000; Belenzinho Full list of author information is available at the end of the article Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Page 2 of 7 A total of 509 women were invited to participate: 24 refused, 163 women previously interested in participat- ing did not return to the post-natal visit, and 322 attended the post-natal visit. However, 18 women were not eligible (11 attended the post-natal visit more than 60 days after delivery, one had HIV positive serology, one had more than one delivery, and five were over 24 years old). Three eligible women were excluded by the investigators: one had an acute infectious disease and two aged <18 years lived in a reformatory and had difficulty obtaining a legal representative’s signature on the informed consent form within the study period. Therefore, 301 primiparous women were included in the analysis. against HPV. However, pregnant adolescents and girls of lower socio-economic status are more likely to drop out of school, therefore, opportunities for catch-up vaccina- tion are valuable to improve coverage among the at risk young female population. Background Then, the success of HPV vaccination programs will require improving awareness regarding HPV, cervical cancer, and the benefits of HPV vaccination for the gen- eral population. Probably, knowledge about HPV has been changing in recent years, given public awareness campaigns around the announcement of the regulatory approval and public announcements of the HPV vaccines. Thereafter, identifying which groups of women remain unfamiliar with these subjects and would, therefore, benefit from educational messages is crucial, particularly in the target age group for HPV vaccination. Study population This study is part of a cross-sectional study carried out from June 2006 to February 2007. The study was con- ducted out at Hospital Maternidade Leonor Mendes de Barros (HMLMB), one of the largest public maternity hospitals in the city of Sao Paulo, Brazil. All primiparous women aged between 15 and 24 years who had been liv- ing in the metropolitan area of Sao Paulo for at least six months and gave birth at this hospital after more than 32 weeks of gestation were eligible for the study. The following exclusion criteria, as previously published [6], were applied: non Brazilian, inability or refuse to give informed consent or immunodeficiency (including AIDS/HIV infection checked in medical records). Answers to open questions were grouped in categories according their similarities to be quantified. Data collection During the routine post-natal visit, women were enrolled and interviewed, in an appropriate setting ensuring priv- acy, by trained interviewers. Using a standardized ques- tionnaire (Additional file 1), information obtained included demographic characteristics, sexual behaviour, reproductive history, contraceptive practice, smoking habits, and awareness and knowledge of HPV, cervical cancer (causes and prevention), vaccines, and HPV vac- cine acceptability. Only women who answered questions about awareness of each one of these subjects affirma- tively were asked the following open questions, respec- tively: Could you explain how HPV may be caught and what it may cause? Could you explain what the causes(s) of cervical cancer are? Could you explain how the exami- nation to prevent cervical cancer or “cytological tests” examination is performed? Could you explain what vac- cines are? Do you know what vaccines you have been given? Hence, the aim of this study was to assess the level of awareness and knowledge of HPV infection, cervical cancer prevention, and vaccines, and factors associated with HPV awareness among low-income young women after first delivery in the city of São Paulo, Brazil. The rationale for studying this population is that first deliv- ery health care services could represent a suitable opportunity to offer HPV vaccines for adolescents and young women who have not been previously vaccinated. Discussion The awareness of HPV among these young primiparous women was low, as only one third of the participants reported having “ever heard about HPV”, despite the fact that the present analysis began 16 days after public news and announcements about the first regulatory approval of one of the HPV vaccines, on June 08, 2006 [10], and the fact that participants in the present study had relatively high levels of education within the Brazi- lian context. When asked about cervical cancer cause(s), very few participants admitted knowing the cause(s) of cervical cancer (8%) and only 6% of women in the sample enum- erated HPV as a cervical cancer cause. Surveys conducted previously and before the regula- tory approval of HPV vaccines also showed a low level of awareness of HPV (30-40%) [11-14]. The results of the present study are in line with other recent studies, carried out after regulatory approval of HPV vaccina- tion, that showed limited levels of awareness of HPV. In the United Kingdom and Italy (where HPV vaccine is free of charge for girls of 12 years of age), only about 24% and 30% of respondents, respectively, reported awareness of HPV [15,16]. Slightly more than a half of the total number of parti- cipants referred to knowing about the examination to prevent cervical cancer or cytological tests. However, only 27% answered that in this test some kind of mate- rial is collected (a sample of cells from the cervix, cells, secretion, fluid, etc.). Seventy-four percent of interviewees mentioned the preventive character of vaccines, and only 57% of them answered correctly concerning vaccines that had been given to them (giving the name of at least one vaccine or the name of the disease that the vaccine targeted). In the present study, awareness of HPV was the first question and we did not ask any open questions con- cerning knowledge of HPV, cervical cancer causes and prevention, or vaccines when the participant was not aware of any of theses subjects, in order to avoid influ- encing the answers to the open questions or obtaining an answer given correctly by luck, as can occur in stu- dies that use multiple-choice answers. g All participants affirmed that they would accept vacci- nation after delivery if the HPV vaccine were available (Table 1). Statistical analysis Levels of awareness and knowledge of HPV, cervical can- cer, and vaccines were expressed as percentages. To estimate the association of HPV awareness with selected factors, prevalence ratios (PR) and 95% confi- dence intervals (CI) were calculated, with HPV awareness as the dependent variable and various factors as indepen- dent variables. Most independent variables were grouped into two or more categories. For ordered categorical independent variables, tests for linear trend (Chi-square for trend) in the PR were conducted by categorizing the independent variables and entering the continuous scores. Variables selected in univariate analysis at a 0.20 significance level were included in the multivariate analy- sis. PR and their 95% CI were estimated using a general- ized linear model (GLM) with binomial distribution and log link function [7]. Statistical significance was assessed The study protocol was submitted and approved by the National Ethical Committee (CONEP) (number 188/2006). Women were recruited to take part in the study dur- ing the post-delivery follow-up period in the hospital. Eligible women were contacted by a health professional (a nurse or physician) and asked whether they wanted to know about a study on prevention of cancer of the cervix; those interested had a post-natal visit scheduled within 43 to 60 days after delivery to be enrolled in the study. Women had to attend the post-natal visit and sign the informed consent form to be included in the study. The study protocol was submitted and approved by the National Ethical Committee (CONEP) (number 188/2006). Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Page 3 of 7 the analysis including the other four variables, years of schooling (P for trend = 0.307) and marital status (P = 0.296) were not significantly associated with HPV awareness. The multivariate analysis revealed that only age (P for trend = 0.021) and previous STI (P < 0.001) were independently associated with HPV awareness. The prevalence ratio of HPV awareness for women aged 19-21 years was 1.32 with a 95% CI of 0.87-2.01, and for women aged 22-24 years it was 1.63 with a 95% CI of 1.07-2.48 (reference group: women 15-18 years old). Women who reported a previous STI were more likely to be aware of HPV (PR = 2.05; 95% CI: 1.46-2.87) com- pared to women who had never had any STI. using the likelihood ratio test [8]. Results The mean age of study participants was 19.9 years (median 20.0 years). Over 60% were white and most women (88%) had at least eight years of schooling (the first grade). About two thirds reported incomes (includ- ing earnings of all family members) of less than four minimum wages per month (equivalent to US$ 479.20 during the study period). Awareness of HPV among these young women after first delivery was low: only one third of them reported that they had ever heard of HPV. Among those who had heard of it, less than a quarter knew that HPV can cause cervical cancer; about half knew that HPV is a sexually transmitted infection (STI), and only two parti- cipants knew that it can cause genital warts. Only 7% (n = 20) of all participants answered both that HPV is an STI and that it can cause cervical cancer. Statistical analysis A two-sided P value of less than 0.05 was considered to indicate statistical signif- icance. All analyses were performed using STATA version 8.2 [9]. Discussion HPV awareness increased with increasing age (P for trend = 0.003) and with increasing years of schooling (P for trend = 0.039) (Table 2). Table 3 shows the distri- bution of HPV awareness according to sexual behaviour, reproductive characteristics, and contraception history. Only ever having had a previous STI was significantly associated with HPV awareness (PR = 2.55; 95% CI: 1.93-3.36). Only 19% and 7% of the present study participants, respectively, knew that HPV is an STI and that it can cause cervical cancer. Another study, carried out in north-eastern Brazil, assessing young women (16- 23 years) showed similar results to the present study: less than 10% of participants acknowledged that HPV might lead to cervical cancer; however, a higher proportion of those women (67%) knew that HPV is sexually trans- mitted [17]. This difference might be explained by the fact that these women had higher educational levels than women in the present study (61% and 50% respectively All selected variables (age, years of schooling, marital status, income and previous STI), were included in the multivariate analysis model. However, when years of schooling and income were included, the model did not converge. Then, the variable income was taken out. In Page 4 of 7 Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Table 1 Answers about awareness and knowledge of HPV, cervical cancer (causes and prevention), and vaccines among 301 participants Total % Had ever heard about HPV 110 37,0 Knowledge about how HPV may be caught and what it may cause 62 20,6 When asked to explain how HPV may be caught and and what it may cause1 HPV is a sexually transmitted disease 58 19,0 HPV can cause cervical cancer 22 7,3 HPV can cause genital warts 2 0,7 Did not know 2 0,7 Knowledge about cervical cancer causes 23 7,6 When asked to explain what they knew about cervical cancer causes1 HPV 19 6,3 Hereditary disease 1 0,3 Sexual transmitted disease 1 0,3 Did not know 2 0,7 Knowledge about the examination to prevent cervical cancer or “Pap smear” 157 52,2 When asked to explain what “Pap smear” is Collection of material, secretion, cells, etc. Discussion 80 26,6 Like a gynecological examination2 41 13,6 Repeated the phrase “examination to prevent cervical cancer’’ 22 7,3 Other 11 3,7 Did not know 3 1,0 Knowledge about vaccines 246 81,7 When asked to explain what they knew about Prevention of disease 223 74,1 Treatment 13 4,3 Other 7 2,3 Did not know 3 1,0 Knowledge about how many vaccines received 183 60,8 When asked to explain which vaccines received Answered at least one vaccine correctly 171 56,8 Other 7 2,3 Did not know 5 1,7 Acceptance of vaccination against HPV after delivery3 301 100,0 1 Participants could give more than one answer. 2 Participant seems to know something about it but it is doubtful that she could distinguish it from another gynecological examination. 3 The interviewer first explained what vaccines are and about HPV vaccine. t awareness and knowledge of HPV, cervical cancer (causes and prevention), and vaccines Table 1 Answers about awareness and knowledge of HPV, cervical cancer (causes and prevention), and vaccines among 301 participants When asked to explain what “Pap smear” is When asked to explain what they knew about When asked to explain which vaccines received g articipant seems to know something about it but it is doubtful that she could distinguish it from another gynecological examination. he interviewer first explained what vaccines are and about HPV vaccine. 2 Participant seems to know something about it but it is doubtful that she could distinguish it from another gynecological 3 The interviewer first explained what vaccines are and about HPV vaccine. had high school education or above). Alternatively, because the authors [17] used five multiple-choice answers per question, a bias cannot be excluded. aware, and whether they were able to recognize the dif- ferences between the ordinal gynecological test and the cytological tests. Based on our data, just 27% of the young primiparous women were able to recognize the differences. We also studied the relationship between HPV aware- ness and several factors. The multivariate analysis high- lights two factors: having had a previous STI and increasing age remained as factors associated with HPV awareness. Accordingly, some authors reported that increasing age (women 14-24 years) [16] and having had a personal, familiar, or friendly history of previous STI or cervical cancer were associated with an increased awareness of HPV and accurate knowledge of the HPV- cervical cancer link [13,14,16]. Discussion Other studies regarding cytological tests estimated whether the women were aware of their purpose, and hence are not suitable for direct comparisons. They reported a variety of results (10-89%) concerning the interviewees’ awareness of the cytological tests’ purposes [11,16,17]. In the present study, the awareness regarding vaccines was high, with 74% of the interviewees mentioning their preventative aspect, and 57% of women could identify at least one of the vaccines they had received; that is, they could name the vaccine or the disease which it was intended to prevent. Half of all participants reported awareness concerning cervical cancer prevention by cytological test, although a smaller number were aware of how it is performed. The query was intended to estimate how many women were Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Page 5 of 7 Table 2 Prevalence ratios and corresponding 95% confidence intervals (CI) for HPV awareness according to selected socio-demographic characteristics and smoking habits among 301 young primiparous women, Sao Paulo, Brazil, 2006-2007 Total HPV awareness Prevalence ratio 95% CI P Age (years) 0.003* 15-18 90 26.7% 1 19-21 119 35.3% 1.32 [0.87-2.02] 22-24 92 47.8% 1.79 [1.20-2.69] Ethnic group 0.923 White 190 37.9% 1 Black 31 32.3% 0.85 [0.50-1.46] Mulatto 77 35.1% 0.93 [0.65-1.32 ] Indian 3 33.3% 0.88 [1.76-4.40 ] Years of schooling 0.039* ≤7 37 29.7% 1 8 67 28.4% 0.95 [0.51-1.78] 9 or 10 64 35.9% 1.21 [0.67-2.19] ≥11 133 42.9% 1.44 [0.85-2.46] Marital status 0.093 Living with partner 245 38.8% 1 Single 56 26.8% 0.69 [0.44-1.10] Income1 0.054* < 1 13 38.5% 1 1-3 188 31.9% 0.83 [0.40-1.70] 4-6 80 45.0% 1.17 [0.56-2.43] 7-10 11 27.3% 0.71 [0.22-2.31] > 10 5 80.0% 2.08 [0.92-4.70] Smoking habits 0.493 Never 231 35.5% 1 Ever2 70 40.0% 1.13 [0.81-1.58] 1 In number of minimum wages per month (one minimum wage = R$250.00 or US$119.80; US$1.00 = R$ 2.09, February 2007). Data missing for 4 participants. 2 Included current and former smokers. Chi f d Table 2 Prevalence ratios and corresponding 95% confidence intervals (CI) for HPV awareness according to selected socio-demographic characteristics and smoking habits among 301 young primiparous women, Sao Paulo, Brazil, 2006 2007 1 In number of minimum wages per month (one minimum wage = R$250.00 or US$119.80; US$1.00 = R$ 2.09, February 2007). Data missing for 4 participants. 2 Included current and former smokers. Chi-square for trend. Discussion illnesses through vaccines and on the HPV prophylactic vaccine was given by the interviewers in the present study. With regard to the acceptability of the vaccine, despite the inadequate knowledge of HPV and cervical cancer, all participants reported that they would accept vaccina- tion after delivery if the HPV vaccine was available. As other studies have reported, there was a generally favourable attitude toward HPV vaccines; despite the low level of knowledge about the link between HPV and cervical cancer, 91% and 88% of women would agree to receive the vaccine in surveys that found that only 15% [18] and 38% [19], respectively, had heard of HPV. In fact, the main factor associated with the acceptance of the HPV prophylactic vaccine in studies carried out prior to its approval was the knowledge of the partici- pants concerning the purpose of vaccines [20,21]. Public acceptance and usage of a prophylactic vaccine are related to the level of knowledge about the disease, which the vaccine will provide protection from [22]. Education about HPV prophylactic vaccines, cervical cancer, and related topics is needed in every country where the vaccine is available. However, it is important to report that the question about the knowledge of vaccines preceded the question about the acceptability of the HPV vaccine, and for the participants who did not know the answer to the first question, one brief explanation on the prevention of The main limitation of this study is that 32% of eligi- ble women who were previously interested in participat- ing did not return to the post-natal visit. The likely Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Page 6 of 7 Rama et al. Conclusions In Brazil, Quadrivalent and Bivalent HPV vaccines have been approved by regulatory authorities for females aged, respectively, 9 to 26 years and 10 to 25 years but they are not yet included in public vaccination programs [23,24]. Despite the implementation of a national cervi- cal cancer screening program based on cytology in Brazil, about 20,000 cervical cancer cases occur each year [25], indicating a need for revised efforts in educa- tion, prevention, and detection. Young women of low socio-economic status after first delivery had low levels of knowledge of HPV and cervi- cal cancer development and prevention. Thus, opportu- nities for educational interventions during the prenatal and postpartum periods are valuable to improve knowl- edge about these subjects. Discussion However, we compared participants and non-respondents using medical hospital records and they did not differ according to several fac- tors: age (P = 0.205), marital status (P = 0.480), smoking habits (P = 0.183), prenatal health care (P = 0.436), and number of prenatal health care visits (P = 0.214). There- fore, this limitation is unlikely to have affected our results significantly. Discussion BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 Page 6 of 7 Table 3 Prevalence ratios and corresponding 95% confidence intervals (CI) for HPV awareness according to selected sexual behaviours, reproductive characteristics, and history of contraception among 301 young primiparous women, Sao Paulo, Brazil, 2006-2007 Total HPV awareness Prevalence ratio 95% CI P Age at first sexual intercourse 0.307 ≤15 132 33.3% 1 > 15 169 39.1% 1.17 [0.86-1.59] Number of lifetime sexual partners 0.238* 1 120 37.5% 1 2-3 116 28.5% 0.76 [0.52-1.10] ≥4 65 49.2% 1.31 [0.94-1.84] Previous STI1 0.002 No 292 34.9% 1 Yes 9 88.9% 2.55 [1.93-3.36] Abortion 0.603 No 282 36.2% 1 Yes 19 42.1% 1.16 [0.67-2.02] Contraception 0.398 No 74 32.4% 1 Yes 227 37.9% 1.17 [0.81-1.69] 1STI: Sexually Transmitted Infection. *Chi-square for trend. Table 3 Prevalence ratios and corresponding 95% confidence intervals (CI) for HPV awareness according to selected sexual behaviours, reproductive characteristics, and history of contraception among 301 young primiparous women, Sao Paulo, Brazil, 2006-2007 Our findings could add information to knowledge of the Latin American scenario reflecting the views of young women of lower socio-economic status after first delivery. Despite the possibility to provide a large number of infor- mation to mothers during the prenatal and postpartum periods, this group of women had low levels of knowledge of HPV and cervical cancer development and prevention. Thus, these women could benefit greatly from educational interventions to encourage participation in primary and secondary cervical cancer prevention programs. reason for the relatively large number of women not attending the post-natal visit at our hospital is the avail- ability of post-natal services in public health care centres near their homes, and therefore women may have pre- ferred those for convenience. However, we compared participants and non-respondents using medical hospital records and they did not differ according to several fac- tors: age (P = 0.205), marital status (P = 0.480), smoking habits (P = 0.183), prenatal health care (P = 0.436), and number of prenatal health care visits (P = 0.214). There- fore, this limitation is unlikely to have affected our results significantly. reason for the relatively large number of women not attending the post-natal visit at our hospital is the avail- ability of post-natal services in public health care centres near their homes, and therefore women may have pre- ferred those for convenience. Additional material Although the possibility of knowledge of HPV can be changing in Brazil, probably it has not been changing significantly, because no large educational campaign about HPV for the population has been improved by the government in the last years. Additional file 1: QUESTIONNAIRE. Structured epidemiologic questionnaire with information about demographic characteristics, sexual behavior, reproductive history, contraceptive practice, smoking habits and questions concerning knowledge of HPV, cervical cancer, and vaccines. Page 7 of 7 Page 7 of 7 Rama et al. BMC Women's Health 2010, 10:35 http://www.biomedcentral.com/1472-6874/10/35 7. McCullagh P, Nelder JA: Generalized linear models. New York: Chapman and Hall;, 2 1989. Author details 1 12. Waller J, McCaffery K, Forrest S, Szarewski A, Cadman L, Wardle J: Awareness of human papillomavirus among women attending a well woman clinic. Sex Transm Infect 2003, 79:320-2. 1Hospital Maternidade Leonor Mendes de Barros, Sao Paulo, Brazil Av. Celso Garcia, 2477; 03015-000; Belenzinho. 2Virology Department of Ludwig Institute for Cancer Research, Sao Paulo, Brazil. 3Formerly with Immunizations, Vaccines and Biologicals Department, World Health Organization, Geneva, Switzerland. 4Faculdade de Medicina da Universidade de Santo Amaro, Sao Paulo, Brazil. 5Laboratório de Investigação Médica (LIM) 14, Departamento de Patologia, Faculdade de Medicina, Universidade de São Paulo, Sao Paulo, Brazil. 6Life and Health Sciences Research Institute (ICVS), School of Health Sciences Universidade do Minho - Campus de Gualtar4710-057 Braga, Portugal. 7Departamento de Medicina Preventiva, Faculdade de Medicina, Universidade de São Paulo, Sao Paulo, Brazil. 13. Nøhr B, Munk C, Tryggvadottir L, Sparén P, Tran TN, Nygård M, Skare GB, Dasbach E, Liaw KL, Kjaer SK: Awareness of human papillomavirus in a cohort of nearly 70,000 women from four Nordic countries. Acta Obstet Gynecol Scand 2008, 87:1048-54. 14. Tiro JA, Meissner HI, Kobrin S, Chollette V: What do women in the U.S. know about human papillomavirus and cervical cancer? Cancer Epidemiol Biomarkers Prev 2007, 16:288-94. 15. Marlow LA, Waller J, Wardle J: Public awareness that HPV is a risk factor for cervical cancer. Br J Cancer 2007, 97:691-4. Received: 11 June 2010 Accepted: 22 December 2010 Published: 22 December 2010 23. Brasil. Ministério da Saúde. Portaria N° 3.124 de 7 de dezembro de 2006: Vacina quadrivalente recombinante contra papilomavírus humanos. Diário Oficial da União; Poder Executivo, de 28 de agosto de; 2006 [ftp://ftp. saude.sp.gov.br/ftpsessp/bibliote/informe_eletronico/2006/iels.dezembro.06/ iels236/U_PT-MS-GM-3124_071206.pdf], (accessed 20 Dec 2010). List of abbreviations used (CI): Confidence Intervals; (GLM): Generalized Linear Model; (HPV): Human Papillomavirus; (PR): Prevalence Ratios; (STI): Sexually Transmitted Infection. (CI): Confidence Intervals; (GLM): Generalized Linear Model; (HPV): Human Papillomavirus; (PR): Prevalence Ratios; (STI): Sexually Transmitted Infection. 8. Breslow NE, Day NE: Statistical methods in cancer research. In The analysis of case-control studies. Publ. no. 32. Volume I. Lyon: International Agency for Research on Cancer; 1980(32). Authors’ contributions 16. Di Giuseppe G, Abbate R, Liguori G, Albano L, Angelillo IF: Human papillomavirus and vaccination: knowledge, attitudes, and behavioural intention in adolescents and young women in Italy. Br J Cancer 2008, 99:225-9. CHR: participated in study design, coordination during field work, acquisition of data, interpretation of data and helped to draft the manuscript. LLV: conceived of the study, participated in its design and helped to draft the manuscript. SP: conceived of the study, participated in its design and helped to draft the manuscript. MAA: have made substantial contributions to acquisition of data, and helped to draft the manuscript. MCC: have made substantial contributions to acquisition of data, and helped to draft the manuscript. ALA: have made substantial contributions to acquisition of data, interpretation of data, and helped to draft the manuscript. ALF: have made substantial contributions to interpretation of data and helped to draft the manuscript. JEN: participated in the design of the study, performed the statistical analysis and helped to draft the manuscript. All authors read and approved the final manuscript. 17. Moreira ED Jr, Oliveira BG, Ferraz FM, Costa S, Costa Filho JO, Karic G: Knowledge and attitudes about human papillomavirus, Pap smears, and cervical cancer among young women in Brazil: implications for health education and prevention. Int J Gynecol Cancer 2006, 16:599-603. 18. Sauvageau C, Duval B, Gilca V, Lavoie F, Ouakki M: Human papilloma virus vaccine and cervical cancer screening acceptability among adults in Quebec, Canada. BMC Public Health 2007, 7:304. 19. Kwan TT, Chan KK, Yip AM, Tam KF, Cheung AN, Lo SS, Lee PW, Ngan HY: Acceptability of human papillomavirus vaccination among Chinese women: concerns and implications. BJOG 2009, 116:501-10. 20. Lazcano-Ponce E, Rivera L, Arillo-Santillán E, Salmerón J, Hernández-Avila M, Muñoz N: Acceptability of a human papillomavirus (HPV) trial vaccine among mothers of adolescents in Cuernavaca, Mexico. Arch Med Res 2001, 32:243-7. Acknowledgements 9. StataCorp: Stata Statistical Software: Release 8.2 College Station, TX, Stata Corporation; 2003. This study was supported by a research grant from Department of Immunization, Vaccines and Biologicals, World Health Organization (ref: V20- 181-13). The authors are grateful to Dr M.T. Aguado for helpful discussions. We are also grateful to Dr Corintio Mariani Neto for the unconditional support provided during field work and to nurses from Hospital Maternidade Leonor Mendes de Barros. 10. USA FDA (2006) FDA News Release 8 June 2006. 2006 [http://www.fda. gov/bbs/topics/NEWS/2006/NEW01385.html], (accessed 24 Jan 2007). 11. Hanisch R, Gustat J, Hagensee ME, Baena A, Salazar JE, Castro MV, Gaviria AM, Sánchez GI: Knowledge of Pap screening and human papillomavirus among women attending clinics in Medellín, Colombia. Int J Gynecol Cancer 2008, 18:1020-6. References 1. Walboomers JMM, Jacobs MV, Manos MM, Bosh FX, Kummer JA, Shah KV, Snijders PJF, Peto J, Meijer CJLM, Muñoz N: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999, 189:12-19. 1. Walboomers JMM, Jacobs MV, Manos MM, Bosh FX, Kummer JA, Shah KV, Snijders PJF, Peto J, Meijer CJLM, Muñoz N: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999, 189:12-19. Snijders PJF, Peto J, Meijer CJLM, Muñoz N: Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999, 189:12-19. 2. Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJ, Meijer CJ: Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003, 348:518-27. 24. Brasil. Ministério da Saúde. Resolução -RE No- 474, DE 21 de fevereiro de 2008: Vacina contra HPV oncogênico (16 e 18, recombinante, com advuvante AS04). Diário Oficial da União; Poder Executivo, de 25 de fevereiro de; 2008 [http://www.anvisa.gov.br/legis/suplemento/ 250208_suplemento_1.pdf], (accessed 02 October 2010). 24. Brasil. Ministério da Saúde. Resolução -RE No- 474, DE 21 de fevereiro de 2008: Vacina contra HPV oncogênico (16 e 18, recombinante, com advuvante AS04). Diário Oficial da União; Poder Executivo, de 25 de fevereiro de; 2008 [http://www.anvisa.gov.br/legis/suplemento/ 250208_suplemento_1.pdf], (accessed 02 October 2010). 3. de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H: Classification of papillomaviruses. Virology 2004, 324:17-27. 25. Brasil. Instituto Nacional de Câncer [INCA]. Coordenação de Prevenção e Vigilância: Estimativa 2008: Incidência de Câncer no Brasil. Rio de Janeiro; 2008 [http://www.inca.gov.br/conteudo_view.asp?id=1793], (accessed 20 Dec 2010). 25. Brasil. Instituto Nacional de Câncer [INCA]. Coordenação de Prevenção e Vigilância: Estimativa 2008: Incidência de Câncer no Brasil. Rio de Janeiro; 2008 [http://www.inca.gov.br/conteudo_view.asp?id=1793], (accessed 20 Dec 2010). 4. Markowitz LE, Dunne EF, Saraiya M, Lawson HW, Chesson H, Unger ER, Centers for Disease Control and Prevention: Quadrivalent human papillomavirus vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2007, 56(RR-2):1-24. 5. Pollack AE, Balkin M, Edouard L, Cutts F, Broutet N, WHO/UNFPA Working Group on Sexual and Reproductive Health and HPV Vaccines: Ensuring access to HPV vaccines through integrated services: a reproductive health perspective. Bull World Health Organ 2007, 85:57-63. Competing interests ll Luisa Lina Villa is a consultant and speaker for the Quadrivalent HPV Vaccine of Merck Sharp & Dohme. José Eluf-Neto has served as a consultant to GlaxoSmithKline in 2006. The others authors have no potential conflicts of interest to disclose. 21. Davis K, Dickman ED, Ferris D, Dias JK: Human papillomavirus vaccine acceptability among parents of 10- to 15-year-old adolescents. J Low Genit Tract Dis 2004, 8:188-94. 22. Baykal C, Al A, Uğur MG, Cetinkaya N, Attar R, Arioglu P: Knowledge and interest of Turkish women about cervical cancer and HPV vaccine. Eur J Gynaecol Oncol 2008, 29:76-9. Received: 11 June 2010 Accepted: 22 December 2010 Published: 22 December 2010 Received: 11 June 2010 Accepted: 22 December 2010 Published: 22 December 2010 Pre-publication history h bl h The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-6874/10/35/prepub 6. Rama CH, Villa LL, Pagliusi S, Andreoli MA, Costa MC, Thomann P, Alves VAF, Longatto-Filho A, Eluf-Neto J: Opportunity for catch-up HPV vaccination in young women after first delivery. J Epidemiol Community Health 2010, 64:610-5. doi:10.1186/1472-6874-10-35 Cite this article as: Rama et al.: Awareness and knowledge of HPV, cervical cancer, and vaccines in young women after first delivery in São Paulo, Brazil - a cross-sectional study. BMC Women's Health 2010 10:35. doi:10.1186/1472-6874-10-35 Cite this article as: Rama et al.: Awareness and knowledge of HPV, cervical cancer, and vaccines in young women after first delivery in São Paulo, Brazil - a cross-sectional study. BMC Women's Health 2010 10:35.
https://openalex.org/W2088655174	http://publikationen.ub.uni-frankfurt.de/files/27431/a02v373a.pdf	English	null	Status and promise of particle interferometry in heavy-ion collisions	Brazilian journal of physics	2,007	cc-by	3,839	are on the level of 20%. The ﬁrst two-pion interferometric measurements of the col- lider era emerged within a year of RHIC becoming opera- tional. To the surprise of many in the ﬁeld, the measurements were remarkably similar to those recorded at the AGS and the SPS. The analyses were inconsistent, both qualitatively and quantitatively, with dynamic models incorporating ﬁrst-order phase transitions in general. In fact, parametric explanations of the data, the forms of which are often motivated by solu- tions to dynamical equations, suggest that the matter explodes violently, growing from a radius of 6 fm to 13 fm in only 10 fm/c. The surprisingly strong acceleration required for such behavior and the associated failure of many of the ﬁeld’s most sophisticated models became known as the “HBT Puzzle”. • Femtoscopic studies are highly multi-dimensional. Even the simplest and most common case of two- identical pion correlations depend on six independent variables, which have only been fully explored within the past few years. This includes extracting character- istic source sizes as function of transverse momentum, rapidity and the angle with respect to the reaction plane for off-axis collisions. Additionally, correlation func- tions have been analyzed as a function of beam energy, centrality and the species of the pair. This accomplish- ment is especially noteworthy when taking the perspec- tive of comparing to the ﬁeld 20 years ago, when ex- tracting a single source dimension was considered state- of-the-art. • Femtoscopic studies are highly multi-dimensional. Even the simplest and most common case of two- identical pion correlations depend on six independent variables, which have only been fully explored within the past few years. This includes extracting character- istic source sizes as function of transverse momentum, rapidity and the angle with respect to the reaction plane for off-axis collisions. Additionally, correlation func- tions have been analyzed as a function of beam energy, centrality and the species of the pair. This accomplish- ment is especially noteworthy when taking the perspec- tive of comparing to the ﬁeld 20 years ago, when ex- tracting a single source dimension was considered state- of-the-art. Five years later, the ﬁeld has made steady progress on a variety of fronts. Theoretically, sophisticated dynamic models have more successfully reproduced experimental results (though the very sophistication of these models has made it difﬁcult to ascertain which aspects of the models are being validated by the comparison). Status and Promise of Particle Interferometry in Heavy-Ion Collisions Selemon Bekele, Fabio Braghin, Zbigniew Chaje¸cki , Paul Chung, John G. Cramer, Tam´as Cs¨org˝o, Hans Eggers, Sean Gavin, Fr´ed´erique Grassi, Yogiro Hama, Adam Kisiel, Che-Ming Ko, Tomoi Koide, Gast˜ao Krein, Roy Lacey, Richard Lednick´y, Michael A. Lisa, Wesley Metzger, Dariusz Mi´skowiec, Kenji Morita, Sandra S. Padula, Scott Pratt, Wei-Liang Qian, Vladislav ˇSimak, Yuri Sinyukov, Michal ˇSumbera, Bernardo M. Tavares, Giuseppe Verde, and Detlef Zschiesche Participants of the II Workshop on Particle Correlations and Femtoscopy (WPCF 2006) Instituto de F´ısica Te´orica - UNESP S˜ao Paulo, Brazil, September 9, 10 & 11, 2006 Participants of the II Workshop on Particle Correlations and Femtoscopy (WPCF 2006) Instituto de F´ısica Te´orica - UNESP S˜ao Paulo, Brazil, September 9, 10 & 11, 2006 are on the level of 20%. New techniques have been applied to the analysis of experimental correlation functions, revealing greater detail about the size and shape of the emission region. During our discussions at the workshop, it was clear that a remarkable consensus had developed among the practitioners of the ﬁeld. Although this agreement by no means represented a ﬁnal conclusion, we found numerous points that could be stated without dissent. In this white paper we will ﬁrst list the points concerning the cur- rent status of the ﬁeld, then further below, enumerate points where participants agreed were important for further progress. • The majority of femtoscopic investigations continues to focus on the correlations of identical pions, but analy- ses involving numerous other pairs (even Ξ −π corre- lations) are becoming more common. Analysis of non- identical particle correlations has allowed the extraction of qualitatively new femtoscopic information about the dynamical source substructure. Thus far, the prelim- inary assessment is that they are consistent with the information gleaned from π −π correlations. Particu- larly, the measured pion-proton and pion-kaon correla- tion asymmetries point rather directly to a strong col- lective ﬂow in heavy ion collisions at SPS and RHIC. • The majority of femtoscopic investigations continues to focus on the correlations of identical pions, but analy- ses involving numerous other pairs (even Ξ −π corre- lations) are becoming more common. Analysis of non- identical particle correlations has allowed the extraction of qualitatively new femtoscopic information about the dynamical source substructure. Thus far, the prelim- inary assessment is that they are consistent with the information gleaned from π −π correlations. Particu- larly, the measured pion-proton and pion-kaon correla- tion asymmetries point rather directly to a strong col- lective ﬂow in heavy ion collisions at SPS and RHIC. • Advanced techniques for angular decomposition and imaging are now being applied to extract shape and size information from any measured correlation. Al- though these analyses are in their nascent stage, it ap- pears they are uncovering quantitative details about the longer-time-scale aspects of particle emission, such as resonance production or surface emission. Brazilian Journal of Physics, vol. 37, no. 3A, September, 2007 Brazilian Journal of Physics, vol. 37, no. 3A, September, 2007 xxxi Received on 24 February, 2007 After ﬁve years of running at RHIC, and on the eve of the LHC heavy-ion program, we highlight the status of femtoscopic measurements. We emphasize the role interferometry plays in addressing fundamental questions about the state of matter created in such collisions, and present an enumerated list of measurements, analyses and calculations that are needed to advance the ﬁeld in the coming years. Keywords: Theoretical and experimental identical-particle correlations; Theoretical and experimental femtoscopy; Particle- antiparticle correlations are on the level of 20%. Achievements: Although smaller latent heats are not yet ruled out (cross-over or second order transitions are also pos- sible), the range of acceptable equations of state would be much broader if not for femtoscopic analyses. Achievements: This presumption has been veriﬁed by measurements of three-pion correlations, which have been shown theo- retically to be sensitive to coherent emission. • A parallel direction has developed, distinct from the study of heavy ion collisions, per se. Extracted knowl- edge of the space-time substructure of the emitting source also allows the femtoscopic program to be run “in reverse.” Assuming known geometries from other correlations analyses, correlations for pairs where the interactions are not well understood (e.g. ΛΛ) are being used to determine details of the interaction between un- stable particles. Since correlation analyses naturally in- volve low-relative momentum pairs, scattering lengths are especially accessible. Despite the enormous progress listed above, signiﬁcant hurdles have yet to be overcome and numerous opportunities have not yet been exploited. Workshop participants felt that enumeration of a “to-do” list for the ﬁeld would be enormously helpful, both for informing the greater heavy-ion community of our plans, and for clarifying, in our own minds, the important needs for our immediate future. These needs encompass both new experimental equipment, measurements, and analyses, along with needed development in theory, and with better integrating interferometric analyses with other families of observables. • The relative success and failure of various dynamic de- scriptions to provide sources that match those observed with interferometry has signiﬁcantly constrained our understanding of the equation of state at high temper- ature. Twenty years ago, bag-model descriptions of the equation of state with latent heats of many GeV/fm3 were common. It is now clear that extremely soft equa- tions of state, i.e., those that have large latent heats, are grossly inconsistent with interferometric measure- ments. Although smaller latent heats are not yet ruled out (cross-over or second order transitions are also pos- sible), the range of acceptable equations of state would be much broader if not for femtoscopic analyses. • The relative success and failure of various dynamic de- scriptions to provide sources that match those observed with interferometry has signiﬁcantly constrained our understanding of the equation of state at high temper- ature. Twenty years ago, bag-model descriptions of the equation of state with latent heats of many GeV/fm3 were common. It is now clear that extremely soft equa- tions of state, i.e., those that have large latent heats, are grossly inconsistent with interferometric measure- ments. Achievements: • Remarkable agreement has been observed between the RHIC experiments, PHENIX, PHOBOS and STAR. All three have produced high-statistics high-quality pion correlations, whose apparent source sizes are consistent to a few tenths of a fm. A similar consistency was ob- served among measurements performed at the top SPS energy; at lower SPS energies the maximum deviations • Without doubt, the dominant dynamical feature of the bulk system created at RHIC is its explosive collective xxxii WPCF 2006 Participants motion (ﬂow). Flow generates a source with character- istic dynamical/geometric substructure, and has impli- cations (e.g. spectral shapes and “v2” anisotropy) when projected onto the momentum-only space. However, with its explicit focus on the space-momentum source substructure, interferometry is the most sensitive and detailed probe of collective ﬂow. The growth of the sideward and longitudinal sizes, along with the lack of signiﬁcant extension of the outward direction, related to a short duration of the particle emission, can only be reproduced with highly explosive dynamics. Paramet- ric descriptions based on thermal emission on the back- ground of large collective outward ﬂow also explain the large radial shift in emission points of different-mass particles and the dependence of the effective source sizes with transverse momentum and direction with re- spect to the reaction plane. Measurements of effective sizes and orientation of the source shape for non-central rapidities have also validated our space-time picture of longitudinal collective ﬂow. Analyses with a wide range of models always come to the same conclusion, qualitatively and quantitatively, that strong longitudinal and transverse ﬂow has developed in central collisions at the SPS and at RHIC. • The connection between the source emission probabil- ity, which is given in coordinate space, and the corre- lation function, measured as a function of relative mo- mentum, is contingent on the assumption of chaotic un- correlated emission sources, whose correlations arise principally from ﬁnal-state two-body interactions. This presumption has been veriﬁed by measurements of three-pion correlations, which have been shown theo- retically to be sensitive to coherent emission. • The connection between the source emission probabil- ity, which is given in coordinate space, and the corre- lation function, measured as a function of relative mo- mentum, is contingent on the assumption of chaotic un- correlated emission sources, whose correlations arise principally from ﬁnal-state two-body interactions. Opportunities and Challenges: A joint experimental and theoret- ical effort will be essential for effectively observing and understanding such correlations, by looking for both an optimized form of the signal and the most promising experimental conditions for measuring it. shape information. It would be of tremendous impor- tance to quantitatively verify, using different classes of ﬁnal-state interactions, the space-time picture of the breakup stage that has emerged from the analysis of identical-pion correlations. Such analyses might re- quire experimental upgrades, such as the STAR time- of-ﬂight wall, which by expanding the range of particle identiﬁcation would allow particles of much different mass to be correlated at low relative velocity. • Interferometric and ﬂow analyses at RHIC have sug- gested that the average speed of sound is in the neigh- borhood of cs ∼0.3 −0.4. It is expected that the LHC region will explore much further above Tc where the matter is predicted to stiffen and the speed of sound ap- proaches cs ∼1/ √ 3 ≈0.58. It is imperative that exper- iments at the LHC have the capability for making high quality correlation measurements for particles with 100 MeV/c ≲pT ≲1GeV/c if this fundamental property of hot matter is to be explored. • The interferometry of penetrating probes has just re- cently become possible. In addition to potentially providing space-time information about pre-breakup stages, γγ correlations can reveal the fraction of low pT photons that do not originate from π0 decays, thus pro- viding a direct photon spectrum at sufﬁciently low pT to yield robust insight into the temperature during ear- lier stages of heavy-ion collisions. Also, recently there has been some theoretical development of correlation functions for lepton pairs, which reiterates the need for measuring correlation data of other penetrating probes, such as lepton-lepton femtoscopy. • In the last few years, lattice calculations have begun to suggest that the QCD phase transition is a cross-over for nearly zero chemical potentials, probed at RHIC with √sNN = 200 GeV collisions, and that it becomes a sec- ond order phase transition at a critical end point in the phase-diagram (TCEP,µCEP). Beyond this critical value, and for higher chemical potentials, µ > µCEP, the tran- sition becomes ﬁrst order. The critical point might be reached for energies just above the AGS range, in low energy runs at the CERN SPS and at the RHIC accelera- tors. Opportunities and Challenges: • Coherent phenomena are intimately associated with correlations. This includes novel Bose effects as well as coherent emission from classical ﬁelds, such as what is often described in dynamic models of the chiral con- densate. In nearly all such cases, the phenomena are expected to be strongest at low pT. Since the momen- tum scale can be estimated as the inverse characteristic source size, measurements at pT ∼50 MeV/c are re- quired to best explore such possibilities. Such measure- ments might require experiments to either run at low magnetic ﬁeld settings or to install special detectors. • The pion source sizes, when analyzed as a function of the collision energy, seem to follow the mean pion cross section for scattering on surrounding particles in the collision ﬁreball. This indicates that the freeze-out happens when the pion mean free path exceeds a cer- tain critical value, in a quantitative analysis estimated to be 1-2 fm. The interferometry data favors the ﬁxed mean free path freeze-out criterion over a freeze-out at a ﬁxed spatial density, or at a ﬁxed phase-space density, or when the mean free path exceeds the system size. • Back-to-back correlations (BBC) have recently been shown to arise if hadronic masses are modiﬁed by in- teractions in a dense medium. These quantum mechan- ical correlations are induced by a non-zero overlap be- tween the in-medium states and free states, which are observed. In particular, medium-modiﬁed bosonic or fermionic ﬁelds can be represented in terms of two- mode squeezed states of the corresponding asymptotic ﬁelds. Both the fermionic and the bosonic BBC lead to positive correlations of unlimited strength. They are more pronounced for large absolute values of the parti- cles’ back-to-back momenta and might survive the ef- • In addition to hints about the equation of state that would manifest themselves through dynamics, and thus through observables such as Rout/Rside, correlations have provided a quasi-model-independent measure of the phase space density and the total entropy observed in heavy ion collisions. Although the ﬁrst estimates are rather rough, this already has provided a signiﬁcant con- straint on the equation of state. Brazilian Journal of Physics, vol. 37, no. 3A, September, 2007 xxxiii xxxiii fect of collective ﬂow. Opportunities and Challenges: As this range will also be covered by the upcoming FAIR facility at GSI and at the planned NICA facility at JINR, it is important that proper detectors are installed for high-resolution measurements of correlations in the critical region. • Meticulous experimental analysis of the structure of the correlation function at very small relative momentum appears to show details of longer-time components of the emission function. Given the connection between long-lived emission and the equation of state, it is im- portant to vigorously pursue such analyses. In some cases, this might require detector upgrades to achieve the ∼2 MeV/c resolution necessary to resolve low rel- ative momentum features in the correlation function. • An analysis of the energy dependence of the averaged particle phase-space density, which is directly related to femtoscopy measurements in current and future ex- periments, is of great interest. This quantity is approxi- mately conserved during the hadronic stage of evolution and therefore is connected with the initial phase-space density of hadronic matter. It provides information about the states of the matter at the end of hadronization stage, or at chemical freeze-out, and thus allows one to search for phase transitions or a limiting Hagedorn tem- perature in relativistic nucleus-nucleus collisions. • The primary motivation of heavy ion experiments is the determination of bulk properties of matter. To achieve this ultimate goal, all relevant observables, including ﬂow and spectra, must be simultaneously analyzed by comparing comprehensive dynamical models with data. Analyses of parametric models have already illustrated the importance of a coordinated study of both correla- tions and spectra. Although numerous dynamic descrip- tions have been tested for their interferometric predic- tions, many models remain untested, or are only tested through rather primitive breakup criteria. The femto- scopic community needs to improve our link with the- orists developing dynamical models such as hydrody- namics. Furthermore, the theory community should be strongly encouraged to develop models which are bet- ter tested, better documented and are more ﬂexible. In particular, femtoscopic conclusions about the equation of state (EoS) have been complicated by the need, in many cases, to compare to the data one group’s calcu- lation using a given EoS, and another group’s calcu- lation using a different EoS. Firmer conclusions about this crucial feature of the matter will be much helped if all groups will produce predictions using a variety of EoS. Opportunities and Challenges: Similar treatment should be adopted with other important factors, such as initial conditions, the list of free parameters considered in each model, etc. Such a • The primary motivation of heavy ion experiments is the determination of bulk properties of matter. To achieve this ultimate goal, all relevant observables, including ﬂow and spectra, must be simultaneously analyzed by comparing comprehensive dynamical models with data. Analyses of parametric models have already illustrated the importance of a coordinated study of both correla- tions and spectra. Although numerous dynamic descrip- tions have been tested for their interferometric predic- tions, many models remain untested, or are only tested through rather primitive breakup criteria. The femto- scopic community needs to improve our link with the- orists developing dynamical models such as hydrody- namics. Furthermore, the theory community should be strongly encouraged to develop models which are bet- ter tested, better documented and are more ﬂexible. In particular, femtoscopic conclusions about the equation of state (EoS) have been complicated by the need, in many cases, to compare to the data one group’s calcu- lation using a given EoS, and another group’s calcu- lation using a different EoS. Firmer conclusions about this crucial feature of the matter will be much helped if all groups will produce predictions using a variety of EoS. Similar treatment should be adopted with other important factors, such as initial conditions, the list of free parameters considered in each model, etc. Such a • Femtoscopy for high pT particles would provide strin- gent tests of theoretical pictures of recombination and fragmentation models such as coalescence. However, femtoscopic measurements have thus far only been made for pairs with low relative momentum, as it is in- herently difﬁcult to gather statistics at high pT, where the phase space density is low. Specialized measure- ments or intensity upgrades might be required for high- quality interferometric measurements in the relevant pT range of several GeV/c. • Analyses of correlations for pairs of particles other than identical pions, such as pp, pK, πp, pΛ···, are in a nascent stage, with new imaging techniques having been recently developed for extracting detailed size and WPCF 2006 Participants xxxiv development is crucial if interferometric data are to be fully exploited. development is crucial if interferometric data are to be fully exploited. of chaotic independent emission. Opportunities and Challenges: In particular, common research projects between heavy ion physicists and ex- perts working on interferometry studies in elementary particle collisions, such as in pp, hp, ¯pp, e+e−, should be strongly encouraged. By better understanding the pT dependence of source sizes in pp collisions, we should attain a quantitative understanding of the effects in AA collisions and provide a systematic error to the underly- ing theory. • Extracting source functions from hydrodynamic mod- els should be better accommodated, which might en- tail providing interfaces between hydrodynamic models and microscopic hadronic codes used for modelling the ﬁnal breakup. Given the possible importance of mean ﬁelds in driving the dynamics or in refracting outgoing trajectories, mean ﬁelds should be included in transport codes. Such codes should be made available to the hy- drodynamic community, along with support for inter- facing the descriptions. A unifying theme of all the points, both in the list of ac- complishments and in the list of upcoming challenges, is the importance of collaboration. This includes sharing knowl- edge, expertise and ideas between collaborations, between experimentalists and theorists, between various segments of the theoretical community, and between different ﬁelds. To that end, there was unanimous consent that the WPCF series of workshops has already been enormously useful. Evidence of discussions and collaborations during the 2005 meeting in Kromˇeˇr´ıˇz was already evident in the results shown in 2006. The continuation of the workshop was enthusiastically en- dorsed by all participants. • Given the increasing sophistication and subtlety of fem- toscopic studies, some collaboration between experi- mentalists and theorists on the data analysis itself might be beneﬁcial. Experimental collaborations may con- sider incorporating more ﬂexibility into rules related to propriety of data, either in general or on a case-by-case basis. • A large fraction of practicing femtoscopists, now focus- ing on relativistic heavy ion collisions, initially worked at lower (sub-AGS) energies. Frequent interactions and exchanges of ideas and techniques has always bene- ﬁtted both communities. However, these interactions may be becoming less frequent due to two factors: (i) the increasing fraction of young people who already began their career at the highest energies, and (ii) the increasingly self-referential nature of relativistic heavy ion physics in general. Continued and enhanced col- laboration between high- and lower-energy femtoscopy should be an explicit consideration in the organization of femtoscopy-oriented symposia and workshops. Acknowledgments The authors gratefully acknowledge the hospitality of the Instituto de F´ısica Te´orica - UNESP, in S˜ao Paulo, for so graciously providing such a pleasant and stimulating environ- ment for the workshop. The organizers would like to thank CAPES (Coordenac¸˜ao de Aperfeic¸oamento de Pessoal de N´ıvel Superior) and FAPESP (Fundac¸˜ao de Amparo `a Pesquisa do Estado de S˜ao Paulo) for partially supporting the WPCF 2006, as well as SBF (the Brazilian Physics Society) for their assistance during the organization of this meeting. • The heavy-ion and the high-energy femtoscopic com- munities should make greater efforts towards commu- nicating. Advancing the understanding of small source (∼1 fm) interferometry will require more careful analy- sis of numerous effects which challenge the assumption
https://openalex.org/W2623238481	https://europepmc.org/articles/pmc5490568?pdf=render	English	null	Cognitive Performance Enhancement Induced by Caffeine, Carbohydrate and Guarana Mouth Rinsing during Submaximal Exercise	Nutrients	2,017	cc-by	12,529	Laura Pomportes 1,2, Jeanick Brisswalter 1, Laurence Casini 3, Arnaud Hays 4 and Karen Davranche 5,* Laura Pomportes 1,2, Jeanick Brisswalter 1, Laurence Casini 3, Arnaud Hays 4 and Karen Davranche 5,* 1 Laboratoire Motricité Humaine Expertise Sport Santé, Université Nice Sophia Antipolis, 06205 Nice, France; laurapomportes@hotmail.fr (L.P.); brisswalter@unice.fr (J.B.) 1 Laboratoire Motricité Humaine Expertise Sport Santé, Université Nice Sophia Antipolis, 06205 Nice, France; laurapomportes@hotmail.fr (L.P.); brisswalter@unice.fr (J.B.) 2 CREPS PACA, 13080 Aix-en-Provence, France 3 Laboratoire de Neurosciences Cognitives, Aix-Marseille Université, CNRS, LNC, 13331 Marseille, France; laurence.casini@univ-amu.fr 3 Laboratoire de Neurosciences Cognitives, Aix-Marseille Université, CNRS, LNC, 13331 Marseille, France; laurence.casini@univ-amu.fr 4 Institut des Sciences du Mouvement, Aix-Marseille Université, UMR 7287, 13288 Marseille, France; arnaud.hays@gmail.com 4 Institut des Sciences du Mouvement, Aix-Marseille Université, UMR 7287, 13288 Marseille, France; arnaud.hays@gmail.com 5 Laboratoire de Psychologie Cognitive, Aix-Marseille Université, CNRS, LPC, 13331 Marseille, France * Correspondence: karen.davranche@univ-amu.fr; Tel.: +33-(0)4-13-55-11-35 5 Laboratoire de Psychologie Cognitive, Aix-Marseille Université, CNRS, LPC, 13331 Marseille, France * Correspondence: karen.davranche@univ-amu.fr; Tel.: +33-(0)4-13-55-11-35 Received: 20 April 2017; Accepted: 6 June 2017; Published: 9 June 2017 Abstract: The aim of this study was to investigate the inﬂuence of serial mouth rinsing (MR) with nutritional supplements on cognitive performance (i.e., cognitive control and time perception) during a 40-min submaximal exercise. Twenty-four participants completed 4 counterbalanced experimental sessions, during which they performed MR with either placebo (PL), carbohydrate (CHO: 1.6 g/25 mL), guarana complex (GUAc: 0.4 g/25 mL) or caffeine (CAF: 67 mg/25 mL) before and twice during exercise. The present study provided some important new insights regarding the speciﬁc changes in cognitive performance induced by nutritional supplements. The main results were: (1) CHO, CAF and GUA MR likely led participants to improve temporal performance; (2) CAF MR likely improved cognitive control; and (3) CHO MR led to a likely decrease in subjective perception of effort at the end of the exercise compared to PL, GUA and CAF. Moreover, results have shown that performing 40-min submaximal exercise enhances information processing in terms of both speed and accuracy, improves temporal performance and does not alter cognitive control. The present study opens up new perspectives regarding the use of MR to optimize cognitive performance during physical exercise. Keywords: nutrition; cognition; perceived exertion; mouth rinse; time-perception task; conﬂict t Keywords: nutrition; cognition; perceived exertion; mouth rinse; time-perception task; conﬂict task nutrients nutrients 1. Introduction Several nutritional supplements are known to possess ergogenic effects on endurance performance (for review, see Reference [1,2]). The most largely studied are carbohydrate (CHO) and caffeine (CAF) assumed to help in limiting fatigue [3–6]. Both supplements have been shown to improve endurance capacity (exercise ≥2 h) but most likely utilizing two different mechanisms of action. After ingestion, CAF, which is thought to act as a central stimulant, is rapidly distributed to all tissues and readily crosses the blood-brain barrier to exert its ergogenic effects on the central nervous system mediated by the antagonism of adenosine receptors, which induces higher dopamine concentrations in the brain [7]. For CHO ingestion, the mechanism behind the ergogenic effect is rather a metabolic response, which would allow for maintaining plasma glucose concentration and high rates of CHO oxidation [8]. However, some studies have also shown that CHO supplementation can improve performance for high intensity exercises (>75% of maximal oxygen uptake (VO2max)) lasting 1 h or less [9,10]. These effects Nutrients 2017, 9, 589; doi:10.3390/nu9060589 www.mdpi.com/journal/nutrients 2 of 17 Nutrients 2017, 9, 589 are difﬁcult to explain by a metabolic hypothesis since CHO availability is not a limiting factor during the ﬁrst hour of exercise [10]. Furthermore, Carter et al. [9] found that rinsing the mouth with a non-sweet maltodextrin solution signiﬁcantly reduced the time to complete a 1-h cycle time trial. For the authors, the beneﬁt observed could be the result of a central effect leading to the improvement of the motor drive or motivation, possibly due to the activation of receptors inside the mouth. p y p These ergogenic effects observed after mouth rinsing (MR) with nutritional supplements open up new perspectives in terms of performance optimization strategies. The fact that similar magnitude beneﬁts have been observed on performance using MR or ingestion [11] incites to investigate thoroughly to deﬁne potential beneﬁts and gainful usage, for example with athletes who are inclined to gastro-intestinal distress or when they need to limit energy intake (weight control). Studies using CHO MR [11–16] have generally reported exercise performance improvement (for review, see [17]), whereas results of the few studies assessing the effect of CAF MR remain inconclusive [12,18]. Concerning CHO MR, the main hypothesis for explaining the ergogenic effect relies upon the link between sweet tastants receptors T1Rs in the oral cavity and esophagus as well as various brain regions [19]. 1. Introduction Supporting this idea, it has been observed that the sole presence of CHO in the mouth, prior to its ingestion, induced an increase in corticomotor pathway excitability [20]. Besides these effects in motor areas, an increase in brain activity of some areas (i.e., the dorsolateral prefrontal cortex, the orbitofrontal cortex and the striatum), known to be involved in attention and reward, has also been observed after MR [13,21]. Because of the key-role of these brain areas in executive functions, performance in cognitive tasks could also be inﬂuenced; this needs to be further investigated. As regards to CAF MR, the rate of caffeine buccal absorption is extremely rapid and leads to a comparable amount of caffeine in the systemic circulation compared to ingestion [22]. Only very few data are available about the inﬂuence of CAF MR on cognitive performance, however it has been recently reported that CAF MR would exert a likely beneﬁcial effect on reaction time (RT) in a task requiring executive control (Stroop task) [21]. By using electroencephalogram signal analysis, the same authors further explored this issue by assessing the effect of 20 s nasal spray with placebo, glucose and CAF on performance in the same cognitive task. While behavioral data reported no effect of nasal spray, electrophysiological data indicated that glucose and CAF nasal spray increased brain activity in some areas known to be engaged in cognitive control [23]. These brain responses could be due to the activation of bitter taste receptors T2Rs respectively in the mouth and the nasal cavity [20,21]. Further studies are required to understand the ergogenic effect of MR on exercise performance and furthermore on cognitive performance. The inﬂuence of nutritional supplements MR during exercise has also been investigated by measuring the rate of perceived exertion (RPE). The RPE could be deﬁned as “the feeling of how hard, heavy and strenuous a physical task is” [24]. Results are mixed but some studies reported that CAF and/or CHO MR lead to a decrease in RPE for the same power output [25–27] which suggests that CHO and CAF MR may induce a decrease in subjective perception of effort, allowing participants to produce more power with the same degree of discomfort. Therefore, for all of these reasons, the ergogenic effect of nutritional substances is a topic of interest for the optimization of sport performance. 1. Introduction Currently, there is a growing interest for guarana (GUA), which is often attributed to CAF content depending on how the extract is prepared [28]. In addition to CAF, guarana seeds are known to harbor a number of other possible stimulants such as ﬂavonoids [29] or other potentially psychoactive components, including saponins and tannins [30,31], which could enhance cognitive function [32–34]. In several studies as well as in sports nutrition strategy, GUA is rarely used alone but mostly associated to multivitamins mineral complex [32,34,35] or to ginseng [33]. We assume that, in addition to the presence of CAF, the other components of guarana seeds could potentially enhance performance via the activation of bitter taste receptors T2Rs [36] or different pathways. Some recent studies have reported that GUA ingestion could also inﬂuence cognitive abilities. More speciﬁcally, it has been reported that ingestion of GUA, could, at rest, induce an improvement in decision-making and alertness [32], improve memory performance [33] and reduce RPE after 30 min of submaximal exercise [34]. 3 of 17 Nutrients 2017, 9, 589 To the best of our knowledge, no studies have investigated the effect of MR on cognitive performance during exercise. In the ﬁeld of physical activity, athletes are faced with strong physiological and cognitive demands to attain goals. Thus, the use of nutritional supplementation may ameliorate or aid to maintain a high performance and avoid adverse consequences on the outcome of sporting events. Consequently, this study aims to investigate the inﬂuence of serial administration of CHO, CAF and GUA MR on cognitive processes during an acute bout of 40-min of exercise. This exercise duration/intensity was chosen to be both appropriate to participants regularly engaged in training and enough intense to theoretically induced performance impairment (for a review see Reference [37]). Potential effects of nutritional supplements are thus expected either in terms of maintaining performance (if any impairment occurred) or ergogenic effect. In the present study, we investigated executive functions, which in a broad sense refer to a number of processes that are necessary to remain adapted to an ever-changing environment. More speciﬁcally, we focused on cognitive control and time estimation, continuously involved in any goal-oriented behavior and we chose two well-established cognitive tasks, respectively a Simon task and a duration-production task. 1. Introduction The Simon task [38] provides information about the ability to inhibit prepotent responses and the temporal task allows you to dissociate the effect on arousal and attention levels [39], two processes that usually remain amalgamated in most studies. The main purpose of the study was to assess whether these cognitive processes would be impacted during exercise by three different nutritional supplements commonly used by athletes and delivered by MR: CHO, CAF and guarana complex (GUAc: GUA + ginseng + vitamins C). Since results of recent studies have suggested that MR could increase brain activity in several areas that have a key-role in executive functions, we assume that both cognitive processes could beneﬁt from supplementation during exercise. 2.2.3. Experimental Design Each participant performed four experimental sessions, two per week, separated by at least 72 h and conducted at the same time of the day. Participants were instructed to keep a food diary during the two days prior to the ﬁrst session and to replicate this diet before each session. They were also required to refrain from alcohol, caffeine, pain medication and nutritional supplements during the 48 h prior to each session. The last meal was taken 3 h and half prior to the start of each experimental session suggesting that participants had 3 h of fast between each session. No food or drinks intake (except water) was allowed between the last meal and the experimental session. The participants were instructed to control their time and hours of sleep the two nights preceding each experimental session. Participants were required to maintain the same training program every week during the experimental protocol. Each experimental session (see Figure 1), corresponding to a different tested nutritional mouth rinse, started with a speciﬁc cycling warm-up consisting of 2 × (2 min 100 W (for males)/80 W (for females) + 2 min 150 W (for males)/130 W (for females) and 3 × (30 s 200 W (for males)/170 W (for females) + 30 s 50 W) and lasting approximately 10 min [40]. Immediately after the warm-up, keeping the seated position on the cycle ergometer, the participants performed a short recall of the two cognitive tasks. The recall was identical to the training session except that the participants could only perform one block of training while cycling. Once the recall was made, the ﬁrst mouth rinse (MR1) was administered during 20 s. Then, the cycling exercise started with a resistance ﬁxed at 60% of the peak power output which was previously recorded for each participant. After one minute of cycling, each participant performed three identical experimental blocks separated by 20 s of MR during which they continued to cycle. Following MR2 and MR3 participants were allowed to rehydrate with as much tap water as they required. During each block, participants performed the duration-production task lasting about three minutes and seven minutes later the Simon task also lasting about three minutes. Finally, at the end of the 40-min exercise, the participants had to ﬁll a visual analogic scale to assess RPE. 2.2. Procedure This study used a cross over design and required the participants to visit the laboratory six times (one preliminary session, one training session and four experimental sessions) at least 72 h apart and at the same time of day. 2.2.2. Training Session Between 48 and 72 h prior to the ﬁrst experimental session, participants underwent a training session with the duration-production task and the Simon task. Details are provided in the cognitive tasks section. 2.2.1. Preliminary Session One week before the start of the ﬁrst experimental session, preliminary testing was performed to collect anthropometric and physiological characteristics. After a standardized warm up, the peak power output was assessed on the cycle ergometer (Cyclus 2®, RBM, Leipzig, Germany) using a progressive test starting at 80 Watt (W) for females and 100 W for males with an increase of 15 W/min until exhaustion. The last completed step was regarded as the peak power output (W). During the test, heart rate (HR) was continuously recorded using a monitor (RS800, Polar®, Helsinki, Finlande) which allows for HRmax assessment (Table 1). 2.1. Participants Twenty-four physically active participants (16 males and 6 females), recruited from a regional training sport center participated in this experiment without being paid (Table 1). All participants were regularly engaged in training (between 3 and 8 h per week) but were not professional cyclists. Participants were not used to drinking nutritional supplementations during training or competition and consumed less than 200 mg of caffeine per day. Before inclusion, the experimental procedure was explained to the participants and they signed an informed consent form approved by the University Ethics Committee (Ile de France, VII, Saint Germain en Laye, France). Table 1. Anthropometrical and physiological characteristics of participants. Mean (Standard Deviation) Variables All Female Male Sample size 24 8 16 Age (years) 26 (8) 30 (10) 24 (6) Height (cm) 174 (10) 164 (7) 179 (7) Body mass (kg) 72 (14) 57 (7) 79 (11) Body mass index (kg·m−2) 23 (3) 21 (2) 25 (3) Peak power output (W) 250 (58) 198 (31) 276 (50) Cycling intensity experimental sessions (W) 150 (35) 119 (18) 166 (30) Heart rate max (bpm·min−1) 189 (8) 185 (10) 190 (7) Table 1. Anthropometrical and physiological characteristics of participants. 4 of 17 Nutrients 2017, 9, 589 Duration-Production Training and Recall Sessions Duration-Production Training and Recall Sessions Duration-Production Training and Recall Sessions Duration-Production Training and Recall Sessions The training session consisted of two parts. For the first ten trials, a 600 Hz tone sounded for 1100 ms. When the sound ended, a red circle appeared in the center of the screen indicating that participants could reproduce the duration of the sound by pressing the right keypress as long as the sound lasted. When the participants released the keypress, an auditory feedback was delivered. Five different feedbacks were used. If the produced interval was correct (less than 7.5% longer or shorter than the target), the feedback “correct” was delivered. If the produced duration was too long or too short (7.5–22.5% longer or shorter than the target), either the word “too long” or “too short” were delivered. If the duration was excessively long or short (more than 22.5% longer or shorter), the words “much too long” or “much too short” were delivered. After the first ten trials, participants performed a second block in which no additional model of target duration was delivered. During the remaining trials, once the red circle appeared on the screen, participants pressed the key for 1100 ms. As in the ten first trials, an auditory feedback was delivered after each response. The participants continued until they produced 12 correct durations through 15 successive trials. A maximum of 50 trials was presented. If participants did not reach the criterion before the end of the block, they began a complete The training session consisted of two parts. For the ﬁrst ten trials, a 600 Hz tone sounded for 1100 ms. When the sound ended, a red circle appeared in the center of the screen indicating that participants could reproduce the duration of the sound by pressing the right keypress as long as the sound lasted. When the participants released the keypress, an auditory feedback was delivered. Five different feedbacks were used. If the produced interval was correct (less than 7.5% longer or shorter than the target), the feedback “correct” was delivered. If the produced duration was too long or too short (7.5–22.5% longer or shorter than the target), either the word “too long” or “too short” were delivered. If the duration was excessively long or short (more than 22.5% longer or shorter), the words “much too long” or “much too short” were delivered. 2.2.3. Experimental Design Pedaling rate was freely chosen and heart rate was recorded throughout the 40 min cycling exercise using a polar RS800 cardiofrequencemeter (Polar Electro, Kempele, Finland). 5 of 17 Nutrients 2017, 9, 589 Figure 1. General procedure of each experimental session. (WU = Warm Up; Recall = Cognitive tasks recall; TEMP = Duration Production task; SIM = Simon task; MR = mouth rinsing; RH = Rehydration; ppo = peak power output; RPE = Rating of Perceived Exertion). Figure 1. General procedure of each experimental session. (WU = Warm Up; Recall = Cognitive tasks recall; TEMP = Duration Production task; SIM = Simon task; MR = mouth rinsing; RH = Rehydration; ppo = peak power output; RPE = Rating of Perceived Exertion). Figure 1. General procedure of each experimental session. (WU = Warm Up; Recall = Cognitive tasks recall; TEMP = Duration Production task; SIM = Simon task; MR = mouth rinsing; RH = Rehydration; ppo = peak power output; RPE = Rating of Perceived Exertion). Figure 1. General procedure of each experimental session. (WU = Warm Up; Recall = Cognitive tasks recall; TEMP = Duration Production task; SIM = Simon task; MR = mouth rinsing; RH = Rehydration; ppo = peak power output; RPE = Rating of Perceived Exertion). Each one of the three 25 mL-MR consisted of either a 7% carbohydrate complex (CHO: fructose (89%) and maltodextrin (11%), ISOXAN® Sport Pro, NHS, Rungis, France), a 67 mg caffeine (CAF, PROLAB® nutrition, Chatswoth, USA) added with orange sugarless syrup, a 0.4 g guarana complex (GUA: 37.5 mg of guarana + 12.5 mg ginseng + 22.5 mg vitamins C, Isoxan Actiflash® Booster, Menarini, NHS, Rungis, France), or a placebo (PL: tap water added with orange sugarless syrup) depending on the experimental session. We decided to use the amount of 7% CHO commonly used in nutrition studies and 67 mg caffeine and 37.5 mg guarana for each MR to reach the dose range of 200 mg caffeine and 300 mg guarana recommended per day. 2.2.3. Experimental Design Each one of the three 25 mL-MR consisted of either a 7% carbohydrate complex (CHO: fructose (89%) and maltodextrin (11%), ISOXAN® Sport Pro, NHS, Rungis, France), a 67 mg caffeine (CAF, PROLAB® nutrition, Chatswoth, USA) added with orange sugarless syrup, a 0.4 g guarana complex (GUA: 37.5 mg of guarana + 12.5 mg ginseng + 22.5 mg vitamins C, Isoxan Actiﬂash® Booster, Menarini, NHS, Rungis, France), or a placebo (PL: tap water added with orange sugarless syrup) depending on the experimental session. We decided to use the amount of 7% CHO commonly used in nutrition studies and 67 mg caffeine and 37.5 mg guarana for each MR to reach the dose range of 200 mg caffeine and 300 mg guarana recommended per day. The study was single-blind and experimental sessions were counterbalanced between participants. Each block lasted about 13 min and each session lasted about 90 min while completing the cognitive tasks for about half total time. The study was single-blind and experimental sessions were counterbalanced between participants. Each block lasted about 13 min and each session lasted about 90 min while completing the cognitive tasks for about half total time. 2.3. Cognitive Tasks 2.3. Cognitive Tasks The cognitive tasks were performed on a cycle ergometer, equipped with two thumb response keys fixed on the top of the right and left handle grips, positioned in front of a screen located 1m away. The cognitive tasks were first performed at rest during both the training and the recall sessions and afterward during the 40-min cycling exercise at 60% of peak power output. The cognitive tasks were performed on a cycle ergometer, equipped with two thumb response keys ﬁxed on the top of the right and left handle grips, positioned in front of a screen located 1m away. The cognitive tasks were ﬁrst performed at rest during both the training and the recall sessions and afterward during the 40-min cycling exercise at 60% of peak power output. 2.3.1. Duration-Production Task 2.3.1. Duration-Production Task The duration-production task consists of pressing a button for a time interval learned during a training phase. The procedure used here was inspired from duration-production tasks used in previous studies [39,41]. The duration-production task consists of pressing a button for a time interval learned during a training phase. The procedure used here was inspired from duration-production tasks used in previous studies [39,41]. Simon Task Experimental Sessions During the whole trial, the participants had to ﬁxate on a white point located in the center of the screen and they were required to respond, as quickly and accurately as possible, by pressing the appropriate response key (with the right or left thumb ﬁnger) according to the shape (square or circle) of a geometric symbol delivered either to the left or to the right of the ﬁxation point. The distance from the center of the white ﬁxation point to the center of the geometric symbol located to either the right or left was 7.5 cm. Participants had to respond according to the shape of the symbol while ignoring its spatial location. The mapping of geometric symbol shape to response key (for example, right response for a square and left response for a circle) was counterbalanced across participants. The task included two equiprobable trial types randomized: the congruent trials (CO) (response side ipsilateral to stimulus side), and the incongruent trials (IN) (response side contralateral to the stimulus side). As soon as a response key was pressed or when a delay of 1.5 s after the stimulus onset had elapsed without a response, the stimulus was removed from the screen and the next trial began. The Simon task, consisting of 64 trials, was carried out at 10 min 30 s, 23 min 30 s and 36 min 30 s during the cycling exercise. Duration-Production Training and Recall Sessions Duration-Production Training and Recall Sessions After the ﬁrst ten trials, participants performed a second block in which no additional model of target duration was delivered. During the remaining trials, once the red circle appeared on the screen, participants pressed the key for 1100 ms. As in the ten ﬁrst trials, an auditory feedback was delivered after each response. The participants continued until they produced 12 correct durations through 15 successive trials. A maximum of 50 trials was presented. If participants did not reach the criterion before the end of the block, they began a complete Nutrients 2017, 9, 589 6 of 17 training session again (10 trials with the sound as the target duration followed by the remaining trials without the sound). The recall session was identical to the training session except that the participants could only perform one block of training while cycling. A recall session was performed prior to each experimental session. Duration-Production Experimental Sessions During the experimental sessions, participants were required to press the right keypress for 1100 ms. Trial onset was initiated by participants once the red circle appeared on the screen. Participants had to maintain the keypress as long as necessary to time the required duration (1100 ms). No feedback on performance was given. One and a half seconds after the release of the key, the red circle appeared again, indicating that the next trial could be initiated. The duration-production task was performed after 60 s, 14 min and 27 min of cycling exercise and lasted approximately 3 min for each block. 2.3.2. Simon Task 2.3.2. Simon Task Simon Training and Recall Sessions In the training phase of the Simon task, all participants performed a minimum of 3 blocks of 64 trials. Two additional blocks were performed, if necessary, until the following learning criteria were achieved in one block: (a) intra-block RT variability below 5%; (b) inter-blocks RT variability with the previous block below 5%; (c) mean RT less than 400 ms; and (d) error rate between 3% and 10%. p A short recall including 12 trials was performed at the beginning of each experimental session while cycling. No learning criterion was used. 2.4. Rating of Perceived Exertion (RPE) A Visual Analogic Scale (VAS) has been chosen since it showed a good reproducibility and sensitivity to assess the subjective effort during exercise [42]. The scale was used under the form of a vertical line, 20 cm of length, anchored by word descriptors on the top (maximal exertion) and on the bottom (no exertion at all) to gauge how hard was the exercise. At the end of the experimental session, participants marked a point on the line that they felt represented the intensity of exercise at the end of the cycling session. The VAS score was determined by the distance in centimeters from the bottom line to the point marked by the participants. 7 of 17 Nutrients 2017, 9, 589 7 of 17 3. Results The two cognitive tasks were assessed during a 40-min cycling exercise at 60% of peak power output. Heart rate during exercise increased to 78% ± 1.2% HRmax in block 1, 82.3% ± 1.3% HRmax in block 2 and 83.8% ± 1.2% HRmax in block 3. 3.1. Cognitive Performance An overview of cognitive results is shown in Table 2. An overview of cognitive results is shown in Table 2. 3.1.1. Duration-Production Task Mean produced duration, measuring the accuracy and variance of produced durations, were calculated for each participant in each of the four conditions. Mean produced duration reﬂects overall lengthening or shortening of produced time. To aid in the interpretation of results, it should be noted that a shortening of produced durations (also called underproduction) means that the criterion value corresponding to the duration learned during the training session was reached earlier. Conversely, a lengthening of produced durations (also called overproduction) means that the criterion value was reached later [41]. The variance of produced durations provides information about individual participant’s variability across trials. The mean and the variance of the produced duration were submitted to separate GLM with block (block 1, block 2 and block 3) as within-subject factors and to magnitude-based inferences for the effects of nutritional supplements. 2.5. Statistical Analysis Results were analyzed with quantitative (General Linear Models, GLM) or qualitative (probabilistic magnitude-based inferences) methods. Firstly, repeated measures within participants GLM were performed on each dependent variable for exercise effect (mean RT, error rate, mean produced duration, temporal variance) and post-hoc Newman-Keuls analyses were conducted for all signiﬁcant effects. Signiﬁcance was set at p < 0.05 for all analyses. Effect sizes for GLM were calculated using partial eta square (ïp2). Secondly, to speciﬁcally test effects of nutritional supplements and RPE, we also reported probabilistic magnitude-based inferences about all the variables using methods described by Hopkins et al. [43] which has been applied in several recent nutritional studies [21,44,45]. Data were log-transformed prior to analyses to reduce bias arising from non-uniformity of error. To compare within-trial changes between trials, we used a modiﬁed statistical spreadsheet that calculates the between-trials differences or effect sizes (ES), 90% conﬁdence interval (CI) using the pooled standard deviation. If the chance of beneﬁt or harm were both >5% the true effect was reported as unclear. Otherwise, chances of beneﬁt or harm were assessed as follows: <1%, almost certainly not; 1–5%, very unlikely; 5–25% unlikely; 25–75% possible; 75–95% likely; 95–99% very likely; >99% most likely [46]. Mean in Temporal Production A signiﬁcant effect of block was observed on mean produced duration (F(2, 44) = 7.6, p = 0.001, ïp2 = 0.26). Participants produced shorter durations in blocks 2 and 3 than in block 1 (respectively p = 0.003 and p = 0.003) (Figure 2a). Thus, although participants globally overproduced the target 1100 ms duration, they underproduced duration when completing the cycling exercise. Nonetheless, magnitude based inferences showed that CHO, CAF and GUAc MR compared to PL lead to a likely decrease on mean produced durations (respectively 80%, 86% and 81%), which would mean that participants globally underproduced duration after MR with CHO, CAF and GUAc when compared with PL (Figure 2b). 8 of 17 Nutrients 2017, 9, 589 Table 2. Cognitive performance as a function of nutritional supplementation and exercise. Mean in Temporal Production Duration Production Task Mean (Standard Errors) Simon Task Mean (Standard Errors) Produced Duration (ms) Variance (ms) Reaction Time (ms) Errors (%) CO IN Total Simon Effect § CO IN Total Simon Effect § Nutritional supplementation PL Block 1 1370.7 (50.9) 197.9 (17.5) 336.0 (8.7) 365.5 (9.7) 350.7 (6.8) 29.4 (5.7) 4.7 (0.8) 9.8 (1.2) 7.2 (0.8) 5.1 (1.2) Block 2 1313.3 (56.8) 201.4 (24.0) 334.3 (7.3) 359.8 (9.0) 347.1 (6.0) 25.5 (5.1) 4.3 (0.9) 8.9 (1.3) 6.6 (0.9) 4.6 (1.3) Block 3 1312.7 (58.6) 185.6 (15.9) 323.9 (7.7) 357.6 (7.9) 340.8 (6.0) 33.7 (3.7) 4.6 (0.6) 8.7 (1.2) 6.6 (0.7) 4.2 (1.5) Total 1332.2 (31.8) 195.0 (11.1) 331.4 (4.6) 361.1 (5.1) 346.2 (3.6) 29.5 (2.8) 4.5 (0.4) 9.1 (0.7) 6.8 (0.5) 4.6 (0.8) CHO Block 1 1348.9 (44.2) 186.8 (14.5) 334.7 (8.1) 362.2 (8.0) 348.4 (6) 27.5 (6.3) 7.0 (1.5) 10.4 (1.7) 8.7 (1.2) 3.4 (2.6) Block 2 1259.2 (41.0) 171.6 14.6) 334.4 (8.4) 361.8 (8.5) 348.1 (6.1) 27.5 (5.5) 3.3 (0.8) 9.5 (0.9) 6.4 (0.8) 6.3 (1.0) Block 3 1249.6 (39.4) 161.5 (11.8) 324.2 (8.0) 356.5 (8.1) 354.6 (6.1) 32.3 (5.5) 4.4 (0.9) 9.8 (1.1) 7.1 (0.8) 5.3 (1.2) Total 1285.9 (24.3) ␈ 173.3 (7.9) ␈▲ 331.1 (4.7) 360.2 (4.7) 345.6 (3.5) 29.1 (3.3) 4.9 (0.7) 9.9 (0.7) 7.4 (0.5) 5.0 (1.0) CAF Block 1 1292.6 (24.5) 187.0 (11.8) 338.0 (8.5) 364.4 (8.9) 351.2 (6.3) 26.4 (5.12) 5.1 (0.9) 10.3 (1.3) 7.7 (0.9) 5.2 (1.6) Block 2 1255.9 (39.1) 187.3 (14.3) 336.8 (8.0) 360.7 (6.7) 348.8 (5.5) 23.9 (4.6) 3.9 (0.9) 10.5 (1.4) 7.2 (0.9) 6.6 (1.6) Block 3 1249.0 (31.5) 167.7 (11.5) 328.4 (7.4) 352.4 (7.4) 340.4 (5.5) 24 (4.7) 4.6 (0.8) 8.5 (1.4) 6.5 (0.9) 3.9 (1.7) Total 1265.8 (18.5) ␈ 180.7 (7.3) ␈ 334.4 (4.6) 359.1 (4.4) 346.8 (3.3) 24.8 (2.8) 4.5 (0.5) 9.8 (0.8) 7.1 (0.5) 5.3 (0.9) GUA Block 1 1311.2 (35.4) 193.7 (15.8) 338.7 (7.9) 370.4 (8.7) 354.6 (6.3) 31.7 (6.4) 5.8 (1.1) 10.5 (1.5) 8.2 (1.0) 4.6 (1.8) Block 2 1249.9 (40.4) 176.5 (13.1) 336.8 (7) 365.8 (7.9) 351.3 (5.7) 29 (5.6) 3.8 (0.9) 13.3 (2.3) 8.6 (1.4) 9.5 (2.6) Block 3 1281.1 (40.2) 170.5 (13.2) 326.9 (7.9) 360.1 (7.6) 343.5 (6.0) 33.2 (5.4) 3.0 (0.7) 12.0 (1.6) 7.5 (1.1) 9.0 (2.8) Total 1280.7 (22.2) ␈ 180.2 (8.1) ␈ 334.1 (4.4) ■ 365.4 (4.6) 349.8 (3.4) 30.0 (3.3) 4.2 (0.6) 11.9 (1.1) 8.1 (0.7) 7.7 (1.2) Exercise Block 1 1331.0 (20.0) $ 191.3 (7.4) $ 336.9 (4.1) 365.5 (4.4) 351.2 (3.2) $ 28.4 (2.9) 5.7 (0.6) 10.2 (0.7) 8.0 (0.5) 8.0 (0.5) Block 2 1269.8 (22.3) * 184.3 (8.5) $ 335.6 (3.8) 362.0 (4.0) 348.8 (2.9) $ 26.2 (2.6) 3.8 (0.4) 10.5 (0.8) 7.1 (0.5) 7.1 (0.5) Block 3 1273.0 (21.7) * 171.3 (6.6) * 325.9 (3.8) 356.6 (3.8) 340.9 (2.9) 30.5 (2.4) 4.1 (0.4) 9.7 (0.7) 6.8 (0.4) ~ 6.8 (0.4) Total 1292.2 (12.4) 182.9 (4.4) 332.9 (2.4) 361.4 (2.3) 347.2 (2.0) 29.95 (1.8) 4.5 (0.3) 10.2 (0.4) 7.4 (0.3) 5.6 (0.5) §Incongruent (IN) minus congruent (CO) trials. Variance in Temporal Production A f ff f bl k Variance in Temporal Production A significant effect of block was observed on the variability (F(2, 44) = 4.68, p = 0.01, ɳp2 = 0.18) The participants were significantly less variable in block 3 in comparison to blocks 1 and 2 (respectively p = 0.01 and p = 0.05). Magnitude based inferences showed that CHO CAF and GUAc MR compared to PL lead to a A signiﬁcant effect of block was observed on the variability (F(2, 44) = 4.68, p = 0.01, ïp2 = 0.18). The participants were signiﬁcantly less variable in block 3 in comparison to blocks 1 and 2 (respectively p = 0.01 and p = 0.05). A significant effect of block was observed on the variability (F(2, 44) = 4.68, p = 0.01, ɳp2 = 0.18) The participants were significantly less variable in block 3 in comparison to blocks 1 and 2 (respectively p = 0.01 and p = 0.05). Magnitude based inferences showed that CHO CAF and GUAc MR compared to PL lead to a A signiﬁcant effect of block was observed on the variability (F(2, 44) = 4.68, p = 0.01, ïp2 = 0.18). The participants were signiﬁcantly less variable in block 3 in comparison to blocks 1 and 2 (respectively p = 0.01 and p = 0.05). Magnitude based inferences showed that CHO, CAF and GUAc MR compared to PL, lead to a likely decrease of variability (respectively 93%, 89% and 92%). Participants were less variable in their temporal production after nutritional supplementation. Furthermore, they were even less variable with CHO when compared to GUAc (likely effect). Magnitude based inferences showed that CHO, CAF and GUAc MR compared to PL, lead to a likely decrease of variability (respectively 93%, 89% and 92%). Participants were less variable in their temporal production after nutritional supplementation. Furthermore, they were even less variable with CHO when compared to GUAc (likely effect). Mean in Temporal Production General Linear Models (GLM): * a signiﬁcant difference was found with block 1; $ a signiﬁcant difference was found with block 3; ~ a trend effect was found with block 1 (p < 0.05). Magnitude based inference: ␈a difference was found with placebo mouth rinsing, PL MR; ▲a difference was found with guarana mouth rinsing, GUA MR; a difference was found with caffeine mouth rinsing, CAF MR; ■a difference was found with carbohydrate mouth rinsing, CHO MR (75–95% likely different). Table 2. Cognitive performance as a function of nutritional supplementation and exercise. Simon Task Mean (Standard Errors) 9 of 17 9 of 17 Nutrients 2017, 9, 589 Nut ie t 2017 9 589 , , Nutrients 2017, 9, 589 9 of (a) (b) Figure 2. Mean produced durations as a function of exercise (a) and supplementation (b) Errors bars represent standard errors of the mean produced duration. * p < 0.05; # 75–95% likely different. Figure 2. Mean produced durations as a function of exercise (a) and supplementation (b) Errors bars represent standard errors of the mean produced duration. * p < 0.05; # 75–95% likely different. (a) (b) (b) (a) Figure 2. Mean produced durations as a function of exercise (a) and supplementation (b) Errors bars represent standard errors of the mean produced duration. * p < 0.05; # 75–95% likely different. Figure 2. Mean produced durations as a function of exercise (a) and supplementation (b) Errors bars represent standard errors of the mean produced duration. * p < 0.05; # 75–95% likely different. 3.1.2. Simon Task 3.1.2. Simon Task The mean RT and the error rate were submitted to separate GLM block (block 1, block 2 and block 3) and congruency (CO and IN) as within-subject factors. Nutritional effect was evaluated with magnitude-based inferences. The mean RT and the error rate were submitted to separate GLM block (block 1, block 2 and block 3) and congruency (CO and IN) as within-subject factors. Nutritional effect was evaluated with magnitude-based inferences. Mean RT A Mean RT 10 of 17 10 f 17 10 of 17 Nutrients 2017, 9, 589 Nutrients 2017, 9, 589 Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 3. Mean reaction time (RT) and errors as a function of supplementation and congruency. Errors bars represent standard errors of mean RT. # 75–95% likely different. Figure 4. Simon Effect for reaction time (RT) and error as a function of supplementation. Errors bars represent standard errors of the mean Δ. # 75–95% likely different. Figure 4. Simon Effect for reaction time (RT) and error as a function of supplementation. Errors bars represent standard errors of the mean Δ. # 75–95% likely different. Figure 4. Simon Effect for reaction time (RT) and error as a function of supplementation. Errors bars represent standard errors of the mean ∆. # 75–95% likely different. Figure 4. Simon Effect for reaction time (RT) and error as a function of supplementation. Errors bars e e e t ta da d e o of the ea Δ # 75 95% likely diffe e t Figure 4. Simon Effect for reaction time (RT) and error as a function of supplementation. Mean RT A Mean RT Errors bars represent standard errors of the mean Δ. # 75–95% likely different. Figure 4. Simon Effect for reaction time (RT) and error as a function of supplementation. Errors bars represent standard errors of the mean ∆. # 75–95% likely different. Mean RT A Mean RT Analyses showed a significant effect of exercise on mean RT (F(2, 44) = 10.1, p = 0.0003, ɳp2 = 0.31). RTs in Block 3 are significantly faster than RTs in blocks 1 and 2 respectively (p = 0.001 and p = 0.002). The classic effect of congruency was found on mean RTs (F(1, 22) = 78.3, p = 0.00, ɳp2 = 0.78) with slower RT in IN trials compared to RT in CO trials. For CO trials magnitude based inference showed that CAF leads to a likely increase of mean RT Analyses showed a signiﬁcant effect of exercise on mean RT (F(2, 44) = 10.1, p = 0.0003, ïp2 = 0.31). RTs in Block 3 are signiﬁcantly faster than RTs in blocks 1 and 2 respectively (p = 0.001 and p = 0.002). The classic effect of congruency was found on mean RTs (F(1, 22) = 78.3, p = 0.00, ïp2 = 0.78) with slower RT in IN trials compared to RT in CO trials. For CO trials, magnitude based inference showed that CAF leads to a likely increase of mean RT compared with PL (80%) and CHO (82%), and that CHO leads to a likely decrease of RT compared with GUAc (89%). On IN trials, CAF leads to a likely decrease of mean RT compared to PL (90%) and GUAc (94%) (Figure 3). More interestingly, magnitude based inference on the Simon effect (i.e., difference between mean IN RT and mean CO RT), which is a direct measure of cognitive control, showed a likely decrease with CAF MR (24 ms) when compared with PL (30 ms, 93%), CHO (29 ms, 85%) and GUAc (79%) (Figure 4). For CO trials, magnitude based inference showed that CAF leads to a likely increase of mean RT compared with PL (80%) and CHO (82%), and that CHO leads to a likely decrease of RT compared with GUAc (89%). On IN trials, CAF leads to a likely decrease of mean RT compared to PL (90%) and GUAc (94%) (Figure 3). More interestingly, magnitude based inference on the Simon effect (i.e., difference between mean IN RT and mean CO RT), which is a direct measure of cognitive control, showed a likely decrease with CAF MR (24 ms) when compared with PL (30 ms, 93%), CHO (29 ms, 85%) and GUAc (79%) (Figure 4). g 3.2. RPE 3.2. RPE 3.2. RPE Magnitude based inferences showed that CHO MR lead to a likely decrease in RPE (11.6 ± 0.7) cm when compared to PL ((12 2 ± 0 8) cm; 87%) GUAc ((12 5 ± 0 8) cm; 91%) and CAF ((12 3 ± 0 7) Magnitude based inferences showed that CHO MR lead to a likely decrease in RPE (11.6 ± 0.7) cm when compared to PL ((12.2 ± 0.8) cm; 87%), GUAc ((12.5 ± 0.8) cm; 91%) and CAF ((12.3 ± 0.7) Magnitude based inferences showed that CHO MR lead to a likely decrease in RPE (11.6 ± 0.7) cm when compared to PL ((12.2 ± 0.8) cm; 87%), GUAc ((12.5 ± 0.8) cm; 91%) and CAF ((12.3 ± 0.7) cm; 91%). 3.2. RPE Magnitude based inferences showed that CHO MR lead to a likely decrease in RPE (11.6 ± 0.7) cm when compared to PL ((12.2 ± 0.8) cm; 87%), GUAc ((12.5 ± 0.8) cm; 91%) and CAF ((12.3 ± 0.7) 91%) Magnitude based inferences showed that CHO MR lead to a likely decrease in RPE (11.6 ± 0.7) cm when compared to PL ((12.2 ± 0.8) cm; 87%), GUAc ((12.5 ± 0.8) cm; 91%) and CAF ((12.3 ± 0.7) cm; 91%). Magnitude based inferences showed that CHO MR lead to a likely decrease in RPE (11.6 ± 0.7) cm when compared to PL ((12.2 ± 0.8) cm; 87%), GUAc ((12.5 ± 0.8) cm; 91%) and CAF ((12.3 ± 0.7) cm; 91%). 4 Discussion Error Rate Error Rate Error Rate Error Rate Analyses showed a significant effect of congruency (F(1, 22) = 33.44 p = 0.00, ɳp2 = 0.60), illustrating the prevalence of more errors in IN trials than in CO trials. The effect of block tended to be significant (F(2, 44) = 3.8, p = 0.07, ɳp2 = 0.11). Errors in block 3 tended to be significantly lower than in block 1 (p = 0.06). No significant difference between block 2 and respectively blocks 1 (p = 0.11) and 3 ( 4 ) f d Analyses showed a significant effect of congruency (F(1, 22) = 33.44 p = 0.00, ɳp2 = 0.60), illustrating the prevalence of more errors in IN trials than in CO trials. The effect of block tended to be significant (F(2, 44) = 3.8, p = 0.07, ɳp2 = 0.11). Errors in block 3 tended to be significantly lower than in block 1 (p = 0.06). No significant difference between block 2 and respectively blocks 1 (p = 0.11) and 3 (p = 0.47) was found. Analyses showed a signiﬁcant effect of congruency (F(1, 22) = 33.44 p = 0.00, ïp2 = 0.60), illustrating the prevalence of more errors in IN trials than in CO trials. The effect of block tended to be signiﬁcant (F(2, 44) = 3.8, p = 0.07, ïp2 = 0.11). Errors in block 3 tended to be signiﬁcantly lower than in block 1 (p = 0.06). No signiﬁcant difference between block 2 and respectively blocks 1 (p = 0.11) and 3 (p = 0.47) was found. 3 (p = 0.47) was found. Magnitude based inference did not show any effect of nutritional supplementations on error rate. 3 (p 0.47) was found. Magnitude based inference did not show any effect of nutritional supplementations on error rate. Magnitude based inference did not show any effect of nutritional supplementations on error rate. 3 2 RPE 4.1. Effect of Physical Exercise on Cognitive Performance Although the effects of exercise on cognition were not the main purpose, innovative results emerged from this study. It therefore seemed interesting to discuss it in more detail in the ﬁrst part of the discussion. It is now classically reported that exercise has a positive effect on cognitive tasks, however, cognitive performance has typically been assessed during short duration exercise (about 20 min) or immediately after exercise while limited research has investigated cognition during longer exercises [47]. In the present study, we observed performance improvement in cognitive tasks throughout the exercise. In the duration-production task, participants produced shorter durations after 14 min, and they were less variable after 27 min of cycling exercise when compared to the beginning of the exercise. Most of the models that have been proposed to explain performance in temporal tasks are based on scalar timing theory [48,49]. According to this theory, people are supposed to estimate time intervals using an internal clock functioning as a stopwatch, with a clock stage composed of a pacemaker-counter device. An interval is speciﬁed by the accumulation of pulses emitted at a regular rate from the pacemaker. The more pulses that are accumulated, the longer the subjective estimation of duration. Within this framework, one hypothesis to explain shorter produced durations (called underproductions) is an acceleration of the pacemaker rate [39,50,51]. When pacemaker rate increases, pulses are emitted and accumulated faster. The number of pulses corresponding to the standard duration they have learned during the training session, would then be accumulated in a shorter time interval, leading to the production of shorter durations. The present data thus suggest that submaximal exercise leads to an acceleration of the pacemaker rate. In literature investigating the psychology of time, several studies show that the acceleration of the pacemaker rate is related to an increase in cortical arousal [39,50]. Moreover, we have observed a decrease in temporal variability, which is also classically explained by an increase in alertness level [52]. This increase in cortical arousal could be induced by an enhancement of the noradrenergic and dopaminergic systems during exercise [53,54]. During exercise the sympathetic nervous system is rapidly stimulated, leading to large increases in the secretion of adrenaline and noradrenaline from the adrenal medulla and noradrenaline from sympathetic nerves. p cm; 91%). cm; 91%). 4. Discussion 4. Discussion Th i f hi d i i h i fl f i l CHO GUA d CAF MR b h 4. Discussion The aim of this study was to investigate the influence of serial CHO, GUAc and CAF MR both The aim of this study was to investigate the inﬂuence of serial CHO, GUAc and CAF MR both on cognitive performance during a 40-min cycling exercise at a submaximal intensity and on RPE at 11 of 17 Nutrients 2017, 9, 589 the end of the exercise. Speciﬁc cognitive processes, such as cognitive control and time perception, considered as being of great importance in goal-oriented behavior, have been investigated through a Simon task and a duration-production task. Several major ﬁndings have emerged from this study: 1/CHO, CAF and GUAc MR likely lead participants to produce shorter durations and to reduce temporal variability, 2/CAF MR likely improved cognitive control and CHO MR led to a likely decrease in RPE after exercise. Moreover, performing 40-min of submaximal exercise enhanced information processing in terms of both speed and accuracy, decreased the variance in temporal production, led participants to produce shorter durations and did not alter cognitive control. 4.2. Mouth Rinse Effect on Cognitive Performance Concerning the MR effects on cognitive performance, magnitude based inferences showed that CHO, CAF and GUAc MR likely lead to shorter produced durations and a decrease in temporal production variability. As previously noticed, temporal underproductions may be explained by an acceleration of the pacemaker rate due to an increase in arousal level [39,50]. This increase of cortical arousal is also consistent with an acceleration in RTs. RTs were likely shorter in the IN trials (with no variation in the error rate) after CAF MR compared to PL and GUAc MR. This improvement resulted in a decrease in the Simon effect (or interference effect), which is a direct measure of cognitive control and highlighted an improvement in cognitive performance. The ergogenic effect of CAF on physical and cognitive performance has already been reported after ingestion [3,68]. It has been proposed that it could be at least partially related to competitive inhibition of adenosine receptors [69]. The effects of CHO ingestion on cognitive performance have not been thoroughly investigated but some studies report a positive effect on cognition. For example, a positive effect of CHO ingestion has been observed on RT after exercise [70,71]. Moreover, Lieberman and colleagues [72] found an improvement in vigilance and mood during sustained physical activity with CHO ingestion. The hypothesis is that CHO intake may affect cognition by modulating the synthesis of certain neurotransmitters. The most cited are acetylcholine, which is dependent upon glucose for its synthesis, and brain serotonin, a neurotransmitter associated with sleepiness (for review, see Reference [69]). The effect of nutritional MR without ingestion was more speciﬁcally investigated in the present study. Several studies highlighted that CHO and CAF MR could have a positive effect on physical [12,17,18,25,73,74] and on cognitive performance [13,15,21]. The present results showed that both CHO and CAF MR lead to underproduced durations and a reduction of temporal variability both considered to be due to an increase in cortical activation level in the ﬁeld of the psychology of time [39,50]. This is consistent with data from electroencephalographic activity (EEG) reported by de Pauw and collaborators [21]. Measures of cortical brain activity were regarded as an index of cortical arousal and served as a sensitive indication of the stimulating effects of CAF on brain functioning. 4.1. Effect of Physical Exercise on Cognitive Performance Even if catecholamines do not cross the blood brain barrier [55], evidence has shown that a peripheral circulating catecholamines increase during exercise is related, by several mechanisms, to an increase in concentrations of brain catecholamines, which activate the prefrontal cortex and other associated areas [56,57]. After 23 min of cycling, a positive effect of exercise was also observed in the Simon task in which we observed faster RTs without an increase of error rate (trend to lower error rates from the 13th min of exercise is even observed). Several meta-analyses and integrative reviews report that cognitive performance can be improved during acute exercise depending on factors like physical exercise intensity and duration, physical ﬁtness of participants and the nature of the cognitive tasks [47,58,59]. This beneﬁt can be explained by an enhancement of sensory sensibility [60], a better efﬁciency of peripheral motor processes [61,62] and/or a reduction in intracortical inhibition accompanied by an increase in corticospinal excitability [53]. The results of the present study are in line with these ﬁndings. Indeed, both the improvement of the information processing and the temporal variability and the underproduction of durations, suggest a possible increase in the brain activation level, which would result from an activation of the reticular-activating system [54]. 12 of 17 12 of 17 Nutrients 2017, 9, 589 However, cognitive processes seem to be differentially sensitive to the effect of exercise. To date, the accumulated evidence is equivocal and provides an unclear picture of the relationship between exercise intensity and cognitive control. Despite some evidence that suggests an impairment of cognitive control (using a Simon task or a similar conﬂict task) during exercise [63,64], others [65,66] fail to observe any deteriorations of selective response inhibition or rather report an improvement despite very high physiological stress [67]. In this study, the lack of exercise effect on the interference effect (as shown by the absence of signiﬁcant block x congruency interaction) suggested that the ability to inhibit prepotent responses remains fully efﬁcient during exercise. 4.2. Mouth Rinse Effect on Cognitive Performance Results report that CAF MR exert a likely beneﬁcial effect on RT in IN trials only in a Stroop Task which is entirely consistent with our results showing that CAF MR only lead to shorter RT with no errors shift in IN trials. The authors proposed that the observed effect was due to the subsequent activation of both orbitofrontal and dorsolateral prefrontal cortices. They also observed that CHO MR induced a higher brain activity within the orbitofrontal cortex but had no effect on RT. Concerning GUAc MR, we found some effects in the temporal task. Participants underproduced durations after GUAc MR compared with PL MR, which suggests that GUAc MR increased brain activation level as did CHO and CAF MR. To our knowledge, this is the ﬁrst time that the effect of GUA MR was observed on cognitive performance. The duration-production task is known to be highly sensitive to arousal manipulation, which could explain why data exhibited no effect in the Simon task. Finally, magnitude based inferences have also revealed that CHO MR leads to a likely decrease in RPE for the same power output compared to PL. This result is very interesting since results are mixed and most studies have failed to observe beneﬁts from CHO MR [9,44,75–77] and CAF MR [18,78] on perception of effort. Only a few studies have reported that CAF and CHO MR could lead to a decrease 13 of 17 Nutrients 2017, 9, 589 in RPE [25–27] suggesting that CHO and CAF MR induce a decrease in subjective perception of effort, allowing participants to produce more power with the same degree of discomfort. This ﬁnding may be explained by the activation of reward areas reported by Silva et al. [17] via stimulation of CHO receptors in the mouth. Indeed, the activation of the insula/frontal operculum, orbitofrontal cortex and striatum, could result in a lowering perceived exertion during exercise. Chambers (2009) [13] suggested that the activation of these brain areas could modify fatigue signals sent from the muscles and result in lower subjective perception of effort for the same power output. Concerning GUAc, our results failed to highlight a positive effect on RPE as observed in a recent study using GUAc ingestion [34]. The authors observed a small but signiﬁcant reduction in RPE during a 30-min treadmill running exercise after ingestion of multivitamin and mineral complex with guarana. 4.2. Mouth Rinse Effect on Cognitive Performance The difference of results could be due, ﬁrst, to the lower amount of GUAc used in our study compared to Veasey and colleagues’ study [34] and, second, to the different administration modes (ingestion versus MR). We can hypothesize that with MR, a larger amount of GUA would be required to impact the RPE. One limitation of the present study is the use of a multivitamin and mineral complex with guarana instead of GUA solely, which did not allow isolate the pure effect of GUA MR on performance. At present, mechanisms accountable of ergogenic effects of GUA ingestion on cognitive performance need to be further investigated. We should also note that some studies suggest that oral contraceptive steroids and estrogen have a signiﬁcant impact on caffeine metabolism [79] and that RT lengthens in the high-progesterone, luteal phase of the menstrual cycle [80]. The fact that the inﬂuence of different phases of menstrual cycle of the female participants has not been controlled in the present study, due to methodological difﬁculties, is also a limitation. Further controlled studies will be necessary to examine potential interactions with mouth rinsing. Magnitude based inferences showed that CHO, CAF and GUAc MR enhance performance. It is still important to note that nutritional supplements beneﬁted only half of the participants. However, when a beneﬁcial effect of MR was observed it was up to two to three times greater than the deterioration (or lack of effect) observed on the other half of the subjects. These ﬁndings suggest that, as for ingestion of nutritional supplementations, the effects of MR on performance are highly variable between individuals. Before using nutritional strategies with athletes during sporting events, it seems essential to determine beforehand whether the athlete is a good or bad responder. 5. Conclusions The present study was the ﬁrst to investigate the effects of different nutritional supplements MR on cognitive performance during exercise. Our data have suggested that the serial administration of CHO, CAF and GUAc MR improves cognitive performance and decreases subjective perception of effort. Moreover, they conﬁrm that submaximal exercise increased arousal levels leading to an improvement in cognitive performance [53,54]. The present study provided important new insights regarding the speciﬁc changes in cognitive processes induced by nutritional supplements and opened up new perspectives regarding the use of MR to optimize cognitive performance during exercise. Obviously, some further studies are needed to conﬁrm these results and to more investigate speciﬁc cognitive processes in more depth. Moreover, while the present study involved a 40-min cycling exercise at a submaximal intensity, which is longer than, most of the previous studies, future studies involving more exhausting exercises, in terms of duration and/or intensity, are needed to further elucidate the relationships between nutrition supplements and fatigue and to uncover whether interactions between these two factors could emerge. Supplementary Materials: The following are available online at www.mdpi.com/2072-6643/9/6/589/s1. Acknowledgments: We thank all the participants for participating to this study, the CREPS PACA and Squash French Federation for ﬁnancial support. Thanks also to Jennifer Joyce for her proofreading of the ﬁnal version of the manuscript. 14 of 17 14 of 17 Nutrients 2017, 9, 589 Author Contributions: J.B., K.D., L.C. and L.P. conceived and designed the experiments; L.P. performed the experiments; J.B., K.D., L.C. and L.P. analyzed the data; A.H contributed reagents/materials/analysis tools; J.B., K.D., L.C. and L.P. wrote the paper. Author Contributions: J.B., K.D., L.C. and L.P. conceived and designed the experiments; L.P. performed the experiments; J.B., K.D., L.C. and L.P. analyzed the data; A.H contributed reagents/materials/analysis tools; J.B., K.D., L.C. and L.P. wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. 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https://openalex.org/W2556921985	https://www.scielo.br/j/jbpsiq/a/F3T4Fw5NLFNzBsdfXdwSM5d/?lang=en&format=pdf	English	null	Psychogenic polydipsia and hyponatremia – A side effect of psychosis: a review with a case report	Jornal Brasileiro de Psiquiatria	2,016	cc-by	3,008	Psychogenic polydipsia and hyponatremia – A side effect of psychosis: a review with a case report ARTIGO ORIGINAL CASE REPORT João Perestrelo1, Bruno Teixeira1 João Perestrelo1, Bruno Teixeira1 Keywords Keywords Primary polydipsia, psychogenic polydipsia, hyponatremia, psychotropic medication, antipsychotics. Keywords Primary polydipsia, psychogenic polydipsia, hyponatremia, psychotropic medication, antipsychotics. ABSTRACT Primary polydipsia, or psychogenic polydipsia, is a condition that results in considerable morbidity and mortality. In psychiatric patients, psychogenic polydipsia and the syndrome of inappropriate antidiuretic hormone secretion may cause hyponatremia. In the 1970s, it was recognized that antipsychotics such as tiotixene and haloperidol could impair the excretion of a free water load. There are also several case reports of drug-induced hyponatremia in patients using atypical drugs suggesting that these probably can also impair water balance and induce hyponatremia. Case report and review of relevant literature are reported in this article. Psychogenic polydipsia is a common cause of hyponatremia among individuals with chronic mental illness. A case of severe hyponatremia caused by psychogenic polydipsia is described involving a female patient with an adult lifelong history of chronic mental illness diagnosed as schizoaffective disorder. After switching her antipsychotic medication to clozapine water ingestion was normalized as well as sodium levels and her psychotic symptoms improved. Primary polydipsia occurs commonly with schizophrenia and other mental diseases and can cause hyponatremia. PPD may present as an acute psychotic state or as inexplicable emergence of seizures. Appropriate, timely clinical assessment with special attention to thirst, fluid intake, and urine output is essential. Proper treatment may include drug withdrawal and fluid and saline restriction. Once corrected, some pharmacological agents can be tried. The article illustrates the importance of the diagnosis of psychogenic polydipsia given its electrolyte disturbances and life threatening situations. INTRODUCTION A 48-year-old female, addressed as Ms. L, presented to our emergency department with altered mental status including disinhibition, dysphoric mood with pressured and coprolalic speech, disaggregated thought and delusional ideas of per- secution, increasing in severity over the past 5 days. The pa- tient had a longstanding history of schizoaffective disorder and had been managed with haloperidol 15 mg/day, hal- dol decanoate 100 mg/month, levomepromazine 150 mg/ day, carbamazepine 200 mg ER twice a day, trihexyphenidyl 4 mg/day, lorazepam 10 mg/day. She was also on carvedilol 25 mg/day, irbesartan 300 mg/day, amlodipine 10 mg/day and indapamide ER 1,5 mg/day to control her blood-pres- sure levels. Psychogenic polydipsia (PPD) is a condition that results in considerable morbidity and mortality1. Psychogenic polydipsia (PPD) is a condition that results in considerable morbidity and mortality1. Hyponatremia is seen in about 4% of chronic schizophrenic patients and sometimes in patients with other psychiatric disorders like psychotic depression, bipolar disorder and mental retardation2. It can lead to serious clinical consequences such as delirium, seizures or rhabdomyolysis and is also an important risk factor for neuroleptic malignant syndrome3. In psychiatric patients, PPD and the syndrome of inappropriate antidiuretic hormone secretion (SIADH) may cause hyponatremia2. PPD is characterized by low plasma sodium in 10% to 20% of those presenting with uncontrollable drinking1. Symptoms are uncommon unless patients continue to drink excessively (> 10 L/d) after they reach their limit of urine dilution (100 mOsm/kg with minimum urine osmolality) and full antidiuretic hormone (ADH) suppression4. A report from the patient’s family revealed that she had increased her fluid intake to up to 6-7 L of water daily. A detailed examination was performed including a blood sample that revealed a serum sodium level of 119 mmol/L, potassium of 3.32mmol/L and chloride level of 79.6 mmol/L. Thyroid hormones, glucose, serum urea and creatinine levels were in the normal range. Urine levels of potassium, sodium and creatinine were also obtained (3.0 mmol/L; 20 mmol/L; < 4.2 mg/dL, respectively) as urine osmolality (43 mOsm/ KgH2O). In the 1970s, it was recognized that antipsychotics such as tiotixene and haloperidol could impair the excretion of a free water load. Later, it was revealed that elderly patients treated with phenothiazines had significantly lower serum sodium levels than patients not treated with these drugs. RESUMO A polidipsia primária ou polidipsia psicogênica (PDP) é uma condição que se traduz em altas taxas de morbilidade e mortalidade. Nos doentes psiquiátricos, a PDP e a síndrome da secreção inapropriada do hormônio antidiurético (SIADH) podem resultar em hiponatremia. Nos anos 1970, foi reconhecido que alguns antipsicóticos como o tiotixeno e o haloperidol 1 Vila Nova de Gaia/Espinho Healtcentre, Department of Psychiatry and Mental Health, Porto, Portugal. Address for correspondence: João Perestrelo Centro Hospitalar de Vila Nova de Gaia Rua Conceição Fernandes, s/n Vila Nova de Gaia 4434-502 – Porto, Portugal Telephone: 227 865 100 Fax: 227 830 209 E-mail: joaomfp@hotmail.com Received in 4/4/2016 Approved in 8/15/2016 DOI: 10.1590/0047-2085000000135 1 Vila Nova de Gaia/Espinho Healtcentre, Department of Psychiatry and Mental Health, Porto, Portugal. Address for correspondence: João Perestrelo Centro Hospitalar de Vila Nova de Gaia Rua Conceição Fernandes, s/n Vila Nova de Gaia 4434-502 – Porto, Portugal Telephone: 227 865 100 Fax: 227 830 209 E-mail: joaomfp@hotmail.com Psychogenic polydipsia and hyponatremia – A side effect of psychosis Case report Case report 301 podiam comprometer a excreção de água livre. Existem ainda diversos casos reportados de hiponatremia iatrogênica em pacientes medicados com antipsicóticos atípicos sugerindo que essa classe farmacológica pode igualmente resultar em hiponatremia. Um caso clínico e uma revisão sobre o tema são reportados neste artigo. A polidipsia psicogênica é uma causa comum de hiponatremia entre os indivíduos com doença mental crônica. Um caso de hiponatremia grave causada por polidipsia psicogênica é descrito envolvendo uma paciente do sexo feminino com antecedentes de perturbação esquizoafetiva. Após alteração da sua medicação antipsicótica para clozapina, observou-se normalização na ingestão hídrica bem como nos níveis de sódio e melhoria da sintomatologia psicótica. A PDP ocorre comumente em doentes esquizofrênicos ou com outras perturbações psiquiátricas e pode resultar em hiponatremia. Um correto diagnóstico com especial atenção a sede, ingestão hídrica e débito urinário é essencial. O tratamento pode incluir a retirada de fármacos e restrição hídrica e salina. Uma vez corrigida, alguns agentes farmacológicos podem ser utilizados. Palavras-chave Polidipsia primária, polidipsia psicogênica, hiponatremia, psicofármacos, antipsicóticos. Palavras-chave Polidipsia primária, polidipsia psicogênica, hiponatremia, psicofármacos, antipsicóticos. J Bras Psiquiatr. 2016;65(3):300-3. DISCUSSION Hyponatremia is the result of an imbalance in water-elec- trolyte homeostasis, usually defined as a lowered serum so- dium level of < 136 mmol/L8. Hyponatremia is the result of an imbalance in water-elec- trolyte homeostasis, usually defined as a lowered serum so- dium level of < 136 mmol/L8. The differential diagnosis of hyponatremia is complex and includes many different etiologies, however it can be simplified into two categories: high (> 295 mOsm/kg) versus low (< 280 mOsm/kg) plasma osmolality. Low plasma osmolality can be further divided into hypo-, hyper-, or normovolemia8. Ms. L was included in this last category. PPD may present as an acute psychotic state or as unexplained emergence of seizures. Some studies show a temporal relation between exacerbations of psychosis and psychogenic polydipsia and water intoxication in psychiatric patients10. Most psychiatric patients with hyponatremia will fit into the normovolemic category and will have low plasma osmolality, lacking clinical indicators of altered volume status. PPD and SIADH belong to this category9. Most cases of PPD with hyponatremia present a very high volume of intake. These cases have very low levels of ADH and urine osmolality is very low (< 100)9 as seen in the presented case. Most inpatients with PPD require evaluation and treatment of their hyponatremia. Symptoms usually occur over 48 hours due to intake far greater than normal levels and resulting cerebral edema. Often with chronic hyponatremia, central nervous cells have adjusted to the chronic state thus, low sodium is asymptomatic. The main goals are diagnosis and treatment of the primary condition and monitoring of electrolytes. Therapeutic fluid restriction is a low-cost form of treatment but given the low insight and noncompliance in psychiatric patients, it may take several days for an effect to be seen12. Clinical manifestations of hyponatremia are commonly related to the osmotic water shift towards the intracellular space, with subsequent increased intracellular fluid volume that can cause cerebral edema. The symptoms are primarily neurological, with their severity related to the speed of onset and the absolute decrease in the serum sodium concentration. Patients may be initially asymptomatic or can experience nausea, malaise or confusion. As the serum concentration continues to fall, additional symptoms including headache, muscle cramps and lethargy can present, increasing confusion and agitation. Caution must be taken in correction of hyponatraemia as central pontine myelinolysis may occur if correction is overly rapid. INTRODUCTION After three-weeks the sodium levels were at the normal range and she started presenting with less delusional ideas and a more adequate behavior interacting more appropriately with the medical team. There are various other theories concerning the etiology of PPD including schizophrenia positive symptoms, compulsive behavior, stress reduction and efforts to counteract the anticholinergic medication side effects1. It was proposed that the increased prevalence of ventricular enlargement in schizophrenic patients may lead to structural impairment of the lateral hypothalamus and disturbances in thirst and osmolality. The polydipsic theory of dopamine supersensitivity is another explanation for the association of polydipsia and psychosis. Dopamine is an important neurotransmitter in the area of the thirst center in the lateral hypothalamus. In animals, a hyperdopaminergic state is associated with increased fluid intake, which can be reversed by dopamine antagonists. High dopamine levels are hypothesized to stimulate thirst centers and dopamine receptor hypersensitivity might explain why PPD often occurs late in the course of schizophrenia, after years of exposure to typical neuroleptics11,12. She was discharged one week later with no psychotic symptoms and an adequate water ingestion. Twelve months after the patient remained free of psychotic or mood symptoms. J Bras Psiquiatr. 2016;65(3):300-3. INTRODUCTION Additionally, it was shown that a greater portion of patients treated with haloperidol had impaired free water excretion and urinary dilution than asymptomatic controls5,6. There are also several case reports of drug-induced hyponatremia in patients using atypical antipsychotics suggesting that these might also impair water balance and induce hyponatremia7. She was immediately hospitalized in our psychiatry unit and performed additional analytic and imaging studies including cortisol (8h) and fasting aldosterone levels, both in the normal range, and a CT scan that didn’t reveal any morphologic change. It was diagnosed a normovolemic hyponatremia and a hypertonic saline solution to correct the electrolytic imbalance was prescribed. This article is designed to present a clinical case of a psychotic patient with psychogenic polydipsia and hyponatremia. A review of the recent literature is presented, based on the best available evidence highlighting the importance of a correct diagnosis and treatment (pharmacologic and behavioral interventions). Levomepromazine, carbamazepine and indapamide were suspended and haloperidol was reduced to 10 mg/day. Oral fluid restriction of 1.5 L/day was instituted although she complained of intense thirst. J Bras Psiquiatr. 2016;65(3):300-3. 302 Perestrelo J, Teixeira B 302 Perestrelo J, Teixeira B Case report Case report 302 Sodium level normalized in 5 days but she was still delusional and dysphoric with no symptom improvement and always trying to sneak to the bathrooms to drink water. Clozapine was initiated as she had failed several trials of first and second generation antipsychotics. The dose was titrated until 400 mg/day with analytic surveillance, without intercurrences. After three-weeks the sodium levels were at the normal range and she started presenting with less delusional ideas and a more adequate behavior interacting more appropriately with the medical team. regulation of the hypothalamic-pituitary axis, leading to dysregulation of antidiuretic hormone (ADH) secretion11. Autoimmune illnesses may also lead to hypothalamic lesions, causing increased thirst. Another theory proposes that patients with PPD present with polydipsia even when ADH is fully suppressed11. Patients exhibit symptomatic hyponatremia when the kidney’s short-term capacity to excrete urine (10 to 15 L/d) is overwhelmed. Sodium level normalized in 5 days but she was still delusional and dysphoric with no symptom improvement and always trying to sneak to the bathrooms to drink water. Clozapine was initiated as she had failed several trials of first and second generation antipsychotics. The dose was titrated until 400 mg/day with analytic surveillance, without intercurrences. DISCUSSION A correction rate of less than 10 mmol/L is advisable over the course of 24 hours10. Atypical antipsychotic agents have been shown in case reports to have some success in alleviating symptoms of PPD. Low-dose risperidone and olanzapine improved polydipsia in a case report1. Clozapine has effects in managing PPD but its efficacy remains unproven in large trials13,14. Although its doubtful efficacy, the patient presented in the clinical case was medicated with clozapine showing great improvements. Beta blockers such as propranolol have been found to be effective8. Another treatment for patients with chronic hyponatremia is demeclocycline, 600 to 1200 mg/d, that directly inhibits ADH action at the distal renal tubules Although PPD is multifactorial, malfunction of the hypothalamic thirst center is seen as a possible cause. Chronic intake of excess fluid may change feedback Case report Psychogenic polydipsia and hyponatremia – A side effect of psychosis 303 REFERENCES 1. Kruse D, Pantelis C, Rudd R, Quek J, Herbert P, McKinley M. Treatment of psychogenic polydipsia: comparison of risperidone and olanzapine, and the effects of an adjunctive angiotensin-II receptor blocking drug (irbesartan). Aust N Z J Psychiatry. 2001;35(1):65-8. 1. Kruse D, Pantelis C, Rudd R, Quek J, Herbert P, McKinley M. Treatment of psychogenic polydipsia: comparison of risperidone and olanzapine, and the effects of an adjunctive angiotensin-II receptor blocking drug (irbesartan). Aust N Z J Psychiatry. 2001;35(1):65-8. This case emphasizes the potentially life-threatening effects of psychogenic polydipsia. The phenomenon is not a new one, but this case highlights a different viewpoint questioning whether hyponatremia is an independent result of psychogenic polydipsia, or whether it leads to a vicious circle, resulting in hyponatremia that may cause psychiatric disturbances. 2. Goldman MB. The assessment and treatment of water imbalance in patients with psycho- sis. Clin Schizophr Relat Psychoses. 2010;4(2):115-23. 2. Goldman MB. The assessment and treatment of water imbalance in patients with psycho- sis. Clin Schizophr Relat Psychoses. 2010;4(2):115-23. 3. Elizalde-Sciavolino C, Racco A, Proscia-Lieto T, Kleiner M. Severe hyponatremia, neurolep- tic malignant syndrome, rhabdomyolysis and acute renal failure: a case report. Mt Sinai J Med. 1998;65(4):284-8. 3. Elizalde-Sciavolino C, Racco A, Proscia-Lieto T, Kleiner M. Severe hyponatremia, neurolep- tic malignant syndrome, rhabdomyolysis and acute renal failure: a case report. Mt Sinai J Med. 1998;65(4):284-8. 4. Dundas B, Harris M, Narasimhan M. Psychogenic polydipsia review: etiology, differential, and treatment. Curr Psychiatry Rep. 2007;9(3):236-41. 4. Dundas B, Harris M, Narasimhan M. Psychogenic polydipsia review: etiology, differential, and treatment. Curr Psychiatry Rep. 2007;9(3):236-41. 5. Peck V, Shenkman L. Haloperidol-induced syndrome of inappropriate secretion of antidi- uretic hormone. Clin Pharmacol Ther. 1979;26(4):442-4. 5. Peck V, Shenkman L. Haloperidol-induced syndrome of inappropriate secretion of antidi- uretic hormone. Clin Pharmacol Ther. 1979;26(4):442-4. INDIVIDUAL CONTRIBUTIONS and reduces urine concentration8. Lithium, which works as a direct competitive antagonist of ADH action, is seldom used as it may be potentially nephrotoxic and thyrotoxic. João Perestrelo – Conception, design and drafting of the article. Another study15 looked at the use of clonidine and enalapril and found improvement in fluid consumption concluding that medications that affect body water balance might decrease excess fluid intake in some patients with history of water abuse. Irbesartan, an angiotensin receptor blocker, was found to be an effective adjunct in the treatment of PPD in a case report1. Lately, newer agents named “aquaretics” that antagonize ADH receptors have been found to increase free water clearance without directly affecting tubular sodium. Conivaptan belongs to this class and has been recently approved by the US Food and Drug Administration for the treatment of euvolemic hyponatremia in inpatients8. Bruno Teixeira – Critical revision of the article and final approval of the version. CONFLICTS OF INTEREST No author has a conflict of interest. CONCLUSION 6. Ajlouni K, Kern MW, Tures JF, Theil GB, Hagen TC. Thiothixene-induced hyponatremia. Arch Intern Med. 1974;134(6):1103-5. 6. Ajlouni K, Kern MW, Tures JF, Theil GB, Hagen TC. Thiothixene-induced hyponatremia. Arch Intern Med. 1974;134(6):1103-5. Primary polydipsia occurs commonly with schizophrenia and other mental diseases and can cause hyponatremia, with acute cerebral edema, coma, and even death. PPD may present as an acute psychotic state or as inexplicable emer- gence of seizures. 7. Bachu K, Godkar D, Gasparyan A, Sircar P, Yakoby M, Niranjan S, et al. Aripiprazole in- duced syndrome of inappropriate antidiuretic hormone secretion (SIADH). Am J Ther. 2006;13(4):370-2. 8. Palmer BF, Gates JR, Lader M. Causes and management of hyponatremia. Ann Pharmaco- ther. 2003;37(11):1694-702. 9. Dundas B, Harris M, Narasimhan M. Psychogenic polydipsia review: etiology, differential, and treatment. Curr Psychiatry Rep. 2007;9(3):236-41. Appropriate, timely clinical assessment with special attention to thirst, fluid intake, and urine output is essential. Proper treatment may include drug withdrawal, fluid and saline restriction which reverse the electrolyte disturbance. Once corrected, some pharmacological agents can be tried. The current literature is not clear as to which treatment is superior, and numerous agents that have been shown some success treating this disturbance are available. In this patient there was a complete recovery after switching her antipsychotic to clozapine. Importantly, at 12-month follow-up the patient has been free of psychotic and affective symptoms. A combination of behavioral treatments and drugs might be the best approach and has been shown to be effective in the long term, but PPD, has a relapsing course and may reoccur without clinician vigilance and appropriate management. 10. Siegel AJ. Hyponatremia in psychiatric patients: update on evaluation and management. Harv Rev Psychiatry. 2008;16(1):13-24. 11. Verghese C, De Leon J, Simpson GM. Neuroendocrine factors influencing polydipsia in psy- chiatric patients: an hypothesis. Neuropsychopharmacology. 1993;9(2):157-66. 12. Kowalski PC, Dowben JS, Keltner NL. Biological perspectives: hyponatremia: a side effect of psychosis. Perspect Psychiatr Care. 2014;50(4):221-3. 13. Meulendijks D, Mannesse CK, Jansen PA, van Marum RJ, Egberts TC. Antipsychotic-induced hyponatraemia: a systematic review of the published evidence. Drug Saf. 2010;33(2): 101-14. 14. Leadbetter RA, Shutty MS Jr. Differential effects of neuroleptic and clozapine on polydipsia and intermittent hyponatremia. J Clin Psychiatry. 1994;55(Suppl B):110-3. 15. Greendyke RM, Bernhardt AJ, Tasbas HE, Lewandowski KS. Polydipsia in chronic psychi- atric patients: therapeutic trials of clonidine and enalapril. Neuropsychopharmacology. 1998;18:272-81. J Bras Psiquiatr. 2016;65(3):300-3.
https://openalex.org/W2078003041	https://zenodo.org/records/1791161/files/article.pdf	German	null	Über die Biologie von Glyphotaelius punctatolineatus Retz. nebst Bemerkungen über das freilebende Puppenstadium der Wasserinsekten	Internationale Revue der gesamten Hydrobiologie	1,910	public-domain	10,067	M i t t eilung en a u s dem biologis c hen Sii Is w a s s erl a b o r at oriurii E'rederiksdal bei Lyngby (Di,nernark). Kr. V. M i t t eilung en a u s dem biologis c hen Sii Is w a s s erl a b o r at oriurii E'rederiksdal bei Lyngby (Di,nernark). Kr. V. Ober die Biologie von 61 y p h o t aeliu s p unc t a t o line a t us Re tz. nebst Bemerkungen uber das freilebende Puppen- stadium der Wasserinsekten. Von Dr. C. Wesenberg-Lund (Hillerod). Mit Tafel I (Figiir 1-32) iind einor dbbildung iiii Text. I. Naturbeobaohtungen iiber Glyphotaelius. An den Ufern des Versuchsteichs I1 findet man in den letzten Tagen des Juni bis cat. 1. August auf der Ufervegetation grofie, fahle Gallert- inassen (Fig. 1); sie sind von langlicher Form, beinahe 30 bis 40 ern lang: ca. 1 cm breit und ca. 3/4 bis 1 cm hoch. Das sind die Eiermassen von Glyphotaelius punctatolineatus Retz., der nach E. Petersen einer unserer seltenen Phryganeen ist (1907, S. 145). Betrachtet man eben ah- gelegte Eiermassen, sieht man, dafi sie quergefurcht sind; spiiter, wenn die Gallerte alter werden, und besonders wenn die Jungen ausgekrocheii sind, geht die Querfurchung verloren. Ich habe leider nie die Eiablage beobachtet, und so vie1 ich w e 8 , hat auch kein anderer sie gesehen. Weil ich aber sehr oft in der Eiablageperiode an den Ufern der Teiche war, recht hiiufig das Tier sah, nie aber eine Eiablage, vermute ich, da8 diese in der A4benddiimnierung oder in der Nacht vor sich geht. Wahr- scheinlich stammt die Querfurchung davon her, daD das Weibchen. wenri die Eier hervortreten, die Spitze des Abdomens von links nach rechts oder umgekehrt bewegt, indem es zugleich larigsarn an dein Stengel auf- oder niederwarts wandert. Ini Jahre 1907 fanden die Eier sich ha. a m 1. Juli, im Jahre 1909 ca. an1 10. Juli in grofien Mengen. Die Eierrnassen waren irnmer uher dem Wasser angebracht, ofters an den Irisblattern, aber auch auf Uares- halmen u. a. Sehr oft standen die Pflanzen direkt im Wasser, oft aber am Ufer wachsend und bis ca. ',IB 111 vom Wasser entfernt. In Trocken- - 94 - perioden werden die Eierinassen milchweiD , und die adersten Gallert- schichten werden dann dick und sehr ziihflussig; ferner beobachtet inan, daD das Yolumen stark abnimmt. In Regenperioden quellen sie stark auf, die Gallertmassen werden beinahe hyalin und sehr fliissig. In den Trocken- perioden hart die Entwickluiig der Eier ganz auf, dagegen entwickeln die Eier sich in den Regenperioden mit merkwurdiger Schnelligkeit. d8. Juni wurden die Blatter, worauf vier wahrscheinlich eben abgelegte Eiermassen saBen, inarkiert und alle drei ‘l’age besichtigt. In den feuchten, recht Vrrsuclisteich 11; die Heiinat der Glypliotaelius, die Wasseroberfllche niit P o t a m o gc t on n a t a n s hcdcckt. (Winge phot.) Vrrsuclisteich 11; die Heiinat der Glypliotaelius, die Wasseroberfllche niit P o t a m o gc t on n a t a n s hcdcckt. I. Naturbeobaohtungen iiber Glyphotaelius. (Winge phot.) kalten Tagen wiichsen sie betriichtlich. 10. Juli enthielten die Klumpeii kleine Larven, die sich noch nicht bewegen konnten. 13. Juli saDen die Gallertmassen noch auf den Blattern, waren aber von Larven ganz en tleer t. Ich habe Gallertmasseii genommen und an eine trockene, von dor Sonne beschienene Stelle gebracht; 14 “age verhielten die Eiermassen sich ganz unverindert. Dann wurden die nun verwelkten Bl5tter mit den Eiermassen in Wasser unter eine Glasglocke gestellt; die Gallerten quollen imf, die fruher weibgelben Eier wurden schwarz, und im Laufe von ffhf Tagen kroehen die Larven aus. 20. his 25. Juli fand ich iibera31 zer- flieDende Gallertmassen. Die Larven halten sich, besonders in Trocken- - 95 - 95 - perioden, in den Gallerten auf, wachsen und niihren sich unzweifelhaft von denselben. Bald findet man in den Gallertmassen Eierschalen , bald nicht; im letzteren Falle sind sie von den Larven verzehrt. In den herabflieDenden Laichmassen sammeln die Larven sich immer it1 den niedrigsten 'I'eilen und fliefien mit denselben larigsam hinunter. Eiermassen, die so aufgehiingt waren, da8 die Larven direkt ins Wasser niedertropfen konnten, habe ich nie gesehen, glaube aber, da8 der Trans- port der Larven besonders in den Morgenstunden, wenn der Tau alles be- deckt, wie auch im Regen vor sich geht. g g Wenn die Eiermassen an vertikalen, direkt im Wasser wachsenden Binsenstengeln abgesetzt wurden , flossen die Larven naturlich mit den Gallerten direkt in das Wasser hinab. Haufig aber konnten die Larven wahrend des passiven Transports nicht das Wasser, sondern nur die feste Erde, und das oft in einer Entferniing von circa 1 m vom Wasser, er- reichen. So weit ich verstehen kann, sind auch die Larven (Fig. 26-27) fur eine direkte Wanderung angepafit ; darauf deuten besonders die relativ sehr langen Beine, von welchen die letzten zwei Paare beinahe eine halbe Korperlange haben. Die Beine stehen vom Korper frei hervor und sind als Schreitbeine sehr brauchbar. Wegen der langen umschlagbaren Klauen spielen sie auch als Klammerorgane eine gro8e Rolle. Die Nachschieber sind sehr stark entwickelt und stehen frei hervor. Cber dem ganzen Korper, besonders am Kopfe ein wenig hinter den Augen, Pronotum und an den Sbdominalsegmenten ragen lange steife Borsten hervor, die wahr- scheinlich wenigstens teilweise im Dienste der Lokomotion stehen. In den Aquarien habe ich gesehen , daD die Larven sich sehr geschickt bewegen. Es ist daher wahrscheinlich, daD dieselben leicht selbst ihren Weg nach dem Wasser finden konnen. I. Naturbeobaohtungen iiber Glyphotaelius. Die hier mitgeteilten Beobachtungen stimmen iibrigens gut mit dem, was Siltala in seiner schonen Arbeit 1906b S. 47 bis 49 und S. 53 iind a. a. 0. berichtet hat; nichts streitet gegen Silt alas Beobachtungen, und diese sind vielleicht ein wenig erweitert worden. Obrigens verweise ich auf seine Arbeit. In den folgenden Stadien verschwinden alle diese Charaktere; bei den ausgewschsenen Larven (Fig. 28) liegen die zwei letzen Beinpaare viel mehr an den Korper angedruckt; sie dienen wie die ersten Beinpaare rnehr als Klammerorgane, sind viel kiirzer und erreichen nicht mehr als der Korperlange; die Klauen sind nicht so lang, und die Nachschieber sind kleiner und nicht so frei hervorstehend, auch die Borsten sind nicht so stark entwickelt. Auf welchem Stadium alle diese Charaktere verloren gehen, wei8 ich nicht mit Sicherheit zu sagen. Die verschiedenen Stadien Silt alas (1907a) - 96 - sind, so lange sie nicht an die Hiiutungen sngekniipft wertlen kijnneri, meirier Meinimg nach nur von ziemlich geringern Wert. Jedenfalls steht es fest, daD die vier ersten Stadieri Siltalas sehr schnell durchlaufen wer- den. Im Sommer 1909 ka,men wahrseheinlich keine Larven eher als ca. 16. .Juli hervor; schon am 10. Aug. sind alle Larven, die ich gesehen habe, im ITierten Stadium (Liinge 10 bis 17 mni), und viele wttren im funften Stadium. Ich bin geneigt zu glauben, daD nicht jedem der vier Stadien Siltalas eine Hiiuturig entspricht, und claD mehrere Hiiutungen zu seinem fiinften und letzten Stadium gehoren. Doch ist es mir unmoglich, hieruber etwtts Sicheres zu sagen. fYenn die jungen Larven das W asser erreichen, bauen sie in wenigen Stuiiden in meirien Aquarien ihr erstes Haus; ich habe hirrzu den Tieren kleine Stiicke von Hdmen usw. angeboten; sie wurden gierig ergriffen und fiir den Bau der Kocher verwendet,. Die Koc.her waren unregelmafiig und interessierten mich nur so lange, bis ich die Larven des ersten St,adiurns in tler Xatiir gefunden hatte. A41s ich nun die Verhaltnisse in der Natur dudierte, fand ich eiries liorgens G allertmassen , die in ihrem Iririern leere Eierschalen und keine Larven enthielten ; in der nachsten Urngebung der Pflmzen (einer Iris) f m d ich nuf den submersen fein geteilten Blattern von Sium latifo lium gro8e Mengen grauer , langsam herunikriechender Strtubyartikeln , die sieh bald als Glyphotaeliuslarven erwiesen. Die Larven (Fig. I. Naturbeobaohtungen iiber Glyphotaelius. 2) waren alle ent- weder in demersten oder zweiten Stadium Siltalas; keine iiber 4 rnin lang und krochen geschickt auf der Vegetation umher. Uer Kocher, der nicht rtiehr als 3 mm niessenden Larven ist ein kleines giillertartiges hyalines Rohr, nur c,a. 4 mm lang und ca. 1 mm breit, in seinem hinteren Teil mit kleinen schwarzbrauneri Wurzelresten bedeckt ; diese liegen Ale mit der Langsachse parallel. Wenn das Rohr nur 2 bis 3 mm lang geworden ist, nimmt das Tier ein Stuck Halm, (Fig. 3 bis 5), dm es wahr- scheinlic~h abbeifit. Dieses Stiick ist ca. 4 bis 6 mm lnng uiid 1 bis 2 mm breit; es wird der Quere iiach an das Rohr a,ngebrxht, immer am vordereri Teil und so, daD es uber dio vordere Rohrenmuiiduiig be.deutend hervor- rsgt; auf diesem Stuc,k baut das Tierchen nun die Seiteiiteile des Rohres weiter uiid fugt in dieselben ganz kleiiie Pflanzenyartikehi. Wenn die Seiteriteile als zwei Walle hervortreteii, wird eiri neues Querstuck auf der entgegengesetzteri Seite aiigebracht uiid immer so, da8 der hintere Rand des iieuen Stucks den vordereri des alteren, schon aigebrachten uberragt. Immer baut das Tier so, da8 das Rohr genau in der Mitte des ganzen Kochers liegt, das heifit so, daD gleich groWe Teile uber das Rohr hinaus- ragen; dieses selbst hat iiur eine Breite \'on einem Drittel des ganzen Kochers; es wird von dem Ra,uplnn des Tieres bedingt, daD die zwei Rreit- fliichen des Kochers , fur jetles neue eingefugte Querstuck wechselweise - 97 - - 97 - 97 Riicken- urid Bauchseite ist. Die Querstiicke werden rnit dem ,,Roden" und dern ,,Deckel" des Rohres zusammengewebt, im allgemeinen jedoch so, daD nur die mittleren Teile init dem Gespinsto zusammenhangen, wahrend die vorderen und hinteren Rander frei hervorragen. Sobald das Tier 3 bis 4 Querstiicke angebracht hat, geht das hintere unbedeckte Ende zugrunde ; wahrscheiiilich fallt es ab; rnoglicherm-eise wird es abgebisseii. So fiigt also das Tier wechselweise bald auf der ,,Rucken"- bald auf der ,,Bauch- suite' ein neues Stuck zu und baut an dasselbe die Seitenwdle des Rohres an. Indem der Kocher an seinem vorderen Ende wiichst, nimmt er in dem hinteren immer ab: SO lange die Kocher noch wachsen, sind die Seiten schwach divergierend, spiiter werden sie parallel. I. Naturbeobaohtungen iiber Glyphotaelius. sowohl daB die Oberflachen der Teiche mit zahlreichen ab- geworfenen Blattstiicken bedeckt sind, als aueh dalS die Hlattstucke des Kochers mindestens bis September sich irnrner frisch grun erhalten. Die groDten Kocher, die ich gemessen habe, sind 55 inm lang und ca. 25 bis 30 min breit. In den Heibstmonaten sieht man den EinfluD der Glyphotaelien an den Potamogetonbliittern sehr deutlich. Der Angriff fangt 1 om Uferrande an, erreicht, aber im Oktober den ZuWersten Rand der Potamo- getonzone. Im Jshre 1908 war die ganze Zone beinahe abgeblattert, iiur die leeren Blattstiele uiid die Rudimente der Blatter waren iibrig geblieben. DaR die Ilarven der Hydromnipen, die iiicht haufig waren, zu diesern fur die Potamogetonpflanzen ttsurigen Resultnte beigetragen hatteu , ist u~i- zweifelhaft ; ihre Hauptentwicklungsperiode f&llt aber in die Monate Juni iind Juli, nicht wie die der Glyphtotaelien in den Spiitherbst. Es war im Oktober merkwurdig , die tausende wid abertausende von Bliittern der t’flanzen abgelost zu sehen und an den Kochern der Glyphotselien wieder zu finden. Die Potamogeton miifiten nicht allein Material fiir die Wohnung der Tiere abgebeii : sie waren zugleich deren Nahrungspflanze; die Unter- seite der Bliitter wurde ahgenagt, die Oberhaut der Oberseite im allgeineinen geschont. Der L)al-mkana,l der Larven war mit dem griinen breiartigen Material vollgepfropft, und als der Angriff am stiirksten war, sah nian uberall die braungriinen Exkremeiite an den Bla ttstielen oder langsam durch das Wasser fallen. Meistens hiingt das Tier mit den Beinen an den Stielen der Potamogeton zirlia 1 em unter dern Wasserspiegel be- festigt; doch oft findet mail auch die Kocher mit einer der Hreitflachen nach oberi frei zwischen den Potaniogetonbliittern herumschwimmen; schlagt man niit den Rudern in die Potamogetori, so verlieren die Tiere ihre hnheftungsstelleii und schwimmen dann an oder direkt unter der Ober- fliiche herum: einzelne siiiken aber aufierordentlich langsum nnd stoDen gewohrilich auf die Potamogetonstengel, ergreifen dieselben und kletterii wieder in die Hohe. Die Tiere selbst sind ubrigens sehr lebhaft, wenn sie an der Oberfliiche in der kleinen pelagischen Region zwischen deli Potamo- getonblattern liegen . strecken sie den T’order korper sehr lnng itus dem Kocher hervor und schlgngeln denselben bald nach rechts bald iiach links; dadurch rudern sie sich dureh das Wasser nahe an der Oberfliiche und erreicheii bald ein anderes Blatt, an welchem sie sich festhaken. I. Naturbeobaohtungen iiber Glyphotaelius. Eine besondere VerschluD- membran am Hinterende findet sich bei den Larven nicht; das Gespinst verschmalert sich hiriten und wird so zusammengesponiieri , daD nur ein kleines Loch, fur den AusstoB der Exkremente bestimmt, zuriiekbleibt. Im allgemeinen ist der Kijcher beinahe zweimal so lang a19 die Larve; wird er aber zu einer Zeit gemessen, wo eine Spitze gerade lange nicht ab- gebrochen ist, kann das Rohr dreimal so lang werden. - So lange das Tier nicht uber ca. 10 bis 17 mm ist (viertes Stadium Siltalas), bestehen die Querstucke gewohnicli aus abgewelkten Gras- und Carexhalmen uiid anderen Vegetabilien, die die Larven am Boden der Gewasser finden. Wenn sie ca. 14 bis 20 mm, uiid die Kocher ca. 20 bis 30 mm erreicht habeii, sirid sie an deli hier besprochenen Lokalitaten nur von einer einzigen Pflanze Potamogeton iiatans aufgebnut, In diesem Stadium verlassen namlich die Tiere den Boden; sie steigen den Stengeln und Blattstielen des Potamo- getons folgend bis an die Oberflache, wo sie sich eine neue Heimat suchen. Das Aufsteigen findet in dem letzten Teil des August statt, und bald sieht man sowohl an der GroDe dsr Kocher wie auch an den Potamogeton- bliittern, da13 ein neues Leben begonrien hat. g Die Larven schneiden sich groDe elliptische Stucke aus den Blatterii der Potamogeton (Fig. 6 bis 13); vom Rande ausgehend geht die Schneide- furche gegen die Mittelrippe hinein und erreicht nur selteii dieselbe. Die Blattabschiiitte werden beinahe immer so gemacht , daD der &uDere Rand des Blattes einen groDeren Teil des Abschnittes begrenzt , sie bekommen daher eine elliptische Form mit einem geraden uiid einem mehr oder weniger gebogenen Rand. Die Stiicke werden am hadigsten SO eingdugt, da8 der gerade Rand sich gegeri das hintere Ende wendet. Die Rander sind immer sehr scharf abgeschnitten, wenn sie gefranzt sind, ruhrt es von spaterer Verwesung her. Die Blattstucke der beiden Seiten sind so ringefugt, daW sie unter recht spitzem Winkel miteinander zusammen- stoBen, so daD ein dreieckiger vom Wasser durchstriimter Kana1 an beiden Yeiteu des Rohres liegt (Fig. 25). 7 7 7 - 98 - Solange das Tier wachst und wahrscheinlich noch eiriige Zeit, nach- dem das Wachstum aufgehort hat, werden immer neue Stiicke ail das vorderste angebracht und die alten am Hinterende abgeworfen. Dies geht darttus hervor, daS die Kocher bis hlitte Oktober inirrier grofier werdeii, ohne daS jedoch die Anzalil der Stucke zunimnit. Weitere Beweise hier- fiiv bind auch. I. Naturbeobaohtungen iiber Glyphotaelius. Oft sieht inan die Larven Luft abgeben; die Lufi liegt in den Rohren; wahrschein- lich ruhrt sie voii d m Narlten der frisch abgebissenen Pflanzenteile, wovon 99 - 99 - der Kocher bedeckt ist, her; es ist aber auch moglich, dafi die Tiere, wenn sie sich an der Oberflache aufhalten, und den Korper schliingelnd aufier- halb des Rohres bewegen, Luft zwischen Korper und Rohr bekornmen. DaD diese Luft fur die %ere, so lange sie ihre Nahrung in den Schwimni- blattern suchen, von Bedeuturig ist , versteht sich von selbst. Hierauf kommen wir spater zuruck. Am Schlusse des September bemerkt man, daD die Rohren nicht mehr die frische grune Farbe besitzen; die Blattstucke sind gelbgrun und tragen Spuren von Vermoderungsprozessen, gleichzeitig begiiineii auch die Blatter der Potamogetonzoiie abzuwelken. 1907 im Olitober, 1908 aber erst im November, sieht iriaii, da8 die a d e r e Form der Kocher sich andert; sie ist nun nie so breit; gewohnlich nur ca. 20 mm, bleibt aber immer ca. 50 mm lanp. Die Deckstucke sind kleiner und bestehen nicht ausschlieDlich am Potamogeton: oft riihren sie von abgewelkten Erlenblattern her, die vou den Erlen am Ufer auf die Seeoberflache niedergefallen sind. Zugleich findet man Kocher , die in ihren hinteren Teilen noch von groDen Potamogetonblattern bedeckt sind, wiihrend solche im vorderen Teile ganz fehlen. Hier entbehrt das Hohr alle deckenden Querstiicke und besteht aus griinen Stucken von BIatt- stielen der Potamogeton (Fig. 14 his 17). Zugleich nimmt das Tier ein ganz neues Bauprinzip an; indem diese Stucke nun nicht quer, sondern parallel der Langsachse des Rohres angebntcht sind. Indem die Tiere von November his Ende Dezember dieses neue Material nach eiiiem neuen Rauprinzip anwenden, entstehen Kocher von einer von den bisherigen ganz verschiedenen Form. Die Kocher werden rohrenformig und gleichen fluchtig gesehen den Kochern von Phryganea grandis, weichen aber von dieseri dadurch ab, daI3 die Stielstucke nicht gleich lang sind; in mehreren Mohren glaubt man sogar eine schraubige Anordnung der Stucke zu finden. Man findet im November alle mogliche Obergiinge (Fig. 17 bis 18) zm7ischen den breiten , aus Blattern und den rohrenformigen aus Stielstucken pebauten Kochern. Es geht aus den Obergangsstadien hervor, dafi die Tiere erst ca. I. Naturbeobaohtungen iiber Glyphotaelius. 30 mm lmge Blattstiele abbeiDen und uber den hinteren Deckstucken parallel mit der Liingsachse des Rohres anbringen; die Stiele sind daher in der ganzen Lange dern Rohre angeklebt, die spateren nur mit den1 vorderen Ende; mit dem hinteren stehen sie wie grobe Bursten ab. Das Material, ztuf dem diese Mitteilungen beruhen, ruhrt 17om 5. bis 20. November 1908 her. Als ich am 26. November die Teiche wieder be- suchte, waren sie zugefroren. Das Eis wurde eiiigebrochen, und es zeigte sich, daS die Glyphotaelien sich noch in der Potamogetonzone befanden. A m 1. Dezember taute der Teich wieder auf, wahrend des Auftauens de5 Eises vermoderten die Bliitter der Potamogeton vollstandig, und sehr viele wurden langsam auf dem Boden abgelagert. Noch sa5en aber die Glypho- - 100 - 100 - taelien in groDer Menge auf den Blattstielen, die noch grun waren. Am 26. Dezember bekamen wir plotzlich Schneesturm; der Teich fror wied tlr zu, wurde aber in1 Gefrierstadiuni vom Boot aus beobachtet ; die Potamo- getonregion war nun beinahe von Glyphotaelien frei, werin man aber niit einem Stangnetze von ca 2 m den Boden untersuchte, fand man ca. 1'/, m bis ' I 2 in vom Ufer zahlreiche Glyphotaelien. Iii den Tagen vom 26. Dezemher bis 28. Dezember fror der Teich zu und v7ar bis 15. April zugefroren. g Die vom 26. Dezember heruhrenden Glyphotaelierikocher (Fig. 19 bis 20) hatten noch die obengenannte , zyliridrische Form und wuren hauptsiicli- sich rzus Potamogetonstielstucken gebaut. Wie schoii oben hemerkt, geht der Obergang von den breiten Rohren init Querstticken zii den schmalen mit nach der Langsachse geordnetrn Stucken im nllgemeinen ganz langsnm vor sich. Ebeii in den letzten Tagen tles Dezember, so wie auch im April, nach dem Auftauen, fand ich auf dem Boden des Wassers sehr oft schone, von Rlattern gebnuie Rohren, die aber alle leer waren. Ich bin daher geiieigt zu glauben, daD die Tiere, deren Rohreii Ende Dezember noch mit Querstuckcii von Blatterri bedeckt sind, diese Rohren verlassen und sich mue bauen. Um so rnehr glaubo ich, daD dies richtig sein kann, weil solche Rohren riach dern Auftuuen des Teiches im April nie niehr z u finden sind. p Wenn die Tiere also Ende Oktober bis November sich nach dern Boden begeben, sind sie graugrun, die schorie griine Farbe des Hochsommers besitzen sie nicht. I. Naturbeobaohtungen iiber Glyphotaelius. Der Darm ist mit Futter vollgepfropft, und eine Ver- schied enheit zwischen dem Darminhalt im August und irn Dezernber ist schwer festzustellen, die Futterpflanze ist in der ganzen Zeit beinahe nur Potamogeton gewesen; es ist wohl moglich, daD die Tiere sich im November uiid Dezember vielmehr von Stengeln als von Bliittern nahren. Vom 26. Dezember his 15. April ist der Teich fest gefroreri. Am 27. Februar, als das Eis ca. 'I2 m dick ist, wird es von einem Stuck des Ufers entfernt; unter dem Eise auf ca. '/* m sehe ich 10 Glyphotaelien i~uf dem Boden liegen oder langsam herumkriechen. Die Rohren (Fig. 21 his 22) sind sehr interessant; an den Seiten oder vom Hinterende hinaus- ragend, liegen noch die l'otamogetonstiele, die Ober- und Uriterseite des Rohres ist aber aus toten Pflanzenmaterialieri , abgewelkten Bliittern der Sparganieii, der Carexarten und sedimentierten Erlenblattern aufgebuut. Alte Stiicke sind parallel der Langsachse des Rohres geordnet und mit deli Seiten des Rohres fest zusaminengesponnen. Daneben findet man aber Rohreii, die in ihrem hinteren Ende breite flugelformig weit uber die Seiten des Rohres hinausrageiide Erleriblatter besitzen. Auch solche mit Potamogetonblattern am hinteren Ende kann man beobachten; diese sind aber so vermodert, da8 sie nicht breiter als das Rohr sind. - 101 - 101 - Am 6. April war der Teich noch fest zugefroren, nur an dem nach Suden gelegenen Ufer hatte die Sonne das Eis sprode gemacht: hier und spater am 15. April (Fig. 23), als der Teich noch zugefroren war, sah ich an dem Sudufer aiif dem Boden zahlreiche Glgphtotaelienkorher. Sie haben hauptsachlich dasselbe Aussehen wie am 27. Februar ; naher besichtigt zeigt es sich jedoch, daO kein einziges Rohr mehr hervorragende quer- geordnete Rlattstucke besitzt , ferner , daD die Ober- und Unterseite des Rohres nur je 1 bis 2 groOere oblonge Stuclre tragen; nirnmt man diese ab, liegen die seidenglanzende Ober- und Unterseite ganz Erei; an den Seiten sind die Kleinstiicke so in Seide eingewebt, da13 es beinahe unmog- lich ist, sie abzunehmen. Der Darmkaml der Tiere enthalt im Februar tvie irn April irnmer Pflanzenteile; diese sind wie im Sommer nicht mehr griin, sondern braun- gelb und bestehen aus abgestorbenen Pflanzenteilen; oft findet man in dem Magen zugleich Algenreste, Dauerkeime und Eier von Bryozoen, d. h. Detritus, der sich teils am Boden, teils auf den Schilfstengeln gesammelt hat. Die Farbe der Larven ist nicht mehr graugrun, sondern gelblich weilj. I. Naturbeobaohtungen iiber Glyphotaelius. Der Darm nimmt kaum eirien so groBen Teil des Korpers ein, wie fruher, dagegen ist der Fettkorper nun sehr dick, wid er gibt der Larve ihre weiDgelbe Farbe. Die Larven mit ihren Kochern sind nun schwere Organisrnen, die, weiin man sie ins Wasser wirft, augenblicklich zu Boden fallen; sie bewegen sich langsam und ungeschickt, und die Hewegung zwischen den Korperringen ist sehr lilein. Am 4. Mai, als ich den Teich wieder besuchte, findet sich beinahe keine Larve am Boden des Wassers mehr; zweifellos hat das Tier sich 1 erpuppt, aber wo ? Nach stundenlanger Untersuchung ohne Resultat, fand ich endlich etwas, das wie zusammengesponnene Carexblatter (Fig. 24), ,Inssah. Es war das Puppengehause, aber in einer vollig verschiedeneii Gestalt von dem, wie ich es mir gedacht hatte. Die Puppengehause sitzen hauptsachlich auf den Carexblattern, nur ca. 1 dcm unter dem Wasser- spiegel. Sie waren schwer zu beobachten: am besten fand man sie, wenn man mit den Handen die Carexhalmen umfaljte: dann merkte man bald die dicken harteri Korper. Noch war das ganze Larvenmaterial iiicht ver- p p p t : einzelne krochen langsam umher. Wenn die Larve sich zixr Verpuppung anschickt, hangt sie sich an einem Carexhalm auf; sie nimmt dann von den herabhiingenden welkeri braungelben Blattern erst kleine und dann spater langere bis ca. 70 mm grol3e Stucke , und spinrit sie in mehreren Schicliten ubereinander; nach uiid nach wird der ganze Kiicher dicker. Die 1 bis 2 em breiten Rliitter 11-erden in ihrer ganzen Lange dem Kocher fest angesponnen; da nin1 die Lllstter vie1 langer sind, als das primare Gehause, ragen sie an beiden Enden ca. 10 bis 20 mm uher dieses heraus; hier stofien die Spitzeii der 102 - 102 aufgesponnenen Bliitter zusammen, so daD das ganze Gehause eine spindel- formige Form bekommt. Wenri das Puppeiigehliuse fertig ist, stellt die Larve noch die Siebmem- branen (Fig. 30 bis 31) an beideii Eriden tles Rohres her; ihr gegenseitiger bbstand ist nur CB. 27 r i m : diese Siebmeinbranen sind wahre Meisterwerke; sie sind init den Rlatt>ern fest zusammengespon~ien , iind das Gespinst des R.andes der Menibran geht. lirnvattenformig an den Bliittern Iiinauf. Sie bestehen aus eiiieiti Edkenperust, dessen Zwiscdienraiume mit gelber Seide ausgefullt sind, nur in den innersten Winkeln, wo die Ralken zusammen- stofien, sind Locher zuruckgeblieben. Die vordere Siebmembran ist immer regelmd3iger gebaut und hat nicht so viele Liicher (ca. 30) als die hintere (30 bis 40). I. Naturbeobaohtungen iiber Glyphotaelius. Diese Siebmenibrarien findet man in grol3en Mengen in Torf- ablagernngen des kleineii Sees; dieselben oder pleichartige sind bekanntlich dlgemeine Fossilien in den alten quartaren Torfmooren; dort liegen sie als beinahe e,inzig bestimmbares Material von de,m Phryganeenleben der Vorzeit. Wenn die Larve die Siebirienibran spiniit, enthalt der Dariri noch recht bedeutende Menge voii Nahrung, uiid in der Puppenwiege dauert drts ruhende Larvenstadiixm noch einige Zeit; die Haut der nun beinahe ganz weiDen Larven birst in der Riickenhaut des Kopfes urid den zwei ersten Thorakalringen, und die Puppe kriecht &us. Puppenlange cat. 20 mm, also ca. 10 mm weniger als die jnnere Lange des Kochers. In den Aquarien habe ich sehr oft viele Puppengehause gehabt; ver- gebens versuchte ich ausfindig zu machen, wie die Puppe ihre Putzapparate benutzt, weder an der vorderen noch an der hinteren Siebmemhran habe ich einen DurchstoD des Putzapparates gesehen (cfr. Thienemann 1905 S. 1). Die Halrne hangen uber dio Siebniembranen heraus uiid bewirken, da,D man nichts sehen kann; nimmt man die Halme weg, verhalt die Puppe sick vollkommen ruhig. In der Natur faiid ic,h die ersten PuppengehRuse am 4. Mai, und die Verpuppung hat wahrscheinlich nicht friiher als am 1. Mai begonnen. Am 14. Mai sind wahrscheinlich alle verpuppt gewesen, am 27. Mai schwammen die ersten Puppeiihaute auf der Oberflache des Wassers, am 9. Juiii in groDen Mengen, und die Tiere saDen an der Ufervegetation. Der Aufenthalt im Puppengehause dauert wahrscheinlich ca. 9 bis 3 Woehen. Die ausgefarbte Puppe (Fig. 29) mit ihrem schori grungefiirbten Hinterleib, den schwarzen Seitenlinien und den rot gefiirbten Fliigeln ist ein sehr schones Tier. 11. Beobaohtungen uber das Pupyenstadium der Wasserinsekten. Lange ehe ieh mich rnit Glyphotaelius beschaftigte, hat rnich dss frei- lebende I’uppenstadium der Phryganeen interessiert. In meinen Aquarien habe ich von vielen verschiedenen Phryganeen, deren Namen mir damals 103 - 103 - 103 wie jetzt unbekannt sind, freilebende Puppen zu hunderten gehabt. Ich erlaube mir hier einige dieser alten Ueobachtungen einzuschieben. wie jetzt unbekannt sind, freilebende Puppen zu hunderten gehabt. Ich erlaube mir hier einige dieser alten Ueobachtungen einzuschieben. Das freilebende Puppeiistadium der Phryganeen ist sehr wenig bekannt ; viele wissen gar nicht, daD ein solches existiert, und in den Lehrbuchern findet man nichts hieruber. In meinem Aquarium dauert es hochstens 34 Stunden; waren die Puppen vor dieser Zeit nicht entwickelt starberi sie itus. Unter naturlichen Verhaltnissen dauert das freilebende Puppenstadium wahrscheinlich nicht so viele Minuten, es hat keine andere Bedeutung als das Tier aus dern Wasser zu bringen, so daD die letzte Verwandlung entweder an der Ober- flache des Wassers oder am Lande vor sich gehen kann. Auf dem Boden meiner Aquarien habe ich oft die ausgekrochenen Puppen auf den Gehausen sitzen sehen. Plotzlich hebt das Tier sich; das erste Beinpaar ist gewohnlich vorwarts gestreckt, das dritte Beinpaar wird parallel dem Korper vollkommen ruhig gehalten, und nur mit dem zwsiten Beinpaar schwimmt das Tier. Dieses Beinpaar ist bekanntlich mit langeii, schwarzen Haarfranzen auf beiden Seiten der Tarsen und oft auf den Schienen besetzt. Die Mittelbeine werden gleichzeitig und in groDen Bogen, wie die Hinterbeine der Notonecten bewegt. Wenn die Tiere sich ruhig verhalten, stehen die Mittelbeine beinahe senkrecht mit grol3en Winkeln zwischen femora und tibia vom Korper ab. Die Schwimmbewegungen dauern nur kurze Zeit, nicht iiber 5 Minuten, dann sinkt das Tier wieder zu Boden; was sie am meisten charakterisiert, ist die Plotzlichkeit womit sie be- ginnen und wieder aufhoren , ferner die erstaunliche Schnelligkeit und groBe Kraft in den einzelnen StoDen, wobei die Bewegung durch das Wasser eine hupfende wird. Falls es dem Tiere in den obgenannten 5 Minuten nicht gelingt, entweder feste Gegenstiinde, die aufierhalb des Wassers her- vorragen oder die Oberflache selbst zu erreichen, sinkt es wieder zu Boden. Nach einem kleinen Ausruhen wird ein neuer Versuch gemscht, und wcnn das Ziel im Laufe von ca. 12 bis 24 Stunden nicht erreicht ist, verweilt das Tier auf dem Boden und stirbt da ab. So lange 0s ruhig sitzt, schlagt der Hinterkorper regelmal3ig auf und nieder, ca. 30 Ychlaige in der Minute (Atmungsbewegungen). 11. Beobaohtungen uber das Pupyenstadium der Wasserinsekten. In zahlreichen Fallen habe ich gesehen, dalj das Tier unmittelbar, nach- dem es ausgekrochen war, auf dieselben Grashalme, die es als Befestigungs- blatt seiner Puppenwiege gebraucht hatte , hinaufkroch , die Wasserober- flgche erreichte und wenige em uber derselben sich verwsndelte ; hier ge- brauchte das Tier also gar nicht seiri Schwimmvermogen. Oft findet rnari, daD die Larven sich nur wenige cm uriter dem Wasserspiegel verpuppen, und daB die Puppengehguse , wenn eine Trockenperiode eintritt und die Teiche sehr seicht sind, uber das Wasser zu hiingen kommen. Dies braucht nicht der weiteren Entwicklung zu schaden, und auch in diesen Fdlen 104 104 - haben die ausgekrochenen Puppen keinen Vorteil von ihrem Schwinim- vermogen. Diese Beobachtungen bezieheri sich wehrscheinlich alle auf Limnophi- liden ; riaher bestimrnt wurden die Tiere leider nicht. Mit den Leptoceriden verhiilt es sich anders. Am Morgen des 14. Juni 1891 Uhr 8,40 sah ich pine Leptoceride aus dem Puppenkocher hervorhrecheri; sie schwamni cu. 4 Minuten herum, erreichte die Oberfliiche und lag dann eiiie Minute ruhig (la, indem sie die Seitenlinie und Tracheen als L4nheftungsapparate an die JTasseroberfliiche brauchte: die ganze Riickenfliiche, die oberhalb des Wassers lag, war trocken, und sah fettartig aus. Schwimmend in der Oberflache inachte das Tier ein paar schnelle Bewegungen mit den Mittelbeinen, dann faltete dir Haut des Kopfes und der Brust sich plotzlich auswarts, wodurch eine flache Schwimmscheibe entstand; eugleich cvurden die Flugelscheideri auswgrts geschlagen und stellten sich in das Wasser tauchend vertikal unter dem Abdomen ein. Die Tarsen der Vorder- und Mittelbeine traten a,ugenblicklich auf die Wasseroberflache, das Tier zog dann die Hinterbeine heraus, die sich auf die Sehwimmscheibe stellten. Dann zog es von unten nach oben die Flugel aus, und in derselben Minute flog es an rnein Fcnster das ganze dauerte nur ce. 2 Minuten; niehrmals und bei verschiedenen Leptoceriden habe ich dasselbe beobachtet; es scheint, als ob jedenfalls niehrere derselben wie die Mucken sich an der Oberfliiche befestigen, und da8 die letzte Mauser eben hier vor sieh geht. Ich habe auch Limnophili- den in Ayuarien stundenlang an der Oberflache hangerid sehen: do& habe ich niemals Tiere in dieser Stellung uuskriechen sehen. - flber die Fainilie der Phryganidae u. a. habe ich leider keine Reobachtungen. Vie1 spater d s ich 1906 S i l t a l a s schone Untersuchungeii bekam, sah ich, daD unsere Beobechtungen (l906a S. 85) gut miteinander iiberein- stimmten. 11. Beobaohtungen uber das Pupyenstadium der Wasserinsekten. Wie schon oben bemerkt, hnl das freilehende Puppenstadiurn keirie andere Bedeutung, als das Tier so schnell wie moglich aus dern Wasser zii bringen. In vielen Fallen lnachte es gar keinen Gebrauch vori seinern Sehwimmvermogeii oder die Schlage, die es mit den Mittelbeinen ausfuhrt, beschranken sich auf ganz wenige. Was mich hier am nieisten interessiert, jst zu sehen, in wie hohem Grade diese wenigen Minuten im Lebeii der I’uppm ihre ganze Organisation so zu sapen gepragt haben, teils ireue Orgaiie ausgebildet, teils alte modifiziert haben. Ich denke hier an die Ent- w-icklung von Schwimmhaaren an den Mittelbeinen besonders den Tarsen; bei eiriigen Phryganeen komrneii auch solche an den Vorderbeinen vor, sind aber hier nur selten so stark eiitwickelt; die Hinterbeine sind bei- nahe ganz kahl. Wir haben es hier mit einem Bewegungsapprat zu tun, der vielleicht nie gebraucht werden soll; hiichstens dann uiid wann ill einer Zeit von 24 Stuiiden und in vielen, wahrscheinlich in den meisten - 105 - 105 - 105 Fiillen, nur wahrend weniger Minuten; man mu8 aimehmen, da13 die Tiere gewohnlich den Bewegungsapparat nicht zu mehr als ein paar ganz ver- einzelten Schlagen brawhen. Ein ererbter Apparat ist es durchaus iiicht, weil iiberhaupt - meines Wissens - kein einziges anderes Insekt die Mittelbeine als Schwimmbeine allein und in dieser Weise braucht; es ist ein fur die Phryganiden spezifisches Bewegungsorgan. , Wie kurz auc,h die Zeit war, in der es gebraucht werden sollte, seine Herstellring war dennoch eine Lebensbedingung fur das Tier, und das Organ wurde her- vorgebracht: wie und wann, das sind Fragen, die ich vorlaufig den Des- candeiizttheoretikern iiberlasse; doch hoffe ich, da8 ich hierauf syater zu- riickkommen kann. Die Puppen haben nuch andere Organe, die in die,ser kurzen Zeit des Lebens und eben nur in dieser eine R'olle h pi el en.^) Bekuintlich ist die Ruckenflache des Abdomen der Puppe rnit besondern Haftapparsten ver- sehen. Der erste Hinterleibsring triigt eiitweder zwei rnit feiiieii Spitzen bedeckte Warzen oder eine sattelformige Erhohung; das 3. bis 7. Segment t,ragt auf der Ruckseit,e ein p a r Chitinpliittchen mit Haken und das 5. Segment ferner ein paar groBere Chitinpliittehen. Man sagt oft, dn(3 dieselben in dem Dienste der Verschiebungshewegungen stehen. Doch haben diese Organe sicherlich nuch eine andere Funktion, und dies ist wahr- scheinlich ihre Hautfunktion. I) Von den eigentiimlicli gcformteii Puppenmandibeln, die keine nndero Rolle spielen, xls die Seitenstr&nge der VerschliiDmeinbrml durcllzusclineiden, und die von den Larven- rnaudihelri ganz verscliieden sind, selie ich liier ah. Das eigentundiclle des Anftretens dieses Organs bei den Puppen wird dedurcli erliebliclt gcstiirlit, da8 die 'l'richoptcrcn :11s imagines bekanntlicli entweder keine oder sehr rudimentare bIandibeln besitzen. - \\*as gebraucht Tverden soll, wird liervorgebraclit; was nicltt, Ts-ird in der Rumpelkammer der Spezics nufniagazinicrt; spater in der Xuliitnit der Spezies lranii es wieder heraus- geuommcn: urn in vielleicht modifiaicrtw Gwtalt nocbntnls m,nge\sandt ZII Iserden. 11. Beobaohtungen uber das Pupyenstadium der Wasserinsekten. Wir finden ganz gleichartige Chitinbildungen bei Puppen einer anderen Insekt8enordnung, w o die Puppen auch dazu bestirnilit siiid an der Ober- fliche horizontad ELI hiingen und sich dort zu verwandeln. Ich denke hier an die Puppen vieler l'ipuliden. Alle diese Chitinbildungeii sind Organe, rnit,telst welcher die Tiere die Wasseroberflache brechen und dann an der Ober- flitche hangen. Wer die an der Oberflache hiingenden Phryganeen und Tipulideiipuppen (z. €3. Pha1a.rocera) beobacht.et hat., Bann sich davon uber- zeugen. Aber nuch die Seitenlinie, die bei den Puppen besonders stark eiitwickelt, ist urid einen in seinern hintereii Teil riiehr als bei der Larve ventralw&$s gebogenen Verlauf hat ist ein Mittel, durch welches die Larve auf der Oberflache ruhi. Jch bin daher geneigt, sowohl in den oben- genannten Chitinfortsiitzen als in der starken Entwicklung der Seiten- linie Mittel zu sehen, wonzit die Puppen sich an der Oberfliche halten, ~ind zu glsuben, dalS diese Funktion eine Crsache zu ihrer Hildung gr- n-esen ist. Die Natur hat augenscheinlich d l e deli Wasserinsekten, die ihre Pup- pen- oder Npphenstadien in) Wasser zubringen, eine schwere Aufgabe pestellt; mit unbenetzten Fliigeln solleii sie sich aus dem feuchten Eleinente direlit iii die Luft hiiiaufschwingeii kiinnen. Zugleich sol1 ein fur das Wssserleben anpepa0tes Respir:ttionsorgaii mit einem fur das Luftle’nm an- gepafiten vertauscht werdeii. Die Aufgabe ist bei deli verschiedenen In- sektenordnungen auf verscahietlene Weise gelost. l;m das frei lebende Puppenstadium der Phrypaueeii [lesser zu verstehen, sei es mir erlaubt, kurz diese verschiedenen Modi zu erwahiien. Wir fiiiden hei vielen der wasserbewoliiienderi Dip t er e n . besonders den meisten Miicken Verhdtnisse, die sehr niit deiieii der Leptocerideii ubereinstimmen; aueh viele Epheme- riden rerhalteii sich in derselhen Weise : doch findeii wir hier zwischeii deni Wasser und Luft bewohnenden Stctdium das merkwimlige, einzig da- steheiide Subimapostudium eiiigeschalt et , Die 0 don a t e n kriechen, wenn sie sich verwaiideln sollen, aus dem Wasser heraus und verwandeln sich in der Luft. Iloch auch hiey gibt es ein merkwiirdiges wenig beachtetes Zwischenstadium, das nicht ohne Iiiteresse ist. Beksnntlich iiffnen sich im letzten Nymphenstadium die groRen Prothorali&tigmen. Ehe die Tiere cfirekt in die Luft hinauf kriechen, siteen sie rnehrere Stunden nur mit dem Kopfe urid dern ersten Prothoraxring oberhalb des Wassers. I11 dieser Stellung atmen sie die atmosphiirische I d 3 durch die Prothorakalstigmen noch vom Wasser benetzt und verlassen erst spitter dieses vollstandig. 11. Beobaohtungen uber das Pupyenstadium der Wasserinsekten. Wenn iiian erst einmal auf dieses Phiinomen nufrnerksam geworden ist, kaiin man an hellen Fruhsoinnieriiior.aeII hunderte von Aeschnen in dieser Stellung an den Grashalmeii und gerade in der Wssseroberfliiche sitzen selien. Was die Schmetterlinge des SuWtvassers anbelangt, gleichen sie in ihreii biologischeri Verhiiltiiissen den Phryganeen sehr; dies gilt vielleicht besoiiders von Hydrocampit nyiriphaeata. Ihre uus zwei Blattern zu- sammengesetzten Pnppengehause sind an Wasserpflanzeii befestigt. Die Larven beiBen einige Locher in dieselhen und fangen die davon aus- stroniende Luft in einem Gespinst, womit sie die Innenseite der zwei Bliitter auskleiden, auf ; etwas gleichartiges finden wir bei den Donacien. D a m verpuppt das Tier sich. Die P u p p ist, eine wahre Schmet terlirigs- puppe ohne Hewegungsvermogen und verniag nicht wie die Phryganeen- puppen durch die wenige Zentimeter dicke Wasserschicht, die sie von der Luft scheidet. zu schwimmen. Wie kann hier die Verwandlung vom Wasserinsekt zum Luftinsekt \-or sich gehen? In den letzten Tagen des Puppenlebens nimmt die Puppenhaut eine fast goldene Farbe an, ein Phiinomen, das man ctnch bei Fliegenpuppen, die in Wasserpflanzen leben, - 107 - beobachten kann. Dies ruhrt von Luft,, die sich unter der braunen Pup- penhaut um die Puppe sammelt, her. Wenn nun das Tier das Puppen- gehause verlassen soll, wird diese Luft von den Flugeln gefangen und das Tier von der Luftkugel blitzschnell durch das Wasser getrieben. Die Fliigel sind , wenn sie aus der Puppenhaut herausgezogen werden, mit einem weil3en Wachsiiberzug bedeckt., und wahrend der blitzschnellen Fahrt durch das Wasser gleitet der Wachsuberzug ab. Daher sieht' man einige Minuten, nachdem das Tier die Oberflache erreicht hat, den senkrechten Weg von der Puppenwiege bis an die Oberflache als eine weifie, silber- gliinzende Saule oder einen Strang markiert; schwebend steht die Saule einige Minute,n, die feinen Staubpartikel gleiten auseinander und verschwin- den. In demselben Augenblicke, wo das Tier die Oberflache erreicht hat, lauft es schnell wie eine Phryganide uber die Wasseroberflache bis an das Ufer, wo das volle Entfalten der Flugel vor sich geht. Die hier mitge- teilten Beobachtungen ruhren von Aquarienstudien vom 15. Juni 1891 her. Ich habe jenes momentsane AusschieBen der Puppe dreimal beobachtet. Wie sich Paraponyx nnd Ac,entropus verhalten, weill ich leider nicht,; Cataclysta befestigt ihre Puppengehause meist oberhalb des Wassers. 11. Beobaohtungen uber das Pupyenstadium der Wasserinsekten. Die obengenannten hufgaben werden also in recht verschiedener Weise gelost; in den verschiedenen Insektenordnungen finden wir an der Schwelle zwischen dem Wasser- und Luftleben eigentiimliche biologisc.he Verha1t'- nisse, die mehr oder weniger die Organisation der Insekten pragen. Das freilebende, nur wenige Minuten, hochstens einige Stunden dauernde Pup- penstadium der Phryganeen mit seinen eigentumlichen, eben fur dieses kurze Lebensintervall eingerichteten hoch spezialisierten Bauverhaltnissen ist in dieser Weise aufzufassen und soll mit den entsprechenden obenge- nannten Insekten Stadien und eigentumlic'hen Strukturverhaltnissen bei anderen verglichen werden. Wir kehren nun nach diesem langen Ausschweifen wieder zu Glypho- taelius zuruck. MTenn das Tier ausgekrochen ist, ist es noch nicht ganz ausgefarbt; dies geschieht, indem es auf den Grashalmen nahe am Ufer ruht. Ich sah die ersten Glyphotaelien am 27. Mai (Fig. 32), die letzten am 15. Juli. Die Zeit, in welcher die Eiablage vor sich geht, ist wahrscheinlich ziemlich lang. Leider sah ich nie eine Paarung. Das Tier war wenigstens bei Tage merkwurdig selten. An einem Somnierabend sah ich ziemlich viele uber die Ufervegetation f'liegen, doch nie in solchen Mengen wie die Hydro- campen die an einzelnen Tagen wie Schneegestober uber dem Wasser- spiegel standen. Nahrungsaufnahme der Imagines sah ich nicht und auf den Rlumen waren sie weiiigstens am Tage nicht zu finden; weil Ihre Lebensdauer ist wahrscheinlich recht kurz. -- 108 - 108 - 108 aber die Teiche weit entfernt T'OD meiner Heimat waren, habe ich nixr wenige dbendbeobachtungen. Am Tage sitzen die Tiere llnnier vollkommen ruhig geschutzt irn Grase des Ufers. aber die Teiche weit entfernt T'OD meiner Heimat waren, habe ich nixr wenige dbendbeobachtungen. Am Tage sitzen die Tiere llnnier vollkommen ruhig geschutzt irn Grase des Ufers. Es scheint, als ob die Glyphotnelien nur wenige Feinde haben; in den Eimassen sah ich nie Schniarotzer; ich suchte solche um so eifriger, als ich in arideren Limnophilideneimassen mehrmals Fliegenlarven gefunden hnbe. Tch besitze Eierhaufen von Lirnnophiliden, die ca. 1 ni uber dem Wasser algesetzt sind (,,tropfende Eiermass"en") und worin 2 bis 3 weil3- gelbe, 1 '/? em lange Fliegenlars-en liegen. Mehrmals sah ich Puppen- kocher, die auf einer der Yeiten ein Loch von en. '/? em Durchmesser liatten; RUS solchen Lochern krochen iinmer Nepheliden aus: inwieweit sie die Jlocher gebohrt huben, wein ich riicht: in solchen Kiichern fanden sich iininer nur Reste von Puppen. Wir haben nun die Biologie der Glyphotaelien auf dieser kleinen nord- w4imdisrhen Lokditat verfolgt. ’) Vereinzelt bis Mitte Juli. 11. Beobaohtungen uber das Pupyenstadium der Wasserinsekten. Auf rneinen zahlreichen Exkursionen habe ich oft die Glyphotaelieri gesehen, iiie aber in solehen Mengen wie in nieineni i7ersuchsteich. Nur selten fanden sie sich an der Oberfliiche und (littin nur da, 1-0 diese beinahe von Bliitteni, besoriders Potamogeton be- deckt war. Oft w-ar das Crehciuse von anderen Blattern als tlenen des Yotarnogeton gebaut, immer wurden jedoch his Oktober grune Blattstucke gebraucht. urid der Bauplari des Kochers war stets derselbe. Im Gripsee waren die Kocher voii Erlen- und Birkenbliittern gebaut, und hier krochen die lmveri inirner auf den Fontinalisteppichsn des Bodens; in tlem Fun- kenteich wurden besonders Teile von Nyrnphaeaceen gebraucht. In der Iiteratur sirid die Glyphotuelien oft erw%hnt, besoiiders bei I) e (ieer (1778, p. 368), Struck (1899, 1'. 1) und Siltala, auf deren Arbeiten ich hinweise. Besonders bei den letztgeriannteri (1902, 1'. 39) fiiidet man eingehende Schilderungen von den Bau der Larven und Puppen. Keine der biologischen Reobachtungen steht in fundamentalern Gegensatz zu den nieinigen, uiid diese haben die fruheren hoffentlich ergiinzt und er- weitert. Wir kiiiinen nun die Haupt,punkte der Riologie voii Glyphotaelius punctatolineatus ini Versuchsteich I1 folgendermafien rekapitulieren. Eicrstadiurn I~arvciistadiuin am Boden des GevuLssers ;LI~ Potamogeton an Potamogeton a. 1%. Juni-1. Aug l..Aug. - 1. Sept 1. Sept. - 1. Nov. I. No\-. - 1. Ilez. K6cher: quergelegte Deck- stucke abgestorbencr Blatt- st iicke. K6eher: quergelegte Deck- stGcke von lebenden Pflan- zen (BlLtter von Potam.). Kiicher von longitiidirial ge- legten Declistiiclten (Sticlc ron Potam.). 109 - 109 Larvenstadiuin am Boden des Gewvjssers am Boden des Gewkssers Puppenstadium Imago 1. Dez. -1. Jan. 1. Jan.-1. Mai 1. Mai - 1. Juni ca. 1. Juni - 1, Jdi Kocher von longitudind gc- legten Deckstkclien (Stiele von Potam.). Kocher von longitudinal gc- ordiietem Bodenmaterial. I) Puppenstadium Imago 111. Theoretische Betrachtungen und Versuche. Was in dieser kleinen Untersuchung am meisten interessiert , sind naturlich die verschiedenen Kocher. Bekanntlich andern viele Phrygmeenlarven im Laufe ihrer Lebenszeit die Raustoffe der Kocher; dagegen wird nur selten die primare rohren- formige Gestalt des Kochers geandert. Wir hahen gesehen, da8 die jungen eben ausgekrochenen Glyphotuelien beinahe augenblicklich die Kocher viel breiter als das Rohr machen, ferner duO sie spater als Oberflachentiere damit eine Zeit fortfahren, dann im November eine neue B a u d mit longi- tudinal gelegten Pflanzenteilen arinehmen und damit fortfahren bis zur Ver- PuPPUng. g Warum fangen sie diese merkwiirdige Bauart an, und warum ver- lassen sie sie wieder? Um diese Frage zu losen, arbeitete ich zuerst viel Knit Aquarienstudien , doch gahen sie alle kein befriedigendes Resultat. Ilann richtete ich wie fruher so oft meine Versuche in der Natur selbst an. An den klaren schonen September- bis Oktober-Tagen studierte ich von meinem Boot uus in der Potamogetonregion von hunderten von Glypho- taelien umgeben, die Verhaltnisse wieder. Bei hellem Soiinenschein konnte ich in einer Tiefe von 2 m noch weiDe Kieselsteine auf dem Boden dcr Teiche beobachten. Nimmt man eine Glyphtotaeliuslarve aus dem Hause, so sinkt sie augenblicklich und schnell hinunter. Die Abfahrt zu dem Boden ca. (2 m) dauert ohne Rohre ca. 20 Sekunden oder weniger. Das Tier weiD, was es gilt, es krummt sich, streckt den Vorderkorper aufwarts, keine Pota- mogetonstengel bieten sich tlls Unterstutzungsflachen dar ; - dem Boden- und in den meisten Fallen einem schnellen Tod in sinem Fischmagen ent- geht es nicht. Niinmt man nun den Kocher allein, d a m schwimmt er, wenn er von Potamogetonblattern gebaut ist, immer an der Oberflache. Der Kocher macht also die Larve leichter, er kann als eine Korkseele betrachtet wer- den. Es fragt sich nun, wie viel leichter er das Tier macht; ist er im- stande, die Larve zu tragen, und den ursprunglichen Bodenorganismus zu - 110 110 eirlem ~'elagischeii Oberfliichenorganismus a#bzuandern? Untersuchen wir Iisher die '&jcher tragenden Larven, so zeigt es sich, da8 einige an der Oberflgche schwimmen, andere a l m zu Boden sinken. 111. Theoretische Betrachtungen und Versuche. Eine sinkt relativ schnell, eine andere langsam , inimer aber ist die Sinkgeschwindigkeit ge- ringer nls bei den Tieren ohne K;iiclie,r; die zwei hIeter werden nie in Tveniger Zeit als 1 Minuten zuruckgelegt,; irnmer fallt das Tier auf einer der Broitseiteri; sehr oft geht der Fall in einer Schraubenlinie vor sich, d a m und nwin in einer rechts und links schaukelnden Rewegung. Mehrere steheri in cam. 1 m Tiefe ulrer 5 Minuten vollkommen ruhig schwebend. Betrachten wir die Oberflache des Wassers, so fiiiden wir zwischen deli Potamogetonbliittern frei schwimmende Kocher mit Lurven; imruer liegen sie an der Oberflache und immer mit einsr der Breitseiten aufwarts. Ich erinnere riiic,h, daD ich Anfang September sehr zahlreiche sc,hwimmende Kocher sah; iin Oktober ist ihre Arimhl nicht so groD, denn jet,ztJ kriechen die meisten Larven an den Blattstiolen. Es fragt sich also nun: Wasum sirzkt die eine, eine andere nicht? Wwum ist d;Ls Sc,hwel;mverinogeri \)ei den Tieren verschieden? Sehon friiher habe ich bemerkt', daIi die Kiicher oft Luft' enthalteii und denke daher, da.13 verschiedene Luft~menge die Ursache ist. Um dieses niiher zu priifen, nahm ic,h Tiere, die ca. 'i2 dcm unter der Oberflache sitzen, niiherte die- selben his an die Oberfliiche nnd driickte die Luft niit den Firigerii unter dem Wasser ails. Es zeigt, sich dann, da8W auch diese Tiere, trotzdem sie keine Luft mehr abgeben, derinocli aufwarts steigen konnen. Dall die Luft- rnengen in dem Kocher na tiirlic'h ein Heruntersinken verhindern, verst,eht sich von selbst, da8 es sich bei den Tieren um ein regelma13iges Herein- schaffen von Luft in die Rohren handelt, glaube ich nicht. Die Gegen- wart von Luft hat vie1 melir das Geprige von etwas zufalligem. DaO die Luftmengen ciennoch nic'ht ga,riz ohne Bedeutung sind, geht daraus hervor, was ich riicht. irnmer, doch wiederholt beobachtet habe -, da13 Tiere, (lie ich greifeii will, Luftblaseri abgeben, worauf sie langsain hinuntersinken. Indem ich nun die Oberfliiche niiher beobachtete, sehe ich eine Larve, die sich ein neue's Deckstuck aus eine,m Potamogetonblatt ausschneidet. Die Pflanzen und der Koc'her der Larve wird mit einem Tropfen weiDer Farbe markiert und nach einer Stunde wieder beobachtet: dann ist das neue Stuck in den Kocher eingefiigt'. Das neue frisch grune Stuck ragt, wenn der Kocher in der Oberfliiche schwirnmt, uber die Wasseroberfliiche empor, wirft ma'n einen Tropfen Wasser darauf, flieDt es wie auf den frischen Potamogetonbliittern ah. 111. Theoretische Betrachtungen und Versuche. Wird das neue Stuck ahge,nommen, sinkt der Kiicher rnit der Larve langsam, aber sic,her zu Boden. Indern ich nun wieder zahlreiche Kocher studiere, zeigt es sich, daD, so larige die Blatter und besonders dns letztmgebrachte Blattstuck frisch ist, das Tier sich an der Oberfliiche halten kann. Wenn das Blatt iilter und bruchig wird, wenn Es fragt sich also nun: Wasum sirzkt die eine, eine andere nicht? Wwum ist d;Ls Sc,hwel;mverinogeri \)ei den Tieren verschieden? Sehon friiher habe ich bemerkt', daIi die Kiicher oft Luft' enthalteii und denke daher, da.13 verschiedene Luft~menge die Ursache ist. Um dieses niiher zu priifen, nahm ic,h Tiere, die ca. 'i2 dcm unter der Oberflache sitzen, niiherte die- selben his an die Oberfliiche nnd driickte die Luft niit den Firigerii unter dem Wasser ails. Es zeigt, sich dann, da8W auch diese Tiere, trotzdem sie keine Luft mehr abgeben, derinocli aufwarts steigen konnen. Dall die Luft- rnengen in dem Kocher na tiirlic'h ein Heruntersinken verhindern, verst,eht sich von selbst, da8 es sich bei den Tieren um ein regelma13iges Herein- schaffen von Luft in die Rohren handelt, glaube ich nicht. Die Gegen- wart von Luft hat vie1 melir das Geprige von etwas zufalligem. DaO die Luftmengen ciennoch nic'ht ga,riz ohne Bedeutung sind, geht daraus hervor, was ich riicht. irnmer, doch wiederholt beobachtet habe -, da13 Tiere, (lie ich greifeii will, Luftblaseri abgeben, worauf sie langsain hinuntersinken. g g g Indem ich nun die Oberfliiche niiher beobachtete, sehe ich eine Larve, die sich ein neue's Deckstuck aus eine,m Potamogetonblatt ausschneidet. Die Pflanzen und der Koc'her der Larve wird mit einem Tropfen weiDer Farbe markiert und nach einer Stunde wieder beobachtet: dann ist das neue Stuck in den Kocher eingefiigt'. Das neue frisch grune Stuck ragt, wenn der Kocher in der Oberfliiche schwirnmt, uber die Wasseroberfliiche empor, wirft ma'n einen Tropfen Wasser darauf, flieDt es wie auf den frischen Potamogetonbliittern ah. Wird das neue Stuck ahge,nommen, sinkt der Kiicher rnit der Larve langsam, aber sic,her zu Boden. Indern ich nun wieder zahlreiche Kocher studiere, zeigt es sich, daD, so larige die Blatter und besonders dns letztmgebrachte Blattstuck frisch ist, das Tier sich an der Oberfliiche halten kann. Wenn das Blatt iilter und bruchig wird, wenn es mit Wasser durchtriinkt ist und seinen Luftinhalt abgegeben hat, rer- mag das Tier es nicht. 111. Theoretische Betrachtungen und Versuche. Ich bemerkte nun awh, was ich fruher ubersehen habe, da8 die Blattstucke immer die Oberseite aufwarts kehren: dies ist leicht begreiflich, weil das frisc,he Blatt,stuc,k nur dabei als Adhiisions- apparat verwendet werden kann. Das Tier mu0 also immer neue Stucke einfugen; wenn die Stucke alt werden, vermag das Tier sich nkht mehr an der 0berflac.he zu halten, es mu13 sich dann damit begniigen auf den Blattstielen und auf den Blattern zu kriechen, und wird es hiervon von Wellen oder Tieren weggeschlagen, muO es hinuntersinken. Es ist nu11 ganz klar, daD die Tiere besser an der Oberflache schwimmen, je groDer die Blattstucke sind; die groDen Blattstucke sind daher als ein sehr brauchbares Bauprinzip anzusehen. T$Ter aber diese groDen Kocher langsam durch da's Wasser und immer mit einer den Breitseiten abwarts, schraubenformig, lnngsa,m verschwinden sieht, bokommt unwillkurlich den Eindruck, daR diese eigentumliche regelmaDige Abflachung des Korpers auch eine andere Bedeutung hat. Was uber das Rohr herausragt, ist als flugel- formige Fortsatze anzusehen, wodurch das Tier seinen Quer- schnittswiderst,and sehr s t a r k vergrofiert. Sie verzogern den Fall, sie andern die Fallrichtung von einer vertika,len zu einer Schraubenlinie, und haben daher fur die Glgphotaeliuslarve dieselbe Bedeutung wie Fallmembranen fur die Planktontiere. 1905, da ic,h zuerst diese groDen Iiiicher an der Ober- flache schwimmen sah, faDte mich augenblicklich der Gedanke: das Bau- i'rinzip cler Larven ist. C)berflachenvergrijlSerung nnd VergrolJerung des [Querschnitts, hervorgebraeht, urn den Fall durch das Wasser zu vermindern. Was die Fallmembra,nen der Planktonorganismen anbelangt , ist unsere 'luffassung dieser hIembranen eigentlich nur eine Theorie , die allerdings sehr wahrsc,heinlich ist; hier war ein Fall, wo ich, der eben damals sehr stark uber Planktonorgariismen arbeitete, diese Theorie esperimentell unter- suchen konnte. m hohem Zylinderglas braucht das Tier, um ohne Kocher den Boden zu erreichen, ca. 4 Sekuriden oder noch weniger Ze,it. Mit un- verletztem Koc'her brauchen die Tiere, die uberhaupt sinkeii kiinnen, eine sehr verschiedene Zeit, wenigstens 10 bis 80 Sekunden. Wenii man nun dem Rohr alle Deckstikke abnimmt', sinkt dams Tier immer zu Boden, und immer in einer Linie, die sich vie1 mehr der vertikalen Linie nahert, als wenn das R'ohr mit, Dec,kstucken bekleidet war. Die Zeit ist auc'h hier sehr verschieden, doch nur wenig liinger als wenn das Tier riackt durch das Wasser sinkt. 111. Theoretische Betrachtungen und Versuche. In eineni DaD die groDen Fliigelfort,slttze an den Seiten des Rohres als horizon- t.ale Fallmembranen wahrend des Falles also verzogernd wirken, ist,, was die Glyphotalienlar.ven anbelr-tngt, uber jedeni Zweifel erhaben; und die - 119 - 119 - Berecht,igung ahnlichen Fdlmeinbranen hei Planktonorganismen diesellie Bedeutung zuzut,eilen ish wieder bekrltftigt worden. g g Warum verld3t nun das Tier iin November --- Dezember diesen Baii- plan, ,zieht die Yallmenibran ein" nnd verfertigt sic'h aus Stielstucken ein zglindrisches Rohr.' Weil es niin ein Bodentier sein sol1 und daher keinen Ge br auch fur Gallinembranen haben kann? Sicherlich nicht. Nur weil die Potamogetonbliitt~er verwesen und weder als Schwimmapparat, noch als Nahrung braiic'hbar sind. Daher werden die LLtrven gezwungeii, ihren Aufenthalt auf den Blat,tstielen - die sich lltriger als die Bliitt,er frisch halten - zu nehmen. Solange diese Blattstiele frisch sind, sind sie so luftpefullt, daD das Tier auch von ihnen sich einen schwimmenden Koc,her konst,ruieren kann; dagegen ist es augenscheinlich unmoglich, dime sc.hmalen zylindrisc,heii Stiicke ill der Querrichtung anzubringeii und sie nach den Priiizipien eiiies vergrofiert'e'n Querschiiit,is zu verwenden. Die Xnderung der Bauprinzipien ist ctaher gar iiicht als eine Zweckm%fiiglieit snzuseheii ; eine solche kommt, iiur scheiribar zustaiide, weil das Tier voii den verschiedenerr Teileri derselbeii Pflaiize zu verschiedeneii Jahreszeiten sowohl seine Baumaterialien als arich seine Nahriiiig - irn Sorrimer Bkt- ter, im Herhst durch die Verhiiltnisse gezwuiigeii nnr Stiele - nimmt.. Etwa,s niideres ist es, ob man iiicht in der Anordiiuiig der Blattst,iic,ke, wobei eiii zylindrisches Rohr zu einer flacheii Scheibe wird. eine Aiipassumg an aufiere Faktoreii suchen darf. DaD dieser Bauplan auf die hier ge- schilderte Lokalitat ~ 7 0 1 1 wirlilic,her Bedeutung fur das Tier ist, betrachte ic.h als uiizweifelhaft. Darnit ist gar nicht' gesagt, daD dieser Bauplaii VOIL eben diesell Verhiiltiiisseii hervorgeriifeii ist. Auf zahlreicheii Lokalitiiteii Nordseelands fiiidet niun Glyphotaelierirohren, die ebeiiso abgeplattet siiid TT-ie die meiiies Versuchsteiches, und die deiinoch, so weit ich geseheii hahe, das game Jahr hindurch ausgesprochene Bodentiere sind; das Prinzip, den Querschnitt,swiderstand des Kochers zu vergronern, ist daher sicherlich iiirht als ein ,,Plaiiktoncharakter" , der voii den iiuI3eren Faktoren der .pelagischeii Region abhiingig ist, anzusehen; er hat, eine aiidere uiis uiibekannte Ur- sache. Dort aber, wo die BuDereri Verhaltnisse den Larveii erlauben an der Oberflache zu leben, wird eiii solcher Bauplaii als Mittel, sich iiahe an derselben zu halter1 mit Vorteil benutzt. Nacht r ag. Ich habe im Winter 1910 an verschiedenen Orten in Nordseeland die Glyphotaelien beobachtet und kann nun mit Sicherheit folgendes hervor- heben. Wo die Kocher im Sommer aus abgewelkten Erlenbliittern und anderen Bodenmaterial verfertigt sind, behalten die Kocher den ganzen Winter hin- durch dieselbe wohlbekannte oben beschriebene breite Form. Am 17. Febr. 1910 wurden die Ufer des Funkenteiches untersucht; der Wasserstand war dann vie1 hoher als im Herbst; iiber den uberschwemmten Ufern, die im Herbst mit abgewelkten Ruchen- und Erlenblattern bedeckt waren, lag nun dickes Eis; nur am nach Suden gelegenen Ufsr war das Eis ver- schwunden. Dort fand ich zahlreiche herumkriechende Glyphotaelien mit ganz gleichartigen Kochern wie im Herbst. Die groljen Locher in den Buchen- und Erlenblattern zeigten, daD die Tiere den ganzen Winter hin- durch an ihren Kochern weiter gebaut haben. Hier in Nordseeland wie 14elleicht an den meisten Lokalitaten sind die Glyphotaelien das ganze Jahr hindurch hauptsiichlich Bodentiere, die auf den Fontinalisteppichen herumkriechen. Wo aber - wie im Versuchsteirh I1 - eine breite Zone von Schwimmbliittern von Potamogeton natans sich befindet , verlassen im Sommerhalbjahr die Tiere den Boden und leben als Oberflachentiere auf den Potamogetonteppichen. DaD die urspriingliche Form des Kochers unter diesen neuen Verhaltnissen den Larven vorteilhaft ist, ist unzweifel- haft. Wenn sie im Spatherbst die Form der Kocher andern, geschieht dies nur, weil es infolge des Mangels an Material (Potsmogetonblatter) zur dringenden Notwendigkeit wird. 111. Theoretische Betrachtungen und Versuche. DaD es die Konstruktion des Kochers ist, die deli Glyphotaelieri erlaubt, sich eiiie iieue Heimat auf den Potamogetonwiesen zu suchen, fiiide ich hochst wa,hrscheinlich. Nie hahe ich auf den Teppichen der Schwimmblatttter verschiedener Pflnneen, die in etwti 3 bis 4 m Tiefe wachsen, andere Phrygmeen gefunden als Glypho- tttelien, Phryganea grandis und Triaenodes bicolor. - Die iibrigen Phryganeen , die ihre Kocher von lebenden Pflanzen bauen, besonders die Liinnophiliden, finden sich nicht hier; ihre Heimat sind ganz kleine mit Lemna und Algenteppichen bedeckt,e Teiche. Die monographische Be- arbeitung der Biologie der Glyphotaelien ist hiermit abgeschlossen; uber Phryganea prandis und Triaenodes bicolor sind die Untersuchungen auch beinahe abgeschlossen, doch sind noch einige Fruhjahrsbeobachtungen notig ehe sie publiziert werden konnen. arbeitung der Biologie der Glyphotaelien ist hiermit abgeschlossen; uber Phryganea prandis und Triaenodes bicolor sind die Untersuchungen auch beinahe abgeschlossen, doch sind noch einige Fruhjahrsbeobachtungen notig ehe sie publiziert werden konnen. Revue d. gas. Hydrobiol. u. Hydrogr. Bd. 111. H. 1 u. 2. Literaturverzeichnis. 1778. De Geer, K., Abhandlungen zur Geschichte der Insekten. Ubersetztvon Gdtze. 11. S. 368. 1907. Petersen, E., Bidrag ti1 Fortegnelse over Danmarks Vaarflner. Ento- molog. Meddelelser. Kdbenhavn 11. R. Vol. 111, 2., p. 145. 1902. Siltala, A., Uber die Metamorphose einiger Phryganeiden und Limno- g , , p philiden. Acta SOC. pro Fauna et Flora h’ennica, Vol. XXI, S. 1. 1906a. Siltala, A., Beohachtungen iiber die Okologie der Trichopterenpuppe. p p Allgem. Zeitschr. f. wiss. Insektenbiol., Bd. 11, S. 88. 906b. Siltala, A., Uber den L a i c h der Trichopteren. Acta SOC. pro Fauna et g , , Flora Fennica, Vol. XXVIII, p. 1. 1907a. Siltala, A., Trichopterologische Untersuchungen. Nr. 2. Zoolog. Jahrb. , , p Supplement., Bd. lX, S. 308. 8 - 114 - 5907b. Siltala, A., Uber die Nahrung der Trichopteren. Acta SOC. pro fsuna et flora Fennica. Vol. XSIX, p. 1. 5907b. Siltala, A., Uber die Nahrung der Trichopteren. Acta SOC. pro fsuna et flora Fennica. Vol. XSIX, p. 1. 5907b. Siltala, A., Uber die Nahrung der Trichopteren. Acta SOC. pro fsuna e flora Fennica. Vol. XSIX, p. 1. 1899. Struck,R., Neue und a l t e Trichopteren-Larvengehiuse. 1llustr.Zeitschr. , p f. Entomol., Bd. IQ, Nr. 8. 1899. Struck,R., Neue und a l t e Trichopteren-Larvengehiuse. 1llustr.Zeitschr , p f. Entomol., Bd. IQ, Nr. 8. 1899. Struck,R., Neue und a l t e Trichopteren-Larvengehiuse. 1llustr.Zeitschr. , p f. Entomol., Bd. IQ, Nr. 8. 1905. Thienemann, A., Biologie der Trichopterenpuppe. Zoolog. Jahrb., Bd. XSII. f. Entomol., Bd. IQ, Nr. 8. Abt. f. Syst., S. 1. 1905. Thienemann, A., Biologie der Trichopterenpuppe. Zoolog. Jahrb., Bd. XSII , Q, Abt. f. Syst., S. 1. 1905. Thienemann, A., Biologie der Trichopterenpuppe. Zoolog. Jahrb., Bd. XSII. , Q, Abt. f. Syst., S. 1. Tafelerkliirung (Tafel I). Alle Figuren sind mit Abbes Zeichenprisms gezeichnet. Wo nichts anderea angegeben ist, sind die Figuren beinahe in natiirlicher GrbBe gezeichnet. Fig. 1. Laichmasse am Irisblatt. ) 2 b b h Fig. 1. Laichmasse am Irisblatt. ) 2 b b h i l i di l h b d k d i i Fig. 1. Laichmasse am Irisblatt. ), 2. Eben ausgebrochene Larve mit longitudinaler Rohrbedeckung und einem einzelnen 4. Kbcher aus quergelegten Caresbl5ttern. 6. ), teils BUS Carex, teils aus Potamogeton natans. quergelegten Deckstiick. VergrGDerung ca. 10. I1 "'1 5. ,, 7-13. ), aus P. natansblkttern Fig. 10 von der ,,Unterseite" gesehen. Fig. 11 nachdem die Deckstticke der einen Seite abgenommen sind. ,, 14-18. teils aus P. natansbkttern teiIs aus P. natansblattstielen gebaut. In Fig. 17 auch Carexblatter, in Fig. 18 auch ErlenblBtter. hauptskchlich von P. natansblattstielen, zugleich (in Fig. 19) mit einem Rest von einern subrnersen Blatt von Scirpus. 19-20. t( 21-22. aus abgewelkten Pflanzeuteilen gemacht. 23. ,, in Qorbereitung zu Puppengehiuse. 25. Querdurchschnitt durch einen K6cher von P. natansblittern. 26. Eben ausgekrochene Larve. QergrbBerung ca. 20. 11 27. Die letzten Segmente mit den Nachschiebern derselben. VergrbBerung ca. 60. ,, 28. Ausgewachsene Larve: Vergleiche die Differenz in der Linge der Beine und ~ 29. Freischwimmende Puppe. ,, 24. Faltiges Puppengehiuse. Nachschieber bei Fig. 26 und 28. 30. Vordere SchlieBmembran des Puppengehiuses. Qergr6Derung ca. 10. 32. Imago. 31. Hintere 17 17 1, VergrbDerung ca. 10. 2. Eben ausgebrochene Larve mit longitudinaler Rohrbedeckung und einem einzelnen quergelegten Deckstiick. VergrGDerung ca. 10. 6. ), teils BUS Carex, teils aus Potamogeton natans. I1 1 5. 7 13 ) P blk Fi 10 d , g ,, 7-13. ), aus P. natansblkttern Fig. 10 von der ,,Unterseite" gesehen. Fig. 11 nachdem die Deckstticke der einen Seite abgenommen sind. hauptskchlich von P. natansblattstielen, zugleich (in Fig. 19) mit einem Rest von einern subrnersen Blatt von Scirpus. 19-20. t( 25. Querdurchschnitt durch einen K6cher von P. natansblittern. ,, g pp g Q 26. Eben ausgekrochene Larve. QergrbBerung ca. 20. Q 26. Eben ausgekrochene Larve. QergrbBerung ca. 20. g Q g g 11 27. Die letzten Segmente mit den Nachschiebern derselben. VergrbBerung ca. 60. g Q g g 27. Die letzten Segmente mit den Nachschiebern derselben. VergrbBerung ca. 60. 28. Ausgewachsene Larve: Vergleiche die Differenz in der Linge der Beine und 27. Die letzten Segmente mit den Nachschiebern derselben. VergrbBerung ca. 60. 28. Tafelerkliirung (Tafel I). Ausgewachsene Larve: Vergleiche die Differenz in der Linge der Beine und Nachschieber bei Fig. 26 und 28. 29. Freischwimmende Puppe. g pp 30. Vordere SchlieBmembran des Puppengehiuses. Qergr6Derung ca. 10. 32. Imago. 31. Hintere 17 17 1, VergrbDerung ca. 10.
https://openalex.org/W3135651539	https://uvadoc.uva.es/bitstream/10324/59965/1/Circulating-AQP4-Levels-in-Patients-with-Cerebral-Amyloid-Angiopathy.pdf	English	null	Circulating AQP4 Levels in Patients with Cerebral Amyloid Angiopathy-Associated Intracerebral Hemorrhage	Journal of clinical medicine	2,021	cc-by	10,716	6 Neurology, Son Espases University Hospital, 07120 Balearic Islands, Spain; silvia.tur@ssib.es 7 Citation: Marazuela, P.; Bonaterra-Pastra, A.; Faura, J.; Penalba, A.; Pizarro, J.; Pancorbo, O.; Rodríguez-Luna, D.; Vert, C.; Rovira, A.; Pujadas, F.; et al. Circulating AQP4 Levels in Patients with Cerebral Amyloid Angiopathy-Associated Intracerebral Hemorrhage. J. Clin. Med. 2021, 10, 989. https://doi.org/10.3390/ jcm10050989 Academic Editor: Peter Sporns Received: 7 January 2021 Accepted: 22 February 2021 Published: 2 March 2021 7 Department of Neurology, Donostia University Hospital, 20080 San Sebastián, Spai mariateresa.martinezzabaleta@osakidetza.eus 8 Department of Neurology, Bellvitge University Hospital, L’Hospitalet de Llobregat, 08907 Barcelona, Spain; pcardonap@bellvitgehospital.cat 9 Stroke Unit, Department of Neurology, Ramon y Cajal University Hospital, 28034 Madrid, Spain; rovera78@hotmail.com 10 Stroke Unit, Virgen del Rocío University Hospital, 41013 Sevilla, Spain; lucia.lebrato.hdez@gmail.com 11 Stroke Program, Department of Neurology, Hospital Clínico Universitario, 47003 Valladolid, Spain; juanfarenillas@gmail.com j g 12 Clinical Neurosciences Research Group, Department of Medicine, University of Valladolid, 47003 Valladolid, Spain p 13 Department of Neurology, Virgen Macarena University Hospital, 41009 Sevilla, Spain; l d d h @ il p 13 Department of Neurology, Virgen Macarena University Hospital, 41009 Sevilla, Spain; soledad.perez.sanchez@gmail.com p g 14 Stroke Research Program, Institute of Biomedicine of Sevilla, IBiS, Virgen del Rocío University Hospital, University of Sevilla, 41009 Sevilla, Spain 14 Stroke Research Program, Institute of Biomedicine of Sevilla, IBiS, Virgen del Rocío University Hospital, University of Sevilla, 41009 Sevilla, Spain * Correspondence: mar.hernandez.guillamon@vhir.org * Correspondence: mar.hernandez.guillamon@vhir.org Abstract: Cerebral amyloid angiopathy (CAA) is a major cause of lobar intracerebral hemorrhage (ICH) in elderly patients. Growing evidence suggests a potential role of aquaporin 4 (AQP4) in amyloid-beta-associated diseases, including CAA pathology. Our aim was to investigate the circulat- ing levels of AQP4 in a cohort of patients who had suffered a lobar ICH with a clinical diagnosis of CAA. AQP4 levels were analyzed in the serum of 60 CAA-related ICH patients and 19 non-stroke sub- jects by enzyme-linked immunosorbent assay (ELISA). The CAA–ICH cohort was divided according to the time point of the functional outcome evaluation: mid-term (12 ± 18.6 months) and long-term (38.5 ± 32.9 months) after the last ICH. Although no differences were found in AQP4 serum levels between cases and controls, lower levels were found in CAA patients presenting speciﬁc hemorrhagic features such as ≥2 lobar ICHs and ≥5 lobar microbleeds detected by magnetic resonance imaging (MRI). Article Circulating AQP4 Levels in Patients with Cerebral Amyloid Angiopathy-Associated Intracerebral Hemorrhage a 1, Anna Bonaterra-Pastra 1, Júlia Faura 1, Anna Penalba 1, Jesús Pizarro 1, Olalla Pancorbo 2, Paula Marazuela 1, Anna Bonaterra-Pastra 1, Júlia Faura 1, Anna Penalba 1, Jesús Pizarro 1, Olalla Pancorbo 2, David Rodríguez-Luna 2, Carla Vert 3, Alex Rovira 3 , Francesc Pujadas 4, M. Mar Freijo 5, Silvia Tur 6, Maite Martínez-Zabaleta 7, Pere Cardona Portela 8, Rocío Vera 9, Lucia Lebrato-Hernández 10, Juan F. Arenillas 11,12, Soledad Pérez-Sánchez 13 , Joan Montaner 1,13,14, Pilar Delgado 1 and Mar Hernández-Guillamon 1,* 1 Neurovascular Research Laboratory, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; paula.marazuela@vhir.org (P.M.); anna.bonaterra@vhir.org (A.B.-P.); julia.faura@vhir.org (J.F.); anna.penalba@vhir.org (A.P.); jesus.pizarro@vhir.org (J.P.); joan.montaner@vhir.org (J.M.); pilar.delgado@vhir.org (P.D.) 2 Stroke Unit, Department of Neurology, Vall d’Hebron Hospital, 08035 Barcelona, Spain; olallaprvhir@gmail.com (O.P.); rodriguezluna@vhebron.net (D.R.-L.) p g g 3 Neuroradiology, Department of Radiology, Vall d’Hebron Hospital, 08035 Barcelona, Spain; carla_vert@hotmail.com (C.V.); alex.rovira.idi@gencat.cat (A.R.) 4 Dementia Unit, Neurology Department, Vall d’Hebron Hospital, 08035 Barcelona, Spain; fpujadas@vhebron.net 5 Neurovascular Group, Biocruces Health Research Institute, 48903 Barakaldo, Spain; mariadelmar.freijoguerrero@osakidetza.net Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine 1. Introduction Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid in the walls of cerebral blood vessels [1]. The most common form of CAA is associated with the accumulation of amyloid-beta (Aβ) peptide in arterioles, capillaries, and leptomeningeal vessels and is frequently found in Alzheimer’s disease (AD) patients [2,3]. AD is the most common form of dementia worldwide, whereas CAA is the main cause of lobar intracerebral hemorrhage (ICH) [4,5]. ICH recurrence is one of the major complications of CAA, leading to substantial mortality and disability [6]. In addition to symptomatic ICH, transient focal neurological episodes and cognitive impairment independent of AD are also common clinical manifestations of CAA [7,8]. Despite the poor prognosis of CAA-related lobar ICH, no effective treatments are available, and a deﬁnitive diagnosis requires histopathologic demonstration by postmortem autopsy [9,10]. However, in clinical practice, the diagnosis of CAA is established following the modiﬁed Boston criteria based on clinical data and the presence of speciﬁc magnetic resonance imaging (MRI) markers [11]. Lobar cerebral microbleeds, cortical superﬁcial siderosis, enlarged perivascular spaces in the centrum semiovale, and white matter hyperintensities are some of the frequent CAA radiological features detected by MRI [12–15]. Pathologically, Aβ peptides are generated by the sequential processing of amyloid precursor protein (APP) by β-secretase and γ-secretase, resulting in peptides consisting of 40 or 42 amino acids (Aβ40 or Aβ42, respectively). Aβ42 mainly accumulates in neuritic plaques in the brain parenchyma of AD patients, while Aβ40 is predominantly deposited on the walls of cerebral vessels, replacing smooth muscle cells and leading to vascular degeneration in CAA [16,17]. Although the cellular pathways explaining Aβ accumulation remain unclear, the most widely accepted theory is based on an imbalance between Aβ production and clearance. Several mechanisms have been described to remove Aβ from the brain, including proteolytic and microglial degradation, active transport across the blood–brain barrier (BBB), and perivascular and lymphatic drainage [17–19]. In fact, several studies have suggested that the perivascular drainage pathway may be impaired in CAA, which would lead to Aβ accumulation in vascular basement membranes [20–22]. Aquaporins are a family of water channel proteins that regulate water transport across cell membranes [23]. Aquaporin 4 (AQP4) is the most abundant water channel of the central nervous system and is mainly expressed by astrocytes and ependymal cells. AQP4 is highly localized to the perivascular astrocytic end-feet surrounding the glial limiting membrane of blood vessels [24,25]. In addition, CAA-related ICH patients who presented a long-term good functional outcome had higher circulating AQP4 levels than subjects with a poor outcome or controls. Our data suggest that AQP4 could potentially predict a long-term functional outcome and may play a protective role after a lobar ICH. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 989. https://doi.org/10.3390/jcm10050989 J. Clin. Med. 2021, 10, 989 2 of 15 Keywords: aquaporin 4; cerebral amyloid angiopathy; intracerebral hemorrhage; magnetic resonance imaging markers; functional outcome 2.1. Study Population The study cohort consisted of 60 patients who presented with symptomatic ICH with a clinical diagnosis of CAA, as well as 19 age- and sex-matched control subjects. CAA–ICH patients had possible, probable or probable CAA with a supporting pathology diagnosis according to the modiﬁed Boston criteria [11]. Controls were healthy participants with no stroke history from the ISSYS (investigating silent strokes in hypertensives, a magnetic resonance imaging study) cohort who underwent brain MRI at a follow-up visit [31]. Controls were chosen for having no hemorrhagic events on their MRI scans. Sixty CAA– ICH patients were recruited in the neurology or stroke units from 10 different Spanish centers. CAA–ICH patients were >55 years old and had suffered at least one lobar ICH. Patients were excluded if they exhibited any deep intracerebral hemorrhage, presented microbleeds in the basal ganglia, internal or external capsule, thalamus or brainstem, or were being treated with anticoagulant therapy. The data obtained from the whole cohort included patient coding, inclusion date, demographic characteristics (age and sex), relevant vascular risk factors (hypertension, diabetes, and dyslipidemia), and brain imaging ﬁndings. The clinical and demographic data of both cohorts are shown in Table 1. Blood samples from the CAA–ICH cohort were obtained in a chronic state of the disease (13.6 ± 17.8 months after the last ICH) to avoid capturing the initial inﬂammatory process. Cognitive impairment was determined at the time of baseline visit (or blood draw) in all patients based on clinical history and neurological examinations. Additionally, functional outcome was assessed using the modiﬁed Rankin Scale (mRS) for neurologic disability. For a more detailed analysis, the CAA–ICH cohort was divided into two subcohorts based on the time point of the last functional outcome evaluation. In subcohort 1 (n = 35), mid- term outcome was assessed at 12 ± 18.6 months after the last ICH, the same time as when blood was drawn. In subcohort 2 (n = 25), blood was collected 15.8 ± 17 months after the last ICH, and long-term outcome was assessed at 34.4 ± 24.8 months after blood collection. A schematic representation describing the time points of recruitment and outcome evaluation for these cohorts is shown in Appendix A Figure A1. Outcomes were dichotomized into good vs. poor: poor outcome was deﬁned as mRS >3, and good outcome was deﬁned as mRS ≤3. Table 1. Demographic, clinical, and radiological characteristics of the total cohort. 2. Methods Demographic, clinical, and radiological characteristics of the total co Variable Control (n = 19) CAA–IC (n = 60 Age, years, median (IQR) 74 (73.5–74) 76.5 (71.5– Sex, female, n (%) 10 (52.6%) 30 (50% Hypertension 19 (100%) 29 (48.3% Diabetes 6 (31.3%) 7 (11.7% Dyslipidemia 16 (78.9%) 17 (28.3% APOE genotype, ε2 carriers 1 (5.3%) 8 (13.3% APOE genotype, ε4 carriers 7 (36.8%) 14 (23.3% Lobar ICH 0 (0.0%) 60 (100% WMH, n (%) 9 (47.4%) 57 (95.0% CMB 0 (0.0%) 40 (66.7% Serum AQP4, ng/mL, median (IQR) 2.12 (1.63–2.67) 2.15 (1.44– CAA, cerebral amyloid angiopathy; IQR, interquartile range; APOE, apolipoprotein hyperintensity; ICH, intracerebral hemorrhage; CMB, cerebral microbleed; AQP4, aqu 0.05 are shown in bold. 1. Introduction Recent evidence suggests that AQP4 plays an essential role in the clearance of solutes between cerebrospinal ﬂuid (CSF) and interstitial ﬂuid (IF) through perivascular drainage, including Aβ clearance [26,27]. In this regard, several studies have described altered AQP4 expression and localization in AD and CAA patients [28–30]. However, most of these studies are based on postmortem analysis of human brain tissue, and the alteration of plasma AQP4 levels in AD or CAA patients is still unexplored. In the present study, we ﬁrst aimed to study whether circulating AQP4 could be an indicative biomarker of CAA pathology and whether its determination would thus contribute to the diagnosis and prognosis of this disease. For this purpose, we analyzed the potential association of AQP4 levels with the main neuroimaging hallmarks of CAA in a multicenter cohort of patients with lobar ICH associated with CAA. We next determined the relationship between circulating AQP4 levels and other functional variables in this cohort. J. Clin. Med. 2021, 10, 989 3 of 15 2. Methods 2. Methods 2.1. Study Population The study cohort consisted of 60 patients who presented with sym a clinical diagnosis of CAA, as well as 19 age- and sex-matched control patients had possible, probable or probable CAA with a supporting p according to the modiﬁed Boston criteria [11]. Controls were healthy p stroke history from the ISSYS (investigating silent strokes in hypert resonance imaging study) cohort who underwent brain MRI at a f Controls were chosen for having no hemorrhagic events on their MR ICH patients were recruited in the neurology or stroke units from 1 centers. CAA–ICH patients were >55 years old and had suffered at Patients were excluded if they exhibited any deep intracerebral hem microbleeds in the basal ganglia, internal or external capsule, thal or were being treated with anticoagulant therapy. The data obtain cohort included patient coding, inclusion date, demographic char sex), relevant vascular risk factors (hypertension, diabetes, and dysli imaging ﬁndings. The clinical and demographic data of both cohorts a Blood samples from the CAA–ICH cohort were obtained in a chronic (13.6 ± 17.8 months after the last ICH) to avoid capturing the initial in Cognitive impairment was determined at the time of baseline visit (o patients based on clinical history and neurological examinations. Add outcome was assessed using the modiﬁed Rankin Scale (mRS) for n For a more detailed analysis, the CAA–ICH cohort was divided into tw on the time point of the last functional outcome evaluation. In subco term outcome was assessed at 12 ± 18.6 months after the last ICH when blood was drawn. In subcohort 2 (n = 25), blood was collecte after the last ICH, and long-term outcome was assessed at 34.4 ± 24.8 collection. A schematic representation describing the time points outcome evaluation for these cohorts is shown in Appendix A Figure dichotomized into good vs. poor: poor outcome was deﬁned as mRS >3 was deﬁned as mRS ≤3. Table 1. 2.2. MRI Protocol and Radiological Data A brain MRI scan was obtained from all participants (1.5 ± 16.4 months after the last ICH). MRI examinations were acquired using a 1.5-T whole-body scanner system. The images obtained included axial T2-weighted turbo spin-echo, axial T1-weighted spin- echo, axial T2-weighted turbo ﬂuid-attenuated inversion recovery (FLAIR), and axial T2-weighted echo-planar gradient-echo sequences. All MRI scans were evaluated by the same neuroradiologist at Hospital Vall d’Hebron, who was blinded to the clinical and biological information. g The radiological characteristics of the CAA–ICH cohort obtained through MRI analysis are presented in Table 2. ICHs were deﬁned as hypointense foci on the T2-weighted images (diameter >5 mm), and their number and location were recorded. The presence, number, and distribution of cerebral microbleeds (CMBs; diameter <5 mm) were evaluated according to the Brain Observer Microbleed Scale [32]. White matter hyperintensities (WMHs) were deﬁned as hyperintense signal lesions in T2-FLAIR or T2-weighted images. Deep and periventricular WMHs were assessed according to the four-point Fazekas rating scale [33]. This scale scores damage from 0 to 4: (0) Absent or isolated foci of 3 mm, (1) foci of less than 5 mm (periventricular caps not included); (2) foci of more than 5 mm (periventricular caps not included); (3) beginning of conﬂuence; (4) large conﬂuent lesions (larger than 20 mm or two or more lesions merged). Severe WMH was deﬁned as a score of 3 or 4. The WMH score was recorded in the hemisphere not affected by hemorrhage, except in cases when both hemispheres were involved. Perivascular or Virchow–Robin spaces are considered CSF-like signal lesions along the course of penetrating arteries. Enlarged perivascular spaces (EPVS) were counted in the basal ganglia and in the centrum semiovale (CSO) and were classiﬁed as moderate (≤20 EPVS) or severe (≥21 EPVS) according to the number found on axial T2-weighted MRI images [13,34]. Cortical superﬁcial siderosis (cSS) was deﬁned as the deposition of hemosiderin in the subpial layers of the cerebral cortex. The distribution and severity of cSS was categorized as focal (restricted to ≤3 sulci) or disseminated (>4 sulci) [11]. cSS contiguous to an ICH was not considered. Total cerebral small vessel disease (SVD) burden deﬁned by Charidimou et al. was assessed using the principal MRI markers of CAA (lobar CMBs, WMH, EPVS, and cSS) [35]. 2.3. Serum AQP4 Determination Peripheral blood was collected in EDTA tubes, and serum was immediately separated by centrifugation at 1500 g for 15 min and stored at –80 ◦C. Only blood samples collected during a follow-up visit (at least 1.5 months after ICH) were considered for the analysis. The total AQP4 levels in serum were determined by enzyme-linked immunosorbent assay (ELISA) using the AQP4 Human Kit (Cusabio Biotech., Wuhan, China) following the manufacturer’s instructions. Optical density was measured at 450 nm in a Synergy™Mx microplate reader (BioTek Instruments Inc., Vermont, USA). All samples were assayed in duplicate, and replicates with a coefﬁcient of variation >20% were discarded from the statistical analyses. 2.1. Study Population Variable Control (n = 19) CAA–ICH (n = 60) p-Value Age, years, median (IQR) 74 (73.5–74) 76.5 (71.5–70) 0.130 Sex, female, n (%) 10 (52.6%) 30 (50%) 1 Hypertension 19 (100%) 29 (48.3%) 0.000 Diabetes 6 (31.3%) 7 (11.7%) 0.063 Dyslipidemia 16 (78.9%) 17 (28.3%) 0.000 APOE genotype, ε2 carriers 1 (5.3%) 8 (13.3%) 0.679 APOE genotype, ε4 carriers 7 (36.8%) 14 (23.3%) 0.251 Lobar ICH 0 (0.0%) 60 (100%) 0.000 WMH, n (%) 9 (47.4%) 57 (95.0%) 0.000 CMB 0 (0.0%) 40 (66.7%) 0.000 Serum AQP4, ng/mL, median (IQR) 2.12 (1.63–2.67) 2.15 (1.44–4.12) 0.626 CAA, cerebral amyloid angiopathy; IQR, interquartile range; APOE, apolipoprotein E; WMH, white matter hyperintensity; ICH, intracerebral hemorrhage; CMB, cerebral microbleed; AQP4, aquaporin 4. p-Values below 0.05 are shown in bold. Table 1. Demographic, clinical, and radiological characteristics of the total cohort. J. Clin. Med. 2021, 10, 989 4 of 15 The study was approved by the Clinical Investigation Ethics Committee of the Vall d’Hebron University Hospital, Barcelona, Spain (PR(AG)326/2014), and had the approval of the ethics committees of all of the participating centers. The study was conducted in accordance with the Declaration of Helsinki. All patients provided signed informed consent before inclusion. 2.2. MRI Protocol and Radiological Data The total SVD burden ranged from 0 to 6 points and was determined by counting the presence and grade of each of these 4 MRI features [35]. In our study, a high SVD burden was deﬁned as a score ≥4. 2.4. Statistical Analyses Statistical analyses were conducted with the SPSS 20.0 package (IBM Corporation, Armonk, NY, USA), and graphs were generated in GraphPad Prism 6 (GraphPad Software, J. Clin. Med. 2021, 10, 989 5 of 15 La Jolla, CA, USA). Descriptive statistics were used to deﬁne the demographic data, clinical variables, and radiological characteristics of the CAA–ICH cohort. The normality of the continuous variables was assessed using the Kolmogorov–Smirnov test. For univariate analysis, the Mann–Whitney U-test and the Kruskal–Wallis test were used to evaluate the signiﬁcant differences in the non-normally distributed variables between groups. Corre- lations were calculated as Spearman’s rho to compare two continuous variables, and the chi-squared test was used to assess the intergroup differences for the categorical variables. Binary logistic regression analysis was performed for ≥2 ICHs and cognitive impairment, including variables signiﬁcantly associated with each end-point in the univariate analysis. A stepwise forward method was used in order to select those variables that ﬁtted in the model. Data are expressed as the mean ± standard error of the mean or median values (interquartile range). A p-value <0.05 was considered statistically signiﬁcant. Table 2. Radiological characteristics of the CAA–ICH cohort. CAA–ICH (n = 60) Boston Criteria Possible 12 (20.0%) Probable 45 (75.0%) Probable with supporting pathology 3 (5.0%) WMH 57 (95.0%) Periventricular 51 (85.0%) Moderate (1–2 Fazekas) 10 (16.7%) Severe (3–4 Fazekas) 41 (68.3%) Deep subcortical WMH 52 (86.7%) Moderate (1–2 Fazekas) 21 (35.0%) Severe (3–4 Fazekas) 31 (51.6%) CMB 40 (66.7%) Lobar CMB 40 (66.7%) 1–5 14 (23.3%) 6–10 9 (15.0%) 10–20 3 (5.0%) >20 14 (23.3%) Deep CMB 0 (0.0%) Cerebellar CMB 4 (6.7%) EPVS 53 (88.3%) EPVS basal ganglia 52 (86.7%) Moderate (1–20) 43 (71.7%) Severe (21 to >40) 9 (15.0%) EPVS CSO 41 (68.3%) Moderate (1–20) 19 (31.7%) Severe (21 to >40) 22 (36.7%) cSS 30 (50.0%) Focal 9 (15.0%) Disseminated 21 (35.0%) Atrophy 23 (38.3%) Small vessel disease burden Low (0–3) 22 (36.7%) High (4–6) 38 (63.3%) Data are expressed as n (%).WMH, white matter hyperintensity; CMB, cerebral microbleed; EPVS, enlarged perivascular space; CSO, centrum semiovale; cSS, cortical superﬁcial siderosis. Table 2. Radiological characteristics of the CAA–ICH cohort. Low (0–3) High (4–6) Data are expressed as n (%).WMH, white matter hyperintensity; CMB, cerebral microbleed; EPVS, enlarged perivascular space; CSO, centrum semiovale; cSS, cortical superﬁcial siderosis. 3. Results 3.1. Baseline Characteristics The demographic, clinical, and radiological characteristics of the 60 CAA-related ICH patients and the 19 healthy control subjects included in the study are described in Table 1. There were no signiﬁcant differences between the two groups in terms of age, sex, or APOE genotype. The control cohort was selected for not having ICH or detectable cerebral J. Clin. Med. 2021, 10, 989 6 of 15 microbleeds by MRI. Some control subjects did show WMHs in both the periventricular and deep regions, but the prevalence was signiﬁcantly higher in CAA-related ICH patients. AQP4 was detected in the serum from the control subjects and the CAA patients, although no signiﬁcant differences were observed between groups. microbleeds by MRI. Some control subjects did show WMHs in both the periventricular and deep regions, but the prevalence was signiﬁcantly higher in CAA-related ICH patients. AQP4 was detected in the serum from the control subjects and the CAA patients, although no signiﬁcant differences were observed between groups. The main MRI radiological characteristics of the CAA–ICH cohort are summarized in Table 2. All patients with lobar ICH fulﬁlled the Boston diagnostic criteria of probable or possible CAA. The majority of these patients (66.7%) had at least one lobar CMB, whereas no CMBs in the deep regions were detected. Overall, a high SVD burden score (63.3%), a high prevalence of severe WMH in the periventricular (68.3%) and deep regions (51.6%), a high degree of EPVS-CSO (36.7%), and disseminated cSS (35%) constituted the principal radiological features of the cohort. 3.2. AQP4 Levels According to Clinical and Radiological Characteristics Circulating levels of AQP4 were evaluated in the CAA-related ICH cohort and ana- lyzed for any correlations with the clinical and radiological characteristics. We determined that lower circulating levels of AQP4 were related to Apoε4 and cognitive impairment in the univariate analysis (Table 3). However, only the presence of WMH in the periventricular areas remained independently associated with cognitive impairment after the adjustment by binary logistic regression analysis (Table A1 and Table 5). On the contrary, patients who had suffered a previous hemorrhagic stroke presented signiﬁcantly lower levels of AQP4, whereas presenting a previous ischemic stroke did not result in changes in serum AQP4 levels (Table 3). Table 3. Univariate analysis of AQP4 levels according to CAA–ICH demographic and clinical characteristics. Variable YES NO p-Value Age r = 0.147 0.264 Sex, female 2.19 (1.46–4.20) n = 30 2.08 (1.41–4.04) n = 30 0.684 Hypertension 1.85 (1.45–3.29) n = 29 2.89 (1.77–4.27) n = 28 0.102 Diabetes 2.60 (2.11–3.58) n = 7 2.14 (1.44–4.27) n = 48 0.435 Dyslipidemia 1.81 (1.41–2.89) n = 17 2.11 (1.44–4.41) n = 36 0.331 APOE genotype, ε2 carriers 1.75 (1.18–3.00) n = 8 2.30 (1.45–4.27) n = 52 0.317 APOE genotype, ε4 carriers 1.46 (1.03–2.60) n = 14 2.41 (1.65–4.20) n = 46 0.028 Cognitive impairment 1.69 (1.27–2.76) n = 30 3.09 (1.81–4.38) n = 30 0.030 Previous stroke 1.28 (0.99–1.67) n = 12 2.68 (1.69–4.35) n = 48 0.002 Previous ischemic stroke 1.53 (1.26–3.27) n = 4 2.68 (1.69–4.35) n = 48 0.261 Previous hemorrhagic stroke 1.12 (0.79–1.61) n = 8 2.68 (1.69–4.35) n = 48 0.001 Data are expressed as median nanograms per mililiter (interquartile range). r, Spearman’s rho correlation, CAA–ICH cohort; n = 60. p-Values below 0.05 are shown in bold. Table 3. Univariate analysis of AQP4 levels according to CAA–ICH demographic and clinical characteristics. Data are expressed as median nanograms per mililiter (interquartile range). r, Spearman’s rho correlation, CAA–ICH cohort; n = 60. p-Values below 0.05 are shown in bold. The neuroimaging analysis of the CAA–ICH cohort revealed a negative correlation between the number of lobar ICHs and AQP4 serum levels (Table 4). In fact, those patients with ≥2 lobar ICHs had signiﬁcantly lower levels of AQP4 than those with only one lobar ICH (Figure 1A). After adjustment for signiﬁcantly associated variables in the univariate J. Clin. Med. 3.2. AQP4 Levels According to Clinical and Radiological Characteristics ICH, intracerebral hemorrhage; WMH, white matter hyperintensity; CMB, cerebral microbleed; EPVS, enlarged perivascular space; CSO, centrum semiovale; cSS, cortical superﬁcial siderosis; SVD, small vessel disease burden. p-Values below 0.05 are shown in bold. 3.2. AQP4 Levels According to Clinical and Radiological Characteristics 2021, 10, 989 7 of 15 analysis (Table 5), the logistic regression analysis conﬁrmed that serum AQP4 levels, Apoε2 allele, high SVD burden, and atrophy were independent predictors of ≥2 lobar ICHs (Table 5). In addition, a tendency (p = 0.052) toward lower AQP4 levels in patients presenting lobar CMBs was detected. When this variable was reclassiﬁed, serum samples from patients presenting with ≥5 CMBs showed lower AQP4 levels (Figure 1B). CAA–ICH patients presenting WMHs in deep regions also had signiﬁcantly lower levels of AQP4 (Table 4). Remarkably, we did not detect an association between serum AQP4 concentration and the total MRI small vessel disease score validated for a pathological CAA presentation with symptomatic ICH [35]. Table 4. Univariate analysis of AQP4 levels according to CAA–ICH radiological characteristics. Variable YES NO p-Value Interval between the last ICH and the date of blood collection r = –0.052 0.703 Number of lobar ICHs r = –0.307 0.017 WMH 2.04 (1.43–3.71) n = 57 4.46 (3.15–5.7) n = 3 0.163 Periventricular 2.02 (1.42–3.69) n = 51 3.29 (1.84–5.02) n = 9 0.092 Deep subcortical WMH 1.88 (1.42–3.67) n = 50 3.41 (2.04–5.19) n = 10 0.045 Lobar CMB 1.83 (1.41–3.79) n = 40 2.84 (1.93–4.25) n = 20 0.052 EPVS 2.26 (1.46–4.04) n = 53 1.81 (1.27–3.78) n = 7 0.718 EPVS basal ganglia 2.30 (1.47–4.12) n = 52 1.63 (1.27–3.78) n = 8 0.521 EPVS CSO 2.45 (1.50–4.36) n = 41 1.84 (1.10–3.00) n = 19 0.144 cSS 2.11 (1.16–4.38) n = 30 2.15 (1.65–2.90) n = 30 0.988 Chronic Infarct 1.53 (1.27–2.02) n = 13 2.52 (1.55–4.19) n = 44 0.146 Atrophy 2.26 (1.43–4.00) n = 23 2.04 (1.50–4.04) n = 37 0.715 High SVD burden (score 4–6) 2.35 (1.27–4.34) n = 23 2.03 (1.81–2.90) n = 37 0.570 Data are expressed as median nanograms per mililiter (interquartile range). r, Spearman’s rho correlation. CAA–ICH cohort: n = 60. ICH, intracerebral hemorrhage; WMH, white matter hyperintensity; CMB, cerebral microbleed; EPVS, enlarged perivascular space; CSO, centrum semiovale; cSS, cortical superﬁcial siderosis; SVD, small vessel disease burden. p-Values below 0.05 are shown in bold. ble 4. Univariate analysis of AQP4 levels according to CAA–ICH radiological characteristics. Data are expressed as median nanograms per mililiter (interquartile range). r, Spearman’s rho correlation. CAA–ICH cohort: n = 60. 3.3. AQP4 and Functional Outcome The results are given as odds ratios (ORs) with 95% conﬁdence intervals (CIs) and p-values. SVD, small vessel disease. p-Values below 0.05 are shown in bold. Figure 2. Association between AQP4 levels and functional neurological outcomes in the CAA–ICH cohort. (A) Boxplot distribution between good (n = 27) and poor (n = 7) mid-term functional outcomes (CAA–ICH subcohort 1) compared to controls (n = 19). (B) Boxplot distribution between good (n = 17) and poor (n = 8) long-term functional outcomes (CAA–ICH subcohort 2) compared to controls (n = 19). Outcomes were dichotomized into good vs. poor, and good outcomes were predeﬁned as 0–3 on the modiﬁed Rankin Scale for Neurologic Disability. ICH, intracerebral hemorrhage; CMB, cerebral microbleed. p < 0.05. Table 5. Binary logistic regression for cognitive impairment and ≥2 ICHs. Regression Cognitive Impairment Regression ≥2 ICHs Variable OR (95% CI) p-value OR (95% CI) p-value AQP4 - 0.520 (0.286–0.976) p = 0.042 APOE genotype, ε2 carriers - 22.536 (1.989–255.296) p = 0.034 WMH periventricular 10.545 (1.227–90.662) p = 0.032 - Atrophy - 6.167 (1.080–35.213) p = 0.004 High SVD burden (score 4–6) - 11.280 (1.109–114.739) p = 0.025 Binary logistic regression analysis was performed with variables associated with cognitive impairment and/or ≥2 ICHs in univariate analysis. The results are given as odds ratios (ORs) with 95% conﬁdence intervals (CIs) and p-values. SVD, small vessel disease. p-Values below 0.05 are shown in bold. Table 5. Binary logistic regression for cognitive impairment and ≥2 ICHs. Regression Cognitive Impairment Regression ≥2 ICHs Variable OR (95% CI) p-value OR (95% CI) p-value AQP4 - 0.520 (0.286–0.976) p = 0.042 APOE genotype, ε2 carriers - 22.536 (1.989–255.296) p = 0.034 WMH periventricular 10.545 (1.227–90.662) p = 0.032 - Atrophy - 6.167 (1.080–35.213) p = 0.004 High SVD burden (score 4–6) - 11.280 (1.109–114.739) p = 0.025 Binary logistic regression analysis was performed with variables associated with cognitive impairment and/or ≥2 ICHs in univariate analysis. The results are given as odds ratios (ORs) with 95% conﬁdence intervals (CIs) and p-values. SVD, small vessel disease. p-Values below 0.05 are shown in bold. Table 5. Binary logistic regression for cognitive impairment and ≥2 ICHs. 3.3. AQP4 and Functional Outcome We next studied the association between circulating AQP4 levels and functional outcome in two subcohorts divided according to the evaluation time point (Appendix A Figure A1). A poor outcome was assessed in seven (20%) of the 35 patients from subcohort 1 and in eight (32%) of the 25 patients from subcohort 2. No statistically signiﬁcant differences were found in the levels of AQP4 between subcohort 1 (1.87 (1.29–3.53) ng/mL) and subcohort 2 (2.38 (1.65–4.38) ng/mL) (p = 0.165). Interestingly, we found that AQP4 levels differed among CAA–ICH patients depending on the time when the outcome was assessed. First, in subcohort 1, when patient serum was obtained in parallel to the functional outcome evaluation, AQP4 levels did not differ between patients classiﬁed with good and poor outcomes (Figure 2A). In contrast, when the long-term functional outcome was assessed, J. Clin. Med. 2021, 10, 989 8 of 15 those CAA–ICH subjects showing good outcomes presented signiﬁcantly higher levels of AQP4 than subjects with a poor outcomes and controls (Figure 2B). In subcohort 2, AQP4 was the only variable associated with poor outcome in the univariate analysis (Appendix A Table A2). Figure 1. Association between AQP4 levels and hemorrhagic events in the CAA–ICH cohort. (A) Box- plot distribution according to the number of symptomatic ICHs (1 ICH, n = 43; ≥2 ICH, n = 17). (B) Boxplot distribution according to the number of lobar CMBs (<5 CMB, n = 34; ≥5 CMB, n = 26). ICH, intracerebral hemorrhage; CMB, cerebral microbleed. Figure 1. Association between AQP4 levels and hemorrhagic events in the CAA–ICH cohort. (A) Box- plot distribution according to the number of symptomatic ICHs (1 ICH, n = 43; ≥2 ICH, n = 17). (B) Boxplot distribution according to the number of lobar CMBs (<5 CMB, n = 34; ≥5 CMB, n = 26). ICH, intracerebral hemorrhage; CMB, cerebral microbleed. Table 5. Binary logistic regression for cognitive impairment and ≥2 ICHs. Regression Cognitive Impairment Regression ≥2 ICHs Variable OR (95% CI) p-value OR (95% CI) p-value AQP4 - 0.520 (0.286–0.976) p = 0.042 APOE genotype, ε2 carriers - 22.536 (1.989–255.296) p = 0.034 WMH periventricular 10.545 (1.227–90.662) p = 0.032 - Atrophy - 6.167 (1.080–35.213) p = 0.004 High SVD burden (score 4–6) - 11.280 (1.109–114.739) p = 0.025 Binary logistic regression analysis was performed with variables associated with cognitive impairment and/or ≥2 ICHs in univariate analysis. 4. Discussion In this study, we have shown for the ﬁrst time the presence of AQP4 in the circulation in CAA-related ICH patients. Although no signiﬁcant differences were found in circulating AQP4 levels between CAA–ICH patients and controls, an association between serum AQP4 levels and cerebral hemorrhagic load in the CAA cohort was detected. Furthermore, our data revealed that AQP4 could predict the long-term functional outcome in CAA patients after lobar ICH. p Interest in AQP4 research has grown considerably over the last few years. AQP4 is the most highly expressed aquaporin in the brain and is involved in the maintenance of the brain water balance under physiological and pathological conditions [24,29]. Several studies have demonstrated its implication in cerebral edema, ischemic stroke, traumatic brain injury, tumors, and neuromyelitis optica, among others [24,28,35]. Various studies have also found that AQP4 is necessary for the clearance of interstitial solutes, including Aβ, through the glymphatic system, suggesting a potential role of AQP4 in the physiopathology of AD and/or CAA [26,36]. To date, the vast majority of studies exploring the role of AQP4 in AD and CAA patients have described an altered distribution of this protein in diseased postmortem brain tissues. Wilcock et al. found reduced AQP4 expression in AD patients with moderate or severe CAA [37]. However, subsequent studies have demonstrated an increase in AQP4 immunoreactivity in AD and CAA brains compared with controls [38–40]. In particular, it was suggested that the AQP4 expression pattern could differ depending on the disease stage, with increased AQP4 immunodetection in CAA cases with the highest AD score [41]. In agreement with that, it has recently been described that the pattern of AQP4 immun- odetection is different in gray matter compared to white matter and that it changes with age and with the severity of CAA [42]. Taking all of this into account, AQP4 expression in the brain may be associated with different factors, such as age, CAA severity, and speciﬁc neuroanatomical area. However, to the best of our knowledge, no studies have reported the modulation of AQP4 levels in the circulation in AD or CAA. Q In this regard, we were able to detect AQP4 in serum from CAA-related ICH patients, although the levels did not differ from those in non-stroke control subjects. 3.3. AQP4 and Functional Outcome Regression Cognitive Impairment Regression ≥2 ICHs Variable OR (95% CI) p-value OR (95% CI) p-value AQP4 - 0.520 (0.286–0.976) p = 0.042 APOE genotype, ε2 carriers - 22.536 (1.989–255.296) p = 0.034 WMH periventricular 10.545 (1.227–90.662) p = 0.032 - Atrophy - 6.167 (1.080–35.213) p = 0.004 High SVD burden (score 4–6) - 11.280 (1.109–114.739) p = 0.025 Binary logistic regression analysis was performed with variables associated with cognitive impairment and/or ≥2 ICHs in univariate analysis. The results are given as odds ratios (ORs) with 95% conﬁdence intervals (CIs) and p-values. SVD, small vessel disease. p-Values below 0.05 are shown in bold. Table 5. Binary logistic regression for cognitive impairment and ≥2 ICHs. Binary logistic regression analysis was performed with variables associated with cognitive impairment and/or ≥2 ICHs in univariate analysis. The results are given as odds ratios (ORs) with 95% conﬁdence intervals (CIs) and p-values. SVD, small vessel disease. p-Values below 0.05 are shown in bold. Figure 2. Association between AQP4 levels and functional neurological outcomes in the CAA–ICH cohort. (A) Boxplot distribution between good (n = 27) and poor (n = 7) mid-term functional outcomes (CAA–ICH subcohort 1) compared to controls (n = 19). (B) Boxplot distribution between good (n = 17) and poor (n = 8) long-term functional outcomes (CAA–ICH subcohort 2) compared to controls (n = 19). Outcomes were dichotomized into good vs. poor, and good outcomes were predeﬁned as 0–3 on the modiﬁed Rankin Scale for Neurologic Disability. ICH, intracerebral hemorrhage; CMB, cerebral microbleed. * p < 0.05. Figure 2. Association between AQP4 levels and functional neurological outcomes in the CAA–ICH cohort. (A) Boxplot distribution between good (n = 27) and poor (n = 7) mid-term functional outcomes (CAA–ICH subcohort 1) compared to controls (n = 19). (B) Boxplot distribution between good (n = 17) and poor (n = 8) long-term functional outcomes (CAA–ICH subcohort 2) compared to controls (n = 19). Outcomes were dichotomized into good vs. poor, and good outcomes were predeﬁned as 0–3 on the modiﬁed Rankin Scale for Neurologic Disability. ICH, intracerebral hemorrhage; CMB, cerebral microbleed. * p < 0.05. Figure 2. Association between AQP4 levels and functional neurological outcomes in the CAA–ICH J. Clin. Med. 2021, 10, 989 9 of 15 4. Discussion In this line, the Apoε2 allele is a well-known risk factor for ICH attributed to CAA, predisposing patients to recurrent bleeding [55–57]. Due to the speciﬁc localization of AQP4 at the astrocyte end-feet, it has been suggested that AQP4 could play a role in maintaining the BBB integrity [58]. Although most studies seem to point to a protective role for AQP4, it is still controversial whether AQP4 deletion alters the integrity of the BBB after ICH [59–62]. Further studies are needed to conﬁrm the exact function of AQP4 in the brain following ICH, and our ﬁndings should be expanded to other cohorts presenting with ICH independently of a CAA etiology. p p g p y gy The present study also revealed that the analysis of serum AQP4 could potentially predict a long-term functional outcome in CAA–ICH patients. Our results showed that patients with a good long-term functional outcome presented higher circulating levels of AQP4 than patients with poor outcomes or healthy controls. In this area, we are aware of only one study that analyzed the levels of AQP4 in the circulation in neurological disorders, reporting higher levels of AQP4 in ischemic stroke patients after the acute phase of the disease [63]. Furthermore, in this same study, AQP4 was found to be an independent predictor of good neurological outcome in stroke patients, which is in agreement with our results [63]. Overall, the increase in circulating AQP4 levels in CAA–ICH patients presenting good long-term outcome and the decreased AQP4 levels in patients with a higher cerebral hemorrhagic load provide suggestive evidence of the potentially protective role of AQP4 after ICH. Q Our study has some limitations. First, the sample size was relatively small, which could mask some associations not detectable after multiple testing corrections. Other limita- tions include the cross-sectional design of the study and the differences in the time points of the recruitment and functional outcome evaluation after the hemorrhagic episode. For this last reason, we decided to divide the total cohort into two subgroups, reducing the sample size even more. Taking all of this into consideration, further research should focus on larger patient cohorts with functional evaluation at different time points to explore whether our ﬁndings could be useful for the clinical prediction of long-term functional outcome in ICH patients. 4. Discussion In agreement with previous studies [43,44], our results revealed that the presence of WMH in periventric- ular areas was also independently associated with cognitive impairment in the CAA–ICH cohort. In addition, we found lower circulating levels of AQP4 in patients with cognitive impairment in the univariate analysis. This result is aligned with ﬁndings in preclinical models, where behavioral tests revealed a cognitive deﬁcit in AQP4 knockout mice [45–47]. Indeed, studies performed in AD and CAA transgenic mouse models have conﬁrmed that AQP4 deletion promotes cognitive deﬁcits and increases Aβ accumulation and synaptic damage, suggesting a possible contribution of AQP4 to Aβ clearance through the brain vasculature [48]. In contrast to these results, in a recent study conducted in 5xFAD mice, an accelerated model of AD with a lower vascular damage contribution, AQP4 deﬁciency did not induce alterations in Aβ accumulation or in memory deﬁcit [49]. Nevertheless, experimental evidence suggests that AQP4 may be involved in the clearance of Aβ through perivascular drainage, thus playing a potential protective role in CAA pathology. However, in our study, we did not ﬁnd an association between circulating AQP4 levels and the global SVD neuroimaging burden score, which has been associated with pathological CAA [35,50]. This result suggests that circulating AQP4 is not an indicative biomarker of CAA pathology. All the same, because AQP4 is related to solute clearance, further research should focus on studying AQP4 protein levels in CSF [26,27]. Remarkably, we found a clear association between serum AQP4 levels and CAA- related hemorrhagic lesions, including symptomatic ICH and CMBs, in the study cohort. Indeed, CAA–ICH patients presenting ≥2 ICHs and/or ≥5 lobar CMBs had lower levels of AQP4 than those patients without those hemorrhagic events, suggesting a potential protective role of this protein. Moreover, we demonstrated a negative correlation between the number of lobar ICHs and AQP4 serum levels, which is in agreement with other studies J. Clin. Med. 2021, 10, 989 10 of 15 10 of 15 where AQP4 deletion worsened the neurological impairment and promoted neuronal death after ICH [51,52]. Indeed, AQP4 genomic variants were recently found to be independent predictors of the outcome after ICH in different populations [53,54]. Furthermore, we found that circulating AQP4 levels, together with the Apoε2 allele, atrophy, and high SVD burden variables, were independent predictors of presenting ≥2 ICHs. 4. Discussion It would also be interesting to study the temporal proﬁle of AQP4, as well as to evaluate the association between circulating AQP4 levels and the progression of neuroimaging markers with a follow-up blood draw and an MRI examination at different time points. Author Contributions: Conceptualization, M.H.-G. and J.M.; methodology, P.M., A.B.-P., J.F., A.P., and J.P.; recruitment of patients, O.P., D.R.-L., F.P., M.M.F., S.T., M.M.-Z., P.C.P., R.V., L.L.-H., J.F.A., S.P.-S., J.M., and P.D.; radiological data analysis, C.V. and A.R.; formal analysis, P.M. and M.H.-G.; writing—original draft preparation, P.M. and M.H.-G.; writing—review and editing, P.M., M.H.-G., 5. Conclusions This study demonstrated, for the ﬁrst time, that AQP4 can be detected in serum samples from CAA-related lobar ICH patients. Our data showed an association between circulating AQP4 levels and speciﬁc hemorrhagic neuroimaging features. In particular, we observed a decrease in AQP4 levels in patients presenting with more than two symp- tomatic lobar ICHs and in patients with more than ﬁve lobar CMBs detected by MRI. These ﬁndings suggest that AQP4 could play a protective role in CAA patients after ICH, potentially preserving BBB functionality, which could provide a possible therapeutic target for this pathology. Furthermore, because we found that circulating APQ4 levels were higher in those patients presenting a good long-term outcome, analysis of this serum biomarker might be considered an interesting candidate to be further investigated to improve the accuracy of the outcome prognosis in patients presenting with lobar ICH. Author Contributions: Conceptualization, M.H.-G. and J.M.; methodology, P.M., A.B.-P., J.F., A.P., and J.P.; recruitment of patients, O.P., D.R.-L., F.P., M.M.F., S.T., M.M.-Z., P.C.P., R.V., L.L.-H., J.F.A., S.P.-S., J.M., and P.D.; radiological data analysis, C.V. and A.R.; formal analysis, P.M. and M.H.-G.; writing—original draft preparation, P.M. and M.H.-G.; writing—review and editing, P.M., M.H.-G., J. Clin. Med. 2021, 10, 989 11 of 15 11 of 15 and P.D.; supervision, M.H.-G.; funding acquisition, M.H.-G. All authors read and agreed to the published version of the manuscript. Funding: This research was funded by the Instituto de Salud Carlos III (ISCIII), (PI17/00275 and PI20/00465), co-ﬁnanced by the European Regional Development Fund FEDER. The Neurovas- cular Research Laboratory is part of the INVICTUS+ network, ISCIII, Spain (RD16/0019/0021). M.H.-G. was supported by the Miguel Servet programme, ISCIII, Spain (CPII17/00010). P.M. held a predoctoral fellowship from the Vall d’Hebron Research Institute. Institutional Review Board Statement: The study protocol was approved by the Clinical Investiga- tion Ethics Committee of the Vall d’Hebron University Hospital, Barcelona, Spain (PR(AG)326/2014), and had the approval of the Ethics Committees of all of the participating centers. The study was conducted conducted according to the guidelines of the Declaration of Helsinki. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data that support this study are available on request from the Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. 5. Conclusions 2021, 10, 989 12 of 15 Table A1. Univariate analysis of the variables associated with cognitive impairment and ≥2 ICHs. Cognitive impairment ≥2 ICHs Variable YES NO p-value YES NO p-Value AQP4 1.69 (1.27–2.76) n = 30 3.09 (1.81–4.38) n = 30 0.030 1.45 (1.08–3.22) n = 17 2.45 (1.69–4.27) n = 43 0.026 Age 77 (73–79) 74 (69–79) 0.406 77 (72–78) 76 (71.5–79) 0.941 Sex, female 18 (60.0%) 12 (40.0%) 0.196 10 (58.8%) 20 (46.5%) 0.567 Hypertension 15 (53.6%) 14 (48.3%) 0.793 10 (66.7%) 19 (45.2%) 0.230 Diabetes 4 (14.3%) 3 (11.1%) 1 2 (14.3%) 5 (12.2%) 1 Dyslipidemia 8 (29.6%) 9 (34.6%) 0.773 8 (53.3%) 9 (23.7%) 0.053 APOE genotype, ε2 carriers 4 (13.3%) 4 (13.3%) 1 5 (29.4%) 3 (7.0%) 0.035 APOE genotype, ε4 carriers 10 (33.3%) 4 (13.3%) 0.125 6 (35.3%) 8 (18.6%) 0.190 Cognitive impairment - - - 9 (52.9%) 21 (48.8%) 1 ≥2 lobar ICHs 9 (30.0%) 8 (26.7%) 1 - - - WMH 29 (96.7%) 28 (93.3%) 1 17 (100%) 40 (93.0%) 0.551 Periventricular 29 (96.7%) 22 (73.3%) 0.026 14 (82.4%) 37 (86.05%) 0.704 Deep subcortical WMH 28 (93.3%) 22 (73.3%) 0.080 15 (88.2%) 35 (81.4) 0.709 Lobar CMB 21 (70.0%) 19 (63.3%) 0.785 13 (76.5%) 27 (62.8%) 0.375 EPVS 25 (83.3%) 28 (93.3%) 0.424 13 (76.5%) 40 (93.0%) 0.092 EPVS basal ganglia 25 (83.3%) 27 (90.0%) 0.706 12 (70.6%) 40 (93.0%) 0.035 EPVS CSO 19 (63.3%) 22 (73.3%) 0.580 11 (64.7%) 30 (69.8%) 0.763 cSS 18 (60.0%) 12 (40.0%) 0.196 13 (76.5%) 17 (39.5%) 0.020 Chronic infarct 10 (33.3%) 3 (11.1%) 0.061 5 (33.3%) 8 (19.0%) 0.294 Atrophy 13 (43.3%) 10 (33.3%) 0.596 12 (70.6%) 11 (25.6%) 0.003 High SVD burden (score 4–6) 21 (55.53%) 17 (44.7%) 0.422 15 (88.2%) 23 (53.5%) 0.017 AQP4, aquaporin 4; ICH, intracerebral hemorrhage; APOE, apolipoprotein E; WMH, white matter hyperintensity; CMB, cerebral microbleed; EPVS, enlarged perivascular space; CSO, centrum semiovale; cSS, cortical superﬁcial siderosis; SVD, small vessel disease. p-Values below 0.05 are shown in bold. nivariate analysis of the variables associated with cognitive impairment and ≥2 ICHs. AQP4, aquaporin 4; ICH, intracerebral hemorrhage; APOE, apolipoprotein E; WMH, white matter hyperintensity; CMB, cerebral microbleed; EPVS, enlarged perivascular space; CSO, centrum semiovale; cSS, cortical superﬁcial siderosis; SVD, small vessel disease. p-Values below 0.05 are shown in bold. Table A2. Univariate analysis for long-term functional outcome in subcohort 2. 5. Conclusions Data Availability Statement: The data that support this study are available on request from the corresponding autor, M.H.-G. Data Availability Statement: The data that support this study are available on request from the corresponding autor, M.H.-G. Conﬂicts of Interest: The authors declare no conﬂict of interest. Figure A1. Schematic representation of the cerebral amyloid angiopathy (CAA)–intracerebral hemor- rhage (ICH) cohort according to the functional outcome evaluation time point. (A) Summary table of the time intervals in the two subcohorts. (B) CAA–ICH subcohort 1 refers to individuals whose functional outcome was assessed at the blood collection date, 12 ± 18.6 months after the last ICH (mid-term outcome). (C) CAA–ICH subcohort 2 refers to individuals in which functional outcome was assessed 34.4 ± 24.8 months after the blood collection date (long-term outcome). Months are expressed as mean ± SD (standard deviation). Parts of this ﬁgure were supported by Servier Medical Art with permission under the Creative Commons Attribution 3.0 Unported License. Figure A1. Schematic representation of the cerebral amyloid angiopathy (CAA)–intracerebral hemor- Figure A1. Schematic representation of the cerebral amyloid angiopathy (CAA)–intracerebral hemor- rhage (ICH) cohort according to the functional outcome evaluation time point. (A) Summary table of the time intervals in the two subcohorts. (B) CAA–ICH subcohort 1 refers to individuals whose functional outcome was assessed at the blood collection date, 12 ± 18.6 months after the last ICH (mid-term outcome). (C) CAA–ICH subcohort 2 refers to individuals in which functional outcome was assessed 34.4 ± 24.8 months after the blood collection date (long-term outcome). Months are expressed as mean ± SD (standard deviation). Parts of this ﬁgure were supported by Servier Medical Art with permission under the Creative Commons Attribution 3.0 Unported License. Figure A1. Schematic representation of the cerebral amyloid angiopathy (CAA)–intracerebral hemor- rhage (ICH) cohort according to the functional outcome evaluation time point. (A) Summary table of the time intervals in the two subcohorts. (B) CAA–ICH subcohort 1 refers to individuals whose functional outcome was assessed at the blood collection date, 12 ± 18.6 months after the last ICH (mid-term outcome). 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CAA–ICH subcohort 2 (n = 25) Variable Poor outcome Good outcome p-Value Periventricular 8 (100%) 13 (76.5%) 0.134 Deep subcortical WMH 8 (100%) 12 (70.6%) 0.086 Lobar CMB 7 (87.5%) 10 (58.8%) 0.152 EPVS 8 (100%) 17 (100%) - EPVS basal ganglia 7 (87.5%) 17 (100%) 0.137 EPVS CSO 8 (100%) 15 (88.2%) 0.312 cSS 3 (37.5%) 9 (52.9%) 0.471 Chronic Infarct 3 (42.9%) 2 (11.8%) 0.088 Atrophy 3 (37.5%) 6 (35.3%) 0.915 High SVD burden (score 4–6) 4 (50.0%) 10 (58.8%) 0.678 AQP4, aquaporin 4; ICH, intracerebral hemorrhage; WMH, white matter hyperintensity; CMB, cerebral mi- crobleed; EPVS, enlarged perivascular space; CSO, centrum semiovale; cSS, cortical superﬁcial siderosis; SVD, small vessel disease. p-Values below 0.05 are shown in bold. 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https://openalex.org/W3127203793	https://jurnal.univpgri-palembang.ac.id/index.php/hon/article/download/5074/pdf_13	Indonesian	null	Pengaruh Latihan Tendangan Menggunakan Ban Karet Terhadap Hasil Tendangan Sabit Pencak Silat	Halaman Olahraga Nusantara	2,021	cc-by-sa	3,661	Abstrak Penelitian ini bertujuan untuk mengetahui pengaruh latihan tendangan menggunakan ban karet terhadap hasil tendangan sabit pencak silat. Metode penelitian ini adalah kuantitaatif dalam bentuk pre eksperimental design. Desain yang di gunakan dalam penelitian ini adalah one group pretest-postest design. Populasi penelitian ini adalah atlet pencak silat putri pra-remaja Kabupaten Pekalongan yang berjumlah 28 orang dengan teknik total sampling. Teknik pengumpulan data menggunakan tes kemampuan tendangan sabit selama 1 menit. Hasil penelitian yang diperoleh yaitu (1) Hasil penelitian pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit pada kaki kiri dengan nilai R-Square sebesar 0,718. Nilai tersebut mengandung arti bahwa latihan berpengaruh terhadap hasil tendangan sabit pada kaki kiri sebesar 71,8%, sedangkan sebesar 28,2% merupakan hasil keterampilan tendangan sabit kaki kiri yang dipengaruhi oleh variabel lain. (2) Hasil penelitian pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit pada kaki kanan dengan nilai R-Square sebesar 0,771. Nilai tersebut mengandung arti bahwa latihan berpengaruh terhadap hasil tendangan sabit pada kaki kanan sebesar 77,1%, sedangkan sebesar 22,9% merupakan hasil keterampilan tendangan sabit kaki kanan yang dipengaruhi oleh variabel lain. Dari hasil tersebut dapat artikan bahwa latihan tendangan dengan ban karet berpengaruh terhadap hasil tendangan sabit pencak silat. Kata Kunci: prestasi, latihan, atlet, tendangan sabit, ban karet PENGARUH LATIHAN TENDANGAN MENGGUNAKAN BAN KARET TERHADAP HASIL TENDANGAN SABIT PENCAK SILAT Yustiana Nabila1 , Marshanda Salsabila Malinda2 , Yusril Ihza Maulana3, Gilang Nuari Panggraita4 Universitas Muhammadiyah Pekajangan Pekalongan 1,2,3,4 nabilayustiana@gmail.com1 , marshanda259@gmail.com2, yusrizihzamaulana2171@gmail.com 3, panggraita@umpp.ac.id4 ISSN 2614-2775 (Print) ISSN 2621-8143 (Online) Volume 4 no I Tahun 2021 Jurnal HalamanOlahraga Nusantara licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. Keywords: achievement, training, athlete, sickle kick, rubber tires Keywords: achievement, training, athlete, sickle kick, rubber tires PENDAHULUAN Olahraga adalah suatu kegiatan yang sistematis dan dapat mendorong, membina, serta mengembangkan potensi jasmani, rohani dan sosial. Olahraga terus berkembang dengan pesat seiring dengan perkembangan modernisasi dan teknologi yang makin canggih dengan adanya berbagai karakteristik dalam bentuk kebutuhan, kesibukan, dan gaya hidup (Lauh et al., 2020). Banyak jenis atau cabang olahraga yang berkembang dan dikenal oleh masyarakat, salah satunya adalah olahraga bela diri yaitu pencak silat. Pencak silat merupakan olahraga yang berasal dari Indonesia. Pencak silat adalah hasil budaya manusia Indonesia untuk membela, mempertahankan, eksistensi (kemandirian) dan integritasnya (ma- nunggal) terhadap lingkungan hidup/alam sekitarnya untuk mencapai keselarasan hidup guna meningkatkan iman dan taqwa (Gristyutawa et al., 2012). Pencak silat menjadi olahraga yang diminati banyak orang, hingga terdapat berbagai klub dan juga padepokan sebagai wadah untu berlatih para pesilat. Seiring perkembangan zaman pencak silat mulai dipertandingkan dalam kejuaraan baik tingkat daerah, nasional maupun internasional. Kejuaraan tersebut juga dapat dijadikan tolak ukur untuk mengetahui sejauh mana tingkat keberhasilan dari proses pelatihan yang dilakukan dalam suatu klub atau padepokan. Ada beberapa aspek yang perlu dilatih kepada seorang pesilat agar dapat memungkinkan pesilat mencapai prestasi maksimal yaitu latihan fisik, latihan teknik, latihan taktik dan latihan mental (Dewi, 2014) Pada gerakan pencak silat terdapat teknik gerakan penyerangan dan pertahanan. Pola gerakan serangan dalam pencak silat ada berbagai macam salah satunya adalah tedangan sabit. Tendangan sabit bisa disebut juga dengan tendangan C karena posisi badan yang meliuk mengikuti ayunan dari tungkai kaki hingga ujung kaki dengan sasaran punggung kaki. Menurut Lubis (2014:35) menyatakan bahwa tendangan busur/sabit adalah tendangan yang lintasannya Abstract This research aims to determine the effect of kick training using a rubber tire on the results of the pencak silat sickle kick. This research method is quantitative in the form of pre experimental design.. The design used in this research was a one group pretest-postest design. The population of this research was 28 female pre-teen martial arts athletes in Pekalongan Regency with total sampling technique. The data collection technique used a sickle kick ability test for 1 minute. The results obtained are (1) The results of the research on the effect of kick training with a rubber tire on the results of a sickle kick on the left foot with an R-Square value of 0.718. This value implies that training has an effect on the results of the sickle kick on the left leg by 71.8%, while 28.2% is the result of the skill of the left foot sickle kick which is influenced by other variables. (2) The results of the research on the effect of kick training with rubber tires on the results of a sickle kick on the right foot with an R-Square value of 0.771. This value implies that exercise has an effect on the results of the sickle kick on the right leg by 77.1%, while 22.9% is the result of the sickle kick skill of the right foot which is influenced by other variables. From these results it can be interpreted that kick practice with rubber tires has an effect on the results of the pencak silat sickle kick. Correspondence author: Yustiana Nabila, Universitas Muhammadiyah Pekajangan pekalongan, Indonesia. E- Mail: nabilayustiana@gmail com Correspondence author: Yustiana Nabila, Universitas Muhammadiyah Pekajangan pekalongan, Indonesia. E- Mail: nabilayustiana@gmail.com Jurnal HalamanOlahraga Nusantara licensed under a Creative Commons Attribution-ShareAlike 4 International License. 77 \| Halaman Olahraga Nusantara Yustiana Nabila, Marshanda Salsabila Malinda, Yusril Ihza Maulana, Gilang Nuari Panggraita (2021) PENGARUH LATIHAN TENDANGAN MENGGUNAKAN BAN KARET TERHADAP HASIL TENDANGAN SABIT PENCAK SILAT Keywords: achievement, training, athlete, sickle kick, rubber tires setengah lingkaran kedalam, sasaran seluruh bagian tubuh, dengan punggung telapak kaki atau jari telapak kaki. Tendangan sabit dilakukan dengan posisi kuda- kuda yang kuat dan benar. Dengan ayunan kaki untuk menendang ke arah lawan dan memantulkan punggung kaki ke bagian sasaran. Perkenaan kaki ke sasaran tepat pada bagian punggung kaki. Posisi kedua tangan ditekuk di depan dada untuk menjaga keseimbangan tubuh. Proses gerakan tendangan sabit dalam olahraga pencak silat dilakukan dengan gerakan pola gerak yang tidak terputus sehingga menjadi suatu rangkaian gerakan yang utuh yaitu mulai dari posisi kuda-kuda, memiringkan badan, mengangkat kaki dengan menendang setinggi lutut dengan sasaran puggung kaki dan meluruskan tungkai dengan gerakan cepat untuk mencapai sasaran pada bagian tubuh lawan. Apabila proses gerakan pencak silat dilakukan dengan tersendat-sendat dan tidak teratur maka dapat mengurangi kelincahan seorang atlet yang melakukan serangan, sehingga sangat mudah untuk ditangkis ataupun ditangkap oleh lawan. Untuk menghasilkan kecepatan bergerak yang cepat diperlukan kecepatan gerak kaki sebagai daya dorong untuk membantu gerakan tungkai pada saat melakukan ayunan (Harliawan & Darminto, 2020). Seorang atlet pencak silat memerlukan kondisi fisik yang tinggi dalam melakukan tendangan sabit dalam pertandingan. Terdapat faktor-faktor yang dapat memicu perkembangan prestasi seorang atlet dalam berolahraga. Seperti harus adanya kualitas dalam pelatihan dan pembinaan olahraga sehingga prestasi olahraga dapat berkembang dengan ilmu yang terkait khususnya pada olahraga pencak silat. Some factors can spur the development of achievements in sports, such as quality improvement in training and sports coaching (Pomatahu, 2018). Pelatih harus mampu membuat program latihan yang benar dengan memahami tahap-tahap latihan sehingga mengetahui berapa besar porsi latihan yang dibutuhkan, karena latihan merupakan faktor utama dalam meningkatkan kualitas otot dan kekuatan otot merupakan modal 79 \| Halaman Olahraga Nusantara 78 \| Halaman Olahraga Nusantara 78 \| Halaman Olahraga Nusantara 79 \| Halaman Olahraga Nusantara untuk mempermudah mempelajari teknik dan mencegah terjadinya cedera dan meperoleh prestasi yang maksimal (Kamarudin, 2015). Menurut Pasau dalam Boihaqi (2017) bahwa orang yang mempunyai fisik tinggi dan besar rata-rata akan mempunyai kemampuan fisik seperti kekuatan, kecepatan, daya tahan jantung dan paru-paru, daya tahan otot dan lain-lain lebih baik daripada orang yang bertubuh kecil dan pendek. Pelaksanaan teknik dasar yang baik dan benar sangat diperlukan dalam pertandingan pencak silat, cara yang harus dilakukan adalah dengan menjalani latihan dengan program latihan yang direncanakan (Pratiwi et al., 2013). Permasalahan kondisi fisik dan kekuatan otot yang lemah membuat tendangan sabit seorang atlet tidak dapat digunakan untuk menyerang dengan optimal. Oleh karena itu dibutuhkan latihan menggunakan metode latihan yang tepat, salah satunya dengan tendangan menggunakan beban dari ban karet yang diikat pada kaki. menit. Tendangan dilakukan sebanyak 2 kali yaitu tendangan kaki kanan dan kaki kiri. Berikut ini adalah norma kemampuan tendangan sabit: menit. Tendangan dilakukan sebanyak 2 kali yaitu tendangan kaki kanan dan kaki kiri. Berikut ini adalah norma kemampuan tendangan sabit: kiri. Berikut ini adalah norma kemampuan tendangan sabit: Tabel 1. Norma tes kemampuan tendangan sabit Tabel 1. Norma tes kemampuan tendangan sabit Kategori Putri Putra Baik Sekali >24 >25 Baik 19-23 20-24 Cukup 16-18 17-19 Kurang 13-15 15-16 Kurang Sekali <12 <14 Analisis data yang digunakan adalah uji normalitas, uji homogenitas dan is regresi menggunakan uji-t. Kategori Putri Putra Baik Sekali >24 >25 Baik 19-23 20-24 Cukup 16-18 17-19 Kurang 13-15 15-16 Kurang Sekali <12 <14 Analisis data yang digunakan adalah uji normalitas, uji homogenitas dan analisis regresi menggunakan uji-t. Analisis data yang digunakan adalah uji normalitas, uji homogenitas dan analisis regresi menggunakan uji-t. 81 \| Halaman Olahraga Nusantara METODE Metode penelitian ini adalah kuantitaatif dalam bentuk pre eksperimental designe. Desain yang di gunakan dalam penelitian ini adalah one group pretest- postest design, yaitu desain penelitian yang terdapat pretest sebelum diberi perlakuan dan posttest setelah diberi perlakuan. Dengan demikian dapat diketahui lebih akurat, karena dapat membandingkan dengan diadakan sebelum diberi perlakuan (Sugiyono dalam Prasetyo, 2015). Populasi penelitian ini adalah atlet pencak silat putri pra-remaja Kabupaten Pekalongan yang berjumlah 28 orang. Dalam penelitian ini menggunakan teknik total sampling, artinya seluruh atlet putri menjadi sampel yang berjumlah 28 orang.Penelitian ini dilakukan di Padepokan perguruan pencak silat kera putih Pekalongan Data yang digunakan dalam penelitian ini adalah data tendangan sabit. Teknik pengumpulan data menggunakan tes kemampuan tendangan sabit selama 1 80 \| Halaman Olahraga Nusantara dengan uji normalitas dan uji homogenitas. Tabel 3. Hasil Uji Normalitas Kelas Kolmogorov-Smirnova Shapiro-Wilk Statistic df Sig. Statistic Df Sig. Hasil Pre Kanan .156 28 .080 .941 28 .119 Post Kanan .196 28 .007 .936 28 .086 Pre Kiri .138 28 .185 .967 28 .495 Post Kiri .110 28 ,200* .961 28 .376 Tabel 3. Hasil Uji Normalitas Berdasarkan tabel 3 dapat dilihat bahwa nilai signifikansi hsail pre-test dan post-test kaki kiri maupuan kanan lebih besar dari 0,05. Sehingga, dapat disimpulkan bahwa data berdistribusi normal. HASIL DAN PEMBAHASAN Pada penelitian ini menggunaan jenis penelitian quasi eksperimental desinge dengan bentuk pre test post test control design dan menggunakan metode kuantitatif Sugiyono (2018:120). Penelitian ini bertujuan untuk mengetahui pengaruh tendangan menggunakan ban karet terhadap tendangan sabit atlet pencak silat putri pra-remaja. Untuk mengetahu hal tersebut dilakukan Pre-test terlebih dahulu, selanjutnya atlet diberikan perlakuan berupa latihan tendangan menggunakan ban karet yang dilaksanakan sebanyak 16 kali pertemuan dengan 3 kali repetisi per 1 menit, dalam 16 kali pertemuan terhitung pada pertemuan awal pretest (latihan awal sebelum di lakukan metode latihan) dan 14 pertemuan selama melakukan metode latihan, serta pertemuan terakhir sebagai posttest (test akhir setelah melakukan metode latihan). 81 \| Halaman Olahraga Nusantara Tabel 2. Hasil Pre-Test dan Post-Test Tendangan Sabit Kategori Rentang Nilai Pre test Post test Kaki kanan Kaki kiri Kaki kanan Kaki kiri Baik Sekali >24 5 Baik 19-23 3 16 19 Cukup 16-18 3 15 8 4 Kurang 13-15 19 9 4 Tabel 2. Hasil Pre-Test dan Post-Test Tendangan Sabit 81 \| Halaman Olahraga Nusantara 81 \| Halaman Olahraga Nusantara Tabel 4. Hasil Uji Homogenitas Tabel 4. Hasil Uji Homogenitas Levene Statistic df1 df2 Sig. 1.948 3 108 .126 Hasil tabel diatas menunjukkan bahwa nilai signifikansi (sig.) sebesar 0,126. Nilai Sig, 0,126>0,05, maka bisa diambil keputusan bahwa varians data tersebut bersifat sama atau homogen. Levene Statistic df1 df2 Sig. 1.948 3 108 .126 Hasil tabel diatas menunjukkan bahwa nilai signifikansi (sig.) sebesar 0,126. Nilai Sig, 0,126>0,05, maka bisa diambil keputusan bahwa varians data tersebut bersifat sama atau homogen. Setelah dilakukan uji normalitas dan homogenitas, selanjutnya dilakukan pengujian hipotesis menggunakan uji analisis regresi. Penelitian ini memiliki satu hipotesis yaitu terdapat pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit. Data yang diperoleh berupa hasil tendangan sabit kaki kiri dan kaki kanan. Maka dilakukan uji hipotesis terhadap masing-masing kaki. Tabel 5. Uji Hipotesis R-square Tendangan dengan Ban Karet terhadap hasil tendangan sabit kaki kiri 82 \| Halaman Olahraga Nusantara Model R R Square Adjusted R Square Std. Error of the Estimate 1 ,718a .515 .506 2.241 82 \| Halaman Olahraga Nusantara 1 Regression 292.571 1 292.571 79.221 ,016b Residual 199.429 54 3.693 Total 492.000 55 Hasil perhitungan nilai F hitung menunjukkan sebesar 79,221 dengan nila signifkansi sebesar 0,016, dimana nilai sigkansi 0,016<0,05. Ini menunjukkan bahwa terdapat pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit pada kaki kanan. PEMBAHASAN Pada dasarnya pembebanan ban karet dalam tendangan sabit melibatkan beberapa unsur yang tentunya untuk menciptakan dan mencapai tujuan atlet yang maksimal. Dalam metode pembebanan tendangan sabit menggunakan ban karet seorang atlet harus juga memiliki keterampilan lebih dan memiliki fisik dan teknik yang cukup memadai. Mulai dari teknik yang benar, daya tahan, dan kekuatan. Selanjutnya Harsono dalam Manullang (2018) mengemukakan bahwa, perkembangan kondisi fisik yang menyeluruh amatlah penting karena tanpa kondisi fisik yang baik atlet tidak akan dapat mengikuti latihan- latihan dengan sempurna. Latihan yang sistematis dapat dilakukan dengan menggunakan beban untuk menambah kekuatan otot. Hal ini juga berfungsi untuk mencapai tujuan yaitu perbaikan kekuatan kondisi fisik, Kesehatan dan pencapaian prestasi dalam suatu cabang olahraga khususnya pencak silat. Komponen kondisi fisik dalam olahraga beladiri pencak silat diantaranya daya tahan (endurance). Daya tahan ini berkaitang dengan waktu pertandingan yang dilakukan dalam satu kali tanding. Seorang pesilat harus bertanding dalam waktu dua menit kali tiga babak dengan teknik yang sempurna untuk bisa memenangkan pertandingan. Selain daya tahan, komponen kondisi fisik lainnya yang tidak kalah penting dalam pertandingan silat adalah kecepatan (speed), kekuatan (strength), daya ledak (power), kelentukan (flexibility) dan kelincahan (agility). Komponen kondisi fisik ini berguna untuk Hasil penelitian pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit pada kaki kiri dengan nilai R-Square sebesar 0,718. Nilai tersebut mengandung arti bahwa latihan berpengaruh terhadap hasil tendangan sabit pada kaki kiri sebesar 71,8%, sedangkan sebesar 28,2% merupakan hasil keterampilan tendangan sabit kaki kiri yang dipengaruhi oleh variabel lain. Tabel 6. Uji Hipotesis F Hitiing Tendangan dengan Ban Karet terhadap hasil tendangan sabit kaki kiri. Tabel 6. Uji Hipotesis F Hitiing Tendangan dengan Ban Karet terhadap hasil tendangan sabit kaki kiri. Model Sum of Squares df Mean Square F Sig. 1 Regression 288.018 1 288.018 57.368 ,011b Residual 271.107 54 5.021 Total 559.125 55 Hasil perhitungan nilai F hitung menunjukkan sebesar 57,368 dengan nilai Hasil perhitungan nilai F hitung menunjukkan sebesar 57,368 dengan nilai signifkansi sebesar 0,011, dimana nilai sigkansi 0,011<0,05. Ini menunjukkan bahwa terdapat pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit pada kaki kiri. Tabel 7. Uji Hipotesis R-square Tendangan dengan Ban Karet terhadap hasil tendangan sabit kaki kanan Model R R Square Adjusted R Square Std. Error of the Estimate 1 ,771a .595 .587 1.922 Hasil penelitian pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit pada kaki kanan dengan nilai R-Square sebesar 0,771. Nilai tersebut mengandung arti bahwa latihan berpengaruh terhadap hasil tendangan sabit pada kaki kanan sebesar 77,1%, sedangkan sebesar 22,9% merupakan hasil keterampilan tendangan sabit kaki kanan yang dipengaruhi oleh variabel lain. Tabel 8. Uji Hipotesis F Hitung Tendangan dengan Ban Karet terhadap hasil tendangan sabit kaki kanan. Model Sum of Squares df Mean Square F Sig. Tabel 7. Uji Hipotesis R-square Tendangan dengan Ban Karet terhadap hasil tendangan sabit kaki kanan Hasil penelitian pengaruh latihan tendangan dengan ban karet terhadap Hasil penelitian pengaruh latihan tendangan dengan ban karet terhadap hasil tendangan sabit pada kaki kanan dengan nilai R-Square sebesar 0,771. Nilai tersebut mengandung arti bahwa latihan berpengaruh terhadap hasil tendangan sabit pada kaki kanan sebesar 77,1%, sedangkan sebesar 22,9% merupakan hasil keterampilan tendangan sabit kaki kanan yang dipengaruhi oleh variabel lain. T b l 8 Uji Hi i F Hi T d d B K h d h il d Tabel 8. Uji Hipotesis F Hitung Tendangan dengan Ban Karet terhadap hasil tendangan sabit kaki kanan. Model Sum of Squares df Mean Square F Sig. 83 \| Halaman Olahraga Nusantara 83 \| Halaman Olahraga Nusantara bergerak keseluruh penjuru gelanggang untuk menyerang dalam gerakan memukul, menendang, menangkis dan mengelak (Kamarudin, 2015) Menurut (Abdurahman et al., 2014) menyatakan bahwa tendangan busur atau tendangan sabit merupakan tendangan yang paling dominan digunakan dalam bertarung khususnya pada kategori tanding karena tendangan ini sangat praktis dan efisien digunakan dalam menyerang maupun belaan. Tendangan busur adalah tendangan yang sering dilakukan saat pertandingan karena tingkat efisiensi yang mudah untuk melakukan serangan atau belaan. Para pesilat serinng kali menggunakan tendangan sabit dikarenakan tendangan ini sedikit sulit diprediksi kecepatan tendangannya, walaupun tendangan sabit merupakan tendangan yang paling mudah ditangkap tetapi jika tendangan ini dilakukan dengan teknik yang benar disertai power yang kuat maka tendangan dapat dijadikan ujung tombak untuk melawan. Keefektifan tendangan sabit dalam pertandingan diperkuat dengan pernyataan Kiswanto dalam Maulana & Wijaya (2018) bahwa tendangan sabit adalah tendangan yang paling mudah di lakukan, yaitu membentuk sabit, setengah lingkaran atau seperti huruf “C” pergerakan dilakukan dari pangkal paha kemudian hentakan bagian lutut hingga ujung kaki dan perkenaanya bagian punggung kaki ke bagian dada atau perut lawan. Berdasarkan kutipan tersebut tendangan sabit bisa disebut juga sebagi tendangan “C”, dimana pergerakan tendangan sabit dimulai dari pangkal paha yang dinaikkan membentuk lengkungan atau huruf “C” dengan perkenaan punggung kaki. Bentuk gerakan tendangan sabit yang melengkung dengan perkenaan punggung kaki memerlukan keterampilan atlet untuk dapat melakukan dengan benar sehingga dapat menyerang lawan tanding. Gerakan sabit juga menggunakan liukan badan agar tendangan sabit diarahkan pada bagian perut dan dada lawan. Peningkatan semua aspek kondisi fisik terutama power pada otot tungkai menjadi pusat dari latihan tendangan sabit. 84 \| Halaman Olahraga Nusantara 84 \| Halaman Olahraga Nusantara Pada proses latihan upaya untuk meningkatkan prestasi seorang atlet tidak ada cara lain selain berlatih, dengan begitu setiap atlet harus belatih untuk mencapai tujuan yang maksimal. Tujuan utama latihan adalah untuk mengembangkan keterampilan dan performa atlet, atlet dibimbing oleh pelatih agar terdapat penambahan beban di setiap program latihannya untuk mencapai keterampilan atlet seperti yang diharapkan. Berdasarkan hasil penelitian latihan menggunakan ban karet dapat menjadi salah satu latihan beban yang efektif dalam meningkatkan hasil tendangan sabit pencak silat. Hal ini dikarenakan ban karet memiliki sifat elastisitas dan gaya pegas. Sifat tersebut juga dapat digunakan dalam suatu proses latihan ketahanan. Melalui pemanfaatan gaya tarik-menarik oleh karet dapat memicu otot yang berkontaksi. Menurut Martens dalam Pranata (2019) menyatakan bahwa kecepatan tendangan dapat ditingkatkan menggunakan latihan beban yaitu dengan latihan gaya pegas sifat elastisitas karet ban dalam. Tahanan karet merupakan alat bantu latihan yang dapat digunakan untuk meningkatkan kecepatan tendangan karena latihan tahanan dapat meningkatkan kecepatan. KESIMPULAN Berdasarkan hasil pengujian hipotesis dalam penelitian ini dapat disimpulkan bahwa terdapat pengaruh latihan pembebanan ban karet pada kaki baik kaki kananan maupun kaki kiri pada hasil tendangan sabit. Latihan menggunakan ban karet dapat mempercepat tendangan sabit dengan latihan yang dilakukan dengan cara yang sistematis dan pembebanan yang dipakai dapat menambah kekuatan otot. Gaya pegas dan sifat elastisitas karet ban dalam menjadi beban yang digunakan dalam melatih kecepatan tendangan. Berbagai penelitian menunjukan bahaw latihan merupakan faktor utama dalam meningkatkan kualitas otot dan kekuatan otot merupakan modal untuk mempermudah mempelajari teknik dan mencegah terjadinya cedera dan meperoleh prestasi yang maksimal. Hal ini berfungsi untuk mencapai tujuan untuk 85 \| Halaman Olahraga Nusantara Yustiana Nabila, Marshanda Salsabila Malinda, Yusril Ihza Maulana, Gilang Nuari Panggraita (2021) PENGARUH LATIHAN TENDANGAN MENGGUNAKAN BAN KARET TERHADAP HASIL TENDANGAN SABIT PENCAK SILAT memperbaiki suatu kondisi fisik, kesehatan, kekuatan, prestasi, dalam cabang olahraga khususnya pencak silat UCAPAN TERIMAKASIH Terimakasih kepada LPPM dan civitas akademik UMPP yang telah memberikan dukungan materi serta motivasi sehingga kegiatan penelitian bisa terlaksana tanpa adanya suatu hambatan apapun. Terimakasih kepada KONI Kabupaten Pekalongan yang telah memberi kesempatan pada peneliti dan memfasilitasi tempat penelitian sehingga peneliti dapat mengetahui hasil dari penelitian. 86 \| Halaman Olahraga Nusantara 86 \| Halaman Olahraga Nusantara Yustiana Nabila, Marshanda Salsabila Malinda, Yusril Ihza Maulana, Gilang Nuari Panggraita (2021) PENGARUH LATIHAN TENDANGAN MENGGUNAKAN BAN KARET TERHADAP HASIL TENDANGAN SABIT PENCAK SILAT memperbaiki suatu kondisi fisik, kesehatan, kekuatan, prestasi, dalam cabang olahraga khususnya pencak silat Lubis, J. (2014). Pencak Silat. Jakarta : PT Raja Grafindo Persada Lubis, J. (2014). Pencak Silat. Jakarta : PT Raja Grafindo Persada Manullang, J. G. (2018). Pengaruh Latihan Split Squat Jump Dan Latihan Maegeri Dari Posisi Jongkok Terhadap Kecepatan Tendangan Maegeri Chudan Pada Atlet Karateka Putera Sabuk Hijau Perguruan Wadokai Dojo Ketsu 1 Palembang. Halaman Olahraga Nusantara (Jurnal Ilmu Keolahragaan), 1(2), 133–262. Maulana, A., & Wijaya, M. R. A. (2018). Pengaruh Latihan Terhadap Kecepatan Tendangan Sabit Pesilat Putri Ekstrakurikuler Pencak Silat Smp Negeri 2 Gunung Guruh Kabupaten Sukabumi 2017 / 2018. Jurnal Repository Ummi, 142–147. Pomatahu, A. (2018). Trends in Sport Sciences. Sport in Sport Sciences, 2, 43. Pranata, L. D. (2019). KINESTETIK : Jurnal Ilmiah Pendidikan Jasmani, 3 (1) 2019 ISSN:2477-331X. 3(1). Prasetyo, H. D. (2015). Perbedaan Pengaruh Latihan Shuttle Run Dan Zig-Zag Run Terhadap Peningkatan Keterampilan Dribbling Siswa Ekstrakurikuler Sepakbola Sma Negeri 1 Banguntapan. Thesis. Fakultas Ilmu Keolahragaan:UNY Pratiwi, R. A., Purnomo, E., & Haetami, M. (2013). Pengaruh Latihan Plyometrik Terhadap Kecepatan Tendangan T Pencak Silat. FKIP UNTAN Pontianak, 1– 8. Sugiyono. (2018). Metode Penelitian Kuantitatif, Kualitatif dan R&D Bandung:Alfabeta DAFTAR PUSTAKA Abdurahman, R. M., Simanjuntak, V., & Purnomo, E. (2014). 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https://openalex.org/W2115202267	https://europepmc.org/articles/pmc2572608?pdf=render	English	null	Recurrent airway obstructions in a patient with benign tracheal stenosis and a silicone airway stent: a case report	Cases journal	2,008	cc-by	1,785	Cases Journal Open Access Abstract Airway stents (silicone and metal stents) are used to treat patients with benign tracheal stenosis, who are symptomatic and in whom tracheal surgical reconstruction has failed or is not appropriate. However airway stents are often associated with complications such as migration, granuloma formation and mucous hypersecretion, which cause significant morbidity, especially in patients with benign tracheal stenosis and relatively normal life expectancy. We report a patient who had frequent critical airway obstructions over 8 years due to granuloma and mucus hypersecretion in a silicone airway stent. The problem was resolved when the silicone stent was removed and replaced with a covered self expanding metal stent. Open Case Report Recurrent airway obstructions in a patient with benign tracheal stenosis and a silicone airway stent: a case report KB Sriram* and PC Robinson Address: Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia Email: KB Sriram* - bajeekrishna@gmail.com; PC Robinson - peter.robinson@health.sa.gov.au * Corresponding author Published: 7 October 2008 Cases Journal 2008, 1:226 doi:10.1186/1757-1626-1-226 Received: 3 August 2008 Accepted: 7 October 2008 This article is available from: http://www.casesjournal.com/content/1/1/226 © 2008 Sriram and Robinson; 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. Published: 7 October 2008 Cases Journal 2008, 1:226 doi:10.1186/1757-1626-1-226 Received: 3 August 2008 Accepted: 7 October 2008 This article is available from: http://www.casesjournal.com/content/1/1/226 © 2008 Sriram and Robinson; 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. Received: 3 August 2008 Accepted: 7 October 2008 © 2008 Sriram and Robinson; 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. BioMed Central BioMed Central Discussion h Benign tracheal stenosis is most often caused by pro- longed intubation and/or tracheostomy. 67–90% of patients who are intubated develop laryngotracheal injury and 12–14% develop tracheal stenosis [3]. Symptomatic benign tracheal stenosis is optimally treated by tracheal reconstruction surgery [4]. However, if this procedure is medically contraindicated or has failed previously then prosthetic stents are used to maintain airway patency. Granulation tissue obstructing distal end of silicone tracheal (Dumon®) Dumon stent Figure 2 Granulation tissue obstructing distal end of silicone tracheal (Dumon®) Dumon stent. Granulation tissue obstructing distal end of silicone tracheal (Dumon®) Dumon stent Figure 2 Granulation tissue obstructing distal end of silicone tracheal (Dumon®) Dumon stent. ing in a smaller internal diameter in most cases [7]. Sili- cone stent-specific complications have raised the question of whether metallic stents might overcome some of these difficulties. Silicone stents are relatively inexpensive, generally do not fracture, resist extrinsic compression (e.g. from scar tissue or tumour) and can be easily repositioned or removed if required. However stent complications have been reported to occur in up to 42% of patients [5]. Long-term complications include migration (17%), granuloma (6%), and mucostasis causing airway obstruction (6%) [5]. One study found that 42% of patients require emer- gency bronchoscopy for stent related complications within 3 months of stent insertion [6]. Provocation of local inflammatory reaction results in growth of granula- tion tissue. Laser resection of in-stent granuloma must be performed with caution as there have been reports of flash fire and stent damage [4]. An additional problem with sil- icone stents is that the ratio of wall thickness to internal diameter is higher when compared to metal stents result- There has been increasing enthusiasm for using covered SEMS such as Ultraflex™ stents, which are built from a sin- gle layer of braided knitted flexible nitinol (nickel-tita- nium alloy) with an outer covering of silicone [1]. They have excellent flexibility, good biocompatibility and the self-expanding force is an important factor that enables the nitinol mesh to attach directly to the tracheal wall. While some reports have shown that there are few compli- cations when SEMS are used to treat benign tracheal sten- osis [8,9] others have found complications occurring in 52–87% of patients [3,4]. These include granuloma, mucus retention and stent fracture [3,4]. Background The patient has remained asymptomatic since, with no further episodes of airway obstruction. Background g Benign tracheal stenosis secondary to prolonged intuba- tion and/or tracheostomy has been treated by airway stenting in selected patients1. A 19 year old woman developed Guillain-Barre syndrome and respiratory failure in 1999, whilst living in a different city. Prolonged intubation followed by tracheostomy was complicated by tracheal stenosis. Tracheal reconstruction surgery was performed but was unsuccessful in maintain- ing airway patency. Immediately afterwards a 12 mm × 40 mm Dumon® silicone stent was inserted. However this stent was complicated by frequent obstructions due to granulation tissue and mucous plugs. There are four categories of airway stents – silicone stents, balloon-dilated metal stents, self-expanding metal stents (SEMS) and covered SEMS. There are no randomized con- trolled trials comparing different stents and each category has its merits and limitations [1]. The silicone Dumon® stent (Novatech, Boston Medical, Massachusetts, USA) is widely popular and often used to treat benign tracheal ste- nosis. Complications include stent migration, granuloma formation and mucostatsis [2]. Covered SEMS, such as the Ultraflex™ stent (Boston Scientific, Massachusetts, USA) are more biocompatible and some reports suggest fewer complication rates compared with silicone stents. We report a patient who developed major complications with a Dumon® stent which were ameliorated when it was replaced by an Ultraflex™ stent. Due to work commitments she relocated to our city in March 2006. In July 2006 she presented to the emergency department of our institution with acute dyspnea and stri- dor. Emergency bronchoscopy revealed extensive granula- tion tissue and mucous plugging within the Dumon® stent. Over the next 16 months she had 9 emergency pres- entations with stent obstructions. Each episode was resolved with bronchoscopic piecemeal removal of granu- lation tissue and suctioning of mucus. Page 1 of 3 (page number not for citation purposes) Page 1 of 3 (page number not for citation purposes) Cases Journal 2008, 1:226 http://www.casesjournal.com/content/1/1/226 Granulation tissue obstructing distal end of silicone tracheal (Dumon®) Dumon stent Figure 2 Granulation tissue obstructing distal end of silicone tracheal (Dumon®) Dumon stent. In Nov 2007 she presented with severe respiratory distress and bronchoscopy revealed that the Dumon® stent had moved proximally (Figure 1) with granulation tissue in the distal end (Figure 2) causing 90% obstruction in mid- trachea. The Dumon® stent was removed and replaced with a 14 mm × 60 mm Ultraflex™ stent. The new stent was successful with resolution of tracheal obstruction. Discussion h Such concerns have prompted the Food and Drug Administration to publish a cautionary advisory on the use of metallic stents in patients with benign airway disease [10]. Silicone tracheal (Dumon®) stent has migrated proximally Figure 1 Silicone tracheal (Dumon®) stent has migrated prox- imally. Our report illustrates the complications associated with silicone airway stents when used to treat benign tracheal stenosis. Interestingly while the patient had frequent air- way obstructions with the Dumon® stent, she did not experience these with the Ultraflex™ stent. Careful patient selection and recognition of complications is paramount before considering patients with benign tracheal stenosis for treatment with prosthetic airway stents. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Silicone tracheal (Dumon®) stent has migrated proximally Figure 1 Silicone tracheal (Dumon®) stent has migrated prox- imally. Page 2 of 3 (page number not for citation purposes) Page 2 of 3 (page number not for citation purposes) Cases Journal 2008, 1:226 http://www.casesjournal.com/content/1/1/226 Authors' contributions KBS reviewed the case notes and prepared the manuscript. KBS reviewed the case notes and prepared the manuscript. PCR read and approved the final manuscript. PCR read and approved the final manuscript. Competing interests p g The authors declare that they have no competing interests. References 1. Chin CS, Litle V, Yun J, et al.: Airway Stents. Ann Thorac Surg 2008, 85:S792-6. 2. Martinez-Ballarin JI, Diaz-Jimenez JP, Castro MJ, Mayo JA: Silicone stents in the management of benign tracheobronchial sten- osis: tolerance and early results in 63 patients. Chest 1996, 109:626-629. 3. Eller RL, Livingston WJ, Elliot Morgan C, et al.: Expandable Tra- cheal Stenting for Benign Disease: Worth the complications? Ann Otol Rhinol Laryngol 2006, 115(4):247-252. y g ( ) 4. Gaissert HA, Grillo HC, Wright CD, et al.: Complication of benign tracheobronchial strictures by self-expanding metal stents. J Thorac Cardiovasc Surg 2003, 126:744-7. g 5. Wood DE, Yun-Hen L, Vallieres E, et al.: Airway stenting for malignant and benign tracheobronchial stenosis. Ann Thorac Surg 2003, 76:167-74. g 6. Matsuo T, Colt HG: Evidence against routine scheduling of sur- veillance bronchoscopy after stent insertion. Chest 2000, 118:1455-1459. 7. Noppen M, Stratakos G, D'Haese J, Meysman M, Vinken W: Removal of covered self-expandable metal stents in benign disorders. Chest 2005, 127:482-487. 8. Wu C-Y, Liu Y-H, Hsieh M-J, et al.: Airway stents in management of tracheal stenosis: have we improved? ANZ J Surg 2007, 77:27-32. 9. Sesterhenn AM, Wagner H-J, Alfke H, et al.: Treatment of Benign Tracheal Stenosis Utilizing Self-Expanding Nitinol Stents. Cardiovasc Intervent Radiol 2004, 27:355-360. 10. Food and Drug Administration: FDA public health notification: complications from metallic tracheal stents in patients with benign airway disorders. [http://www.fda.gov/cdrh/safety/ 072905-tracheal.html]. Accessed 27th July 2008. References Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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https://openalex.org/W3033215693	https://archive.lstmed.ac.uk/15421/1/nutrients-12-01963.pdf	English	null	Factors Affecting Vitamin C Status and Prevalence of Deficiency: A Global Health Perspective	Nutrients	2,020	cc-by	13,271	Received: 3 June 2020; Accepted: 26 June 2020; Published: 1 July 2020 Received: 3 June 2020; Accepted: 26 June 2020; Published: 1 July 2020 Abstract: A recent review of global vitamin C status has indicated a high prevalence of deﬁciency, particularly in low- and middle-income countries, as well as in speciﬁc subgroups within high-income countries. Here, we provide a narrative review of potential factors inﬂuencing vitamin C status globally. The in vivo status of vitamin C is primarily aﬀected by dietary intake and supplement use, with those who supplement having a higher mean status and a lower prevalence of deﬁciency. Dietary intake can be inﬂuenced by cultural aspects such as traditional cooking practices and staple foods, with many staple foods, such as grains, contributing negligible vitamin C to the diet. Environmental factors can also aﬀect vitamin C intake and status; these include geographic region, season, and climate, as well as pollution, the latter partly due to enhanced oxidative stress. Demographic factors such as sex, age, and race are known to aﬀect vitamin C status, as do socioeconomic factors such as deprivation, education and social class, and institutionalization. Various health aspects can aﬀect vitamin C status; these include body weight, pregnancy and lactation, genetic variants, smoking, and disease states, including severe infections as well as various noncommunicable diseases such as cardiovascular disease and cancer. Some of these factors have changed over time; therefore, we also explore if vitamin C status has shown temporal changes. Overall, there are numerous factors that can aﬀect vitamin C status to diﬀerent extents in various regions of the world. Many of these factors are not taken into consideration during the setting of global dietary intake recommendations for vitamin C. Keywords: vitamin C; vitamin C status; vitamin C deﬁciency; global health; dietary intake; obesity; smoking; communicable disease; infection; noncommunicable disease Anitra C. Carr 1,* and Sam Rowe 2 Anitra C. Carr 1,* and Sam Rowe 2 1 Nutrition in Medicine Research Group, Department of Pathology & Biomedical Science, University of Otago Christchurch 8011, New Zealand 2 Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool L35QA, UK; sam.rowe@lstmed.ac.uk * Correspondence: anitra.carr@otago.ac.nz; Tel.: +64-3364-0649 * Correspondence: anitra.carr@otago.ac.nz; Tel.: +64-3364-0649 nutrients nutrients Nutrients 2020, 12, 1963; doi:10.3390/nu12071963 Factors Aﬀecting Vitamin C Status and Prevalence of Deﬁciency: A Global Health Perspective Anitra C. Carr 1,* and Sam Rowe 2 1. Introduction Due to random genetic mutations that have occurred during our evolution, humans have lost the ability to synthesize ascorbate in our livers [1]. Therefore, instead of being a normal liver metabolite, as is the case for most other animal species, ascorbate has instead become a vitamin and must be obtained through the diet to prevent hypovitaminosis C and outright deﬁciency [2]. In a recent review, we described the global status of vitamin C and the prevalence of deﬁciency as assessed by plasma concentrations [3]. Whilst vitamin C concentrations are normally tightly controlled in humans with adequate intake, a number of studies suggest that hypovitaminosis C and deﬁciency are not uncommon worldwide and may be very common in some settings. Vitamin C is well absorbed in small quantities. Uptake occurs primarily through the sodium-dependent vitamin C transporter-1 (SVCT-1), which is rapidly saturated, allowing relatively limited absorption of vitamin C per serving [4]. Vitamin C is then accumulated at varying concentrations in diﬀerent body tissues [5]. The vitamin is excreted unchanged Nutrients 2020, 12, 1963; doi:10.3390/nu12071963 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 19 Nutrients 2020, 12, 1963 in the urine, but reuptake occurs in the renal tubules [6]. Further loss of vitamin C occurs through the oxidation of ascorbic acid to dehydroascorbic acid which may then be recycled to ascorbic acid or undergo further oxidative decomposition [7]. Thus, in vivo, vitamin C concentrations are aﬀected by a range of factors that include dietary intake, absorption, distribution, oxidative decomposition, and elimination. Vitamin C acts as an important antioxidant and plays a myriad of functions in optimal health and prevention of disease [8,9]. Even mild insuﬃciency or hypovitaminosis C can be associated with symptoms such as low mood [10]. More severe deﬁciency can be associated with a range of clinical presentations [11]. Prolonged severe deﬁciency results in the clinical syndrome of scurvy, a condition that continues to be diagnosed in individuals and occasionally in public health outbreaks to this day [12]; if left untreated, scurvy is a fatal disease. The optimum intake and concentrations of vitamin C, however, have been subject to debate and recommended values are not universal [13]. Due to a range of beneﬁts, a number of international authorities have increased their dietary recommendations for vitamin C from those previously recommended on the basis of preventing scurvy [13]. 1. Introduction Although these recommendations generally take into account variations in requirements based on age, sex, pregnancy and lactation, and sometimes smoking status, there are many other factors that aﬀect vitamin C status that are not taken into consideration by these global authorities. In this review, we highlight the numerous factors that impact on vitamin C status and prevalence of deﬁciency globally. 2.1. Dietary Intake Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). e e s ses c e ey e s, c c pe s, g e s, p o e s, soy e s, e s, pe s, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, ﬂax seeds, pumpkin seeds, sunﬂower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). Suboptimal diet is an important preventable risk factor for noncommunicable diseases, and a low intake of fruit was found to be the third leading dietary risk for deaths (two million deaths) and disability-adjusted life-years (65 million DALYs) globally [31]. The World Health Organization recommends at least 400 g (i.e., five portions) of fruit and vegetables per day, excluding potatoes, sweet potatoes, cassava, and other starchy roots [32]. Historically, scurvy has been reported in refugee camps in Ethiopia; the relief food distributed to the refugees was almost completely deficient in vitamin C, and the environment where the camps were located precluded an adequate supply of fresh food [33]. However, vitamin C deficiency may even be common in fertile areas of Africa, such as Uganda [34]. There have been a number of outbreaks of clinical scurvy in recent years, including Suboptimal diet is an important preventable risk factor for noncommunicable diseases, and a low intake of fruit was found to be the third leading dietary risk for deaths (two million deaths) and disability-adjusted life-years (65 million DALYs) globally [31]. The World Health Organization recommends at least 400 g (i.e., ﬁve portions) of fruit and vegetables per day, excluding potatoes, sweet potatoes, cassava, and other starchy roots [32]. Historically, scurvy has been reported in refugee camps in Ethiopia; the relief food distributed to the refugees was almost completely deﬁcient in vitamin C, and the environment where the camps were located precluded an adequate supply of fresh food [33]. However, vitamin C deﬁciency may even be common in fertile areas of Africa, such as Uganda [34]. 2.1. Dietary Intake Dietary intake of vitamin C is a key determinant of body status, with the amount consumed and the frequency of consumption correlating with plasma status and prevalence of deﬁciency (Table 1) [14,15]. Fresh fruit and vegetables are the major dietary source of vitamin C, with fruit intake correlating more strongly with plasma vitamin C status [16,17]. Foods rich in vitamin C include oranges and other citrus fruit, guavas, kiwifruit, cranberries, strawberries, papayas, mangoes, melons, cantaloupe, spinach, Swiss chard, tomatoes, asparagus, and Brussels sprouts (Figure 1) [18]. In contrast, grains (e.g., rice, millet, wheat/couscous, corn), some starchy roots and tubers, meat (other than liver), eggs, and dairy contain very little vitamin C. Table 1. Dietary factors determining vitamin C status. Factor Summary and Comments References Dietary intake Dietary intake, particularly fruit intake, correlates with improved vitamin C status and decreased prevalence of deﬁciency; is dependent on the amount consumed, frequency of consumption, and type of food consumed as the vitamin C content of food varies. High dietary fat and sugar intake are associated with decreased vitamin C intake and status. [14–17,19–24] Staple foods Staple foods such as grains (e.g., rice, millet, wheat/couscous, corn) and some starchy roots and tubers are low in vitamin C; populations who consume these staples can have lower overall vitamin C intake. [18,25] Traditional cooking practices Through boiling or steaming, water-soluble vitamins may be leached from food and prolonged cooking of food can destroy vitamin C; this could lead to decreased vitamin C status in certain social or ethnic groups. Drying of leafy vegetables also decreases water-soluble vitamins. [26,27] Supplement use Supplement users have signiﬁcantly higher vitamin C status and negligible prevalence of deﬁciency. Non-users have a 2–3 fold odds ratio of insuﬃcient and deﬁcient vitamin C status. [14,15,19,28–30] Table 1. Dietary factors determining vitamin C status. 3 of 19 of 19 Nutrients 2020, 12, 1963 Nutrients 2020 12 x utrients 2020, 12, 1963 3 of Nutrients 2020, 12, x FOR PEER REVIEW 3 of 19 Figure 1. Estimated vitamin C content of selected fruit and vegetables. Data derived from the United States Department of Agriculture (https://fdc.nal.usda.gov/). Note that vitamin C content can vary depending on the plant variety, and cooking may decrease the vitamin C content to variable extents. 2.1. Dietary Intake Pulses include kidney beans, chickpeas, mung beans, pinto beans, soybeans, lentils, peanuts, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, flax seeds, pumpkin seeds, sunflower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). Figure 1. Estimated vitamin C content of selected fruit and vegetables. Data derived from the United States Department of Agriculture (https://fdc.nal.usda.gov/). Note that vitamin C content can vary depending on the plant variety, and cooking may decrease the vitamin C content to variable extents. Pulses include kidney beans, chickpeas, mung beans, pinto beans, soybeans, lentils, peanuts, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, ﬂax seeds, pumpkin seeds, sunﬂower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). d f l d f d bl d d f h Figure 1 Estimated vitamin C content of selected fruit and vegetables Data derived from the United Figure 1. Estimated vitamin C content of selected fruit and vegetables. Data derived from the Figure 1. Estimated vitamin C content of selected fruit and vegetables. Data derived from the United States Department of Agriculture (https://fdc.nal.usda.gov/). Note that vitamin C content can vary depending on the plant variety, and cooking may decrease the vitamin C content to variable extents. Pulses include kidney beans, chickpeas, mung beans, pinto beans, soybeans, lentils, peanuts, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, flax seeds, pumpkin seeds, sunflower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). Figure 1. Estimated vitamin C content of selected fruit and vegetables. Data derived from the United States Department of Agriculture (https://fdc.nal.usda.gov/). 2.1. Dietary Intake Note that vitamin C content can vary depending on the plant variety, and cooking may decrease the vitamin C content to variable extents. Pulses include kidney beans, chickpeas, mung beans, pinto beans, soybeans, lentils, peanuts, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, ﬂax seeds, pumpkin seeds, sunﬂower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). Figure 1. Estimated vitamin C content of selected fruit and vegetables. Data derived from the United States Department of Agriculture (https://fdc.nal.usda.gov/). Note that vitamin C content can vary depending on the plant variety, and cooking may decrease the vitamin C content to variable extents. Pulses include kidney beans, chickpeas, mung beans, pinto beans, soybeans, lentils, peanuts, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, flax seeds, pumpkin seeds, sunflower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). Figure 1. Estimated vitamin C content of selected fruit and vegetables. Data derived from the United States Department of Agriculture (https://fdc.nal.usda.gov/). Note that vitamin C content can vary depending on the plant variety, and cooking may decrease the vitamin C content to variable extents. Pulses include kidney beans, chickpeas, mung beans, pinto beans, soybeans, lentils, peanuts, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, ﬂax seeds, pumpkin seeds, sunﬂower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. Animal products, such as meat (other than liver), eggs, and milk contain negligible vitamin C. Dotted lines: lower line indicates daily intake to prevent scurvy (10 mg/d); upper line indicates daily intake for optimal health (200 mg/d). Pulses include kidney beans, chickpeas, mung beans, pinto beans, soybeans, lentils, peanuts, split peas; nuts include hazelnuts, pistachios, macadamia nuts, pecans, walnuts, brazil nuts, cashew nuts; seeds include chia, flax seeds, pumpkin seeds, sunflower seeds, sesame seeds; grains include rice, millet, wheat/couscous, cornmeal. 2.2. Cultural Aspects: Staple Foods and Traditional Cooking Practices 2.2. Cultural Aspects: Staple Foods and Traditional Cooking Practices 2.2. Cultural Aspects: Staple Foods and Traditional Cooking Practices Food consumption is inﬂuenced by a number of factors, such as geographical, economic, social, and cultural [25]. It is well known that economic factors, such as poverty, can limit the consumption of healthy foods. Furthermore, tradition and social customs may inﬂuence the consumption of a variety of foods. For example, a study in Nepal highlighted cultural beliefs around menstruation, pregnancy, and lactation that decreased micronutrient intake and intrahousehold disfavouritism towards women in the distribution of micronutrient-rich foods [27]. In low- and middle-income countries (LMICs), geographic considerations and agricultural practices inﬂuence the production and consumption of staple foods, which can greatly inﬂuence vitamin C intake. For example, countries whose populations consume grains such as rice and millet as staple foods (e.g., in Asia and parts of Africa) tend to have lower intakes of vitamin C [18,25]. In contrast, some areas of Africa and Latin America where yams and sweet potato are staple foods, vitamin C intakes may be higher. However, the vitamin C content of food can also vary depending on the time of harvest, transportation and storage conditions, and food preparation conditions. For example, the vitamin C content of some staple foods, e.g., cassava, is signiﬁcantly depleted by diﬀerent processing methods, which could lead to inaccuracies in estimated vitamin C intake [38]. Vitamin C is heat-labile and can be destroyed by cooking; the lower vitamin C status of Indians and Malays living in Singapore is thought to be partly due to its destruction by more prolonged cooking [26]. 2.1. Dietary Intake There have been a number of outbreaks of clinical scurvy in recent years, including in tribesmen in Kenya and during the Afghanistan winter [35,36], and cases of clinical scurvy continue to be diagnosed related to poor intake even in high-income countries [12,37]. Nutrients 2020, 12, 1963 4 of 19 2.3. Supplement Use An optimal vitamin C intake (i.e., 200 mg/d) can be obtained from ﬁve-plus daily servings of fresh fruit and vegetables, providing that at least one or two servings are high vitamin C foods, however, this is not always possible for many people around the world. Therefore, taking supplements in addition to dietary intake can help to maintain optimal vitamin C status. There are many diﬀerent forms of vitamin C supplements; however, research indicates that these generally have the same bioavailability as food-derived vitamin C [39]. Numerous studies have found that even in developed countries, where there is no shortage of fresh fruit and vegetables, those who consume supplements have signiﬁcantly higher vitamin C status and/or lower prevalence of deﬁciency (Table 2). Two large health surveys carried out in Canada (CHMS) and the USA (NHANES IV) reported 20% supplement use in Canada and 37 to 47% supplement use, for men and women, respectively, in the USA [14,15]. It should be noted, however, that in these surveys, supplement use was considered to be as little as once in the previous month. Nevertheless, both studies found vitamin C status was at least 20 µmol/L higher in supplement users, with a low prevalence of deﬁciency (0–2%) in those who supplemented [14,15]. Two studies in the UK indicated that people who did not supplement had a two-fold odds ratio of having a vitamin C status of <28 µmol/L and a three-fold odds ratio of their vitamin C status being <11 µmol/L, relative to concentrations >28 µmol/L [19,28]. Table 2. Eﬀect of socioeconomic factors on vitamin C status. Factor Summary and Comments References Socioeconomic status/deprivation Individuals with lower socioeconomic status or higher deprivation have lower vitamin C status and a higher prevalence of deﬁciency; this is partly due to the higher cost of good quality, nutrient-dense food. [15,19,21,24,28,40–45] Education and social class Similarly, individuals with lower education and manual occupations have lower vitamin C status. [14,17,20,28,42] Institutionalized Institutionalized elderly, and other institutionalized individuals (e.g., priests, prisoners, boarding school children) have lower vitamin C status and a higher prevalence of deﬁciency; this is partly due to a lower dietary intake. [41,46–48] Table 2. Eﬀect of socioeconomic factors on vitamin C status. 2.3. Supplement Use Factor Summary and Comments References Socioeconomic status/deprivation Individuals with lower socioeconomic status or higher deprivation have lower vitamin C status and a higher prevalence of deﬁciency; this is partly due to the higher cost of good quality, nutrient-dense food. [15,19,21,24,28,40–45] Education and social class Similarly, individuals with lower education and manual occupations have lower vitamin C status. [14,17,20,28,42] Institutionalized Institutionalized elderly, and other institutionalized individuals (e.g., priests, prisoners, boarding school children) have lower vitamin C status and a higher prevalence of deﬁciency; this is partly due to a lower dietary intake. [41,46–48] Table 2. Eﬀect of socioeconomic factors on vitamin C status. 5 of 19 Nutrients 2020, 12, 1963 3.1. Geographic Region Geographic diﬀerences in vitamin C status have been reported (Table 3); there are likely many factors that contribute to the observed diﬀerences. For example, signiﬁcant diﬀerences in vitamin C status and prevalence of deﬁciency were observed between Finland and neighboring Russia (where strikingly low plasma concentrations were observed) [17]. Geographical diﬀerences in the consumption of fresh fruit and vegetables were apparent, which were associated with deﬁciency. Low educational status was also noted to be associated with deﬁciency on the Russian side of the border [17]. Another study carried out in ﬁve countries across Europe indicated a diﬀerence in the vitamin C status of women, with the biggest diﬀerences being observed between Northern Ireland and the Republic of Ireland [49]. No diﬀerences in vitamin C status were observed for men from these diﬀerent countries, whilst women had higher vitamin C status than the men in three of the ﬁve countries. Mosdol et al. [19] reported that low-income participants living in Scotland and Northern Island had a lower prevalence of deﬁciency than those living in England, whilst those living in Wales had a higher prevalence of deﬁciency. As part of the SU.VI.MAX trial, France was divided into seven regions, and vitamin C status was found to be signiﬁcantly lower in the Northern region [50]. The reasons for these regional diﬀerences are unknown. In a large study of elderly people in India, a higher prevalence of deﬁciency was observed in the north (74% deﬁciency) compared with the south (46% deﬁciency) [24]. Similarly, in Mexico, a higher prevalence of deﬁciency was observed in children in the north and south regions (~26% deﬁciency) compared with those living in Mexico City (12% deﬁciency) [40]. This could reﬂect diﬀerences in the socioeconomic status between urban and rural environments. Table 3. Environmental factors aﬀecting vitamin C status. Factor Summary and Comments References Geographical region Vitamin C status varies by geographical region, both within and between countries; this could partly reﬂect diﬀerences in socioeconomic status and available foods. [17,19,24,40,41,49–51] Season Vitamin C status varies seasonally between countries, likely reﬂecting diﬀerent crops and thus the types and/or amounts of vitamin C-rich foods consumed. [24,30,52,53] Climate Drought and harsh winter climates have been associated with outbreaks of clinical scurvy. [36] Pollution Exposure to environmental pollutants, e.g., smoke, can deplete vitamin C status; this is partly due to enhanced oxidative stress. [24,42,54–58] d l Table 3. Environmental factors aﬀecting vitamin C status. 3.3. Pollution Currently, over half of the world’s population lives in urban areas, and WHO data indicate that more than 80% of people living in urban areas are exposed to air quality levels that exceed WHO guideline limits, with LMICs suﬀering from the highest exposures [60,61]. Air pollution causes an estimated seven million premature deaths worldwide every year, primarily resulting from increased mortality from strokes, heart disease, chronic obstructive pulmonary disease, lung cancer, and acute respiratory infections [61]. Environmental air pollution, such as smoke derived from burning biomass, can comprise reactive species that potentially aﬀect in vivo antioxidant status [24]. Another example is environmental tobacco smoke, which is an underestimated pollutant in many parts of the world. Exposure to environmental tobacco smoke is associated with depleted vitamin C status in both nonsmoking adults and children [42,54–58]. Tribble et al. [54] found that the vitamin C status of passive smokers was signiﬁcantly lower than that of nonexposed nonsmokers, despite comparable dietary intakes of the vitamin. Hypovitaminosis C was observed in 12% of passive smokers, but not in nonexposed nonsmokers. Analysis of the data of children from the NHANES III and IV surveys revealed a dose–response relationship between levels of tobacco exposure and serum vitamin C concentrations [55,56]. Not all studies in adults, however, have shown eﬀects of passive smoking on vitamin C status [62–65]. Nevertheless, supplementation with vitamin C and other antioxidants was found to decrease oxidative biomarkers in participants exposed to environmental tobacco smoke [62,66]. 3.2. Season and Climate Since fresh fruit and vegetables are the major source of vitamin C for most people, it is perhaps not surprising that there have been reports of seasonal diﬀerences in vitamin C status (Table 3). Surprisingly, however, studies carried out in England and China have indicated that vitamin C status tends to be highest in winter and lowest in autumn, with up to 10 µmol/L diﬀerence between these seasons [30,52]. Another study in China indicated a much higher vitamin C status in winter relative to spring [53]. This likely reﬂects the types or amounts of vitamin C-rich foods being consumed in winter. Similar trends were observed in northern India, with less deﬁciency being observed in the winter months; however, in southern India, the winter months were associated with a higher prevalence of deﬁciency [24]. This likely reﬂects the diﬀerent climatic and agricultural patterns across the subcontinent. It is possible that seasonal variances in plasma vitamin C concentrations are aﬀected by baseline vitamin C status, i.e., whether the individual is already saturated or not. Bates et al. [59] reported seasonal variations in participants with higher intakes and blood status at baseline compared with little variation in those with low intakes and status, the latter likely due to a depleted body pool 6 of 19 Nutrients 2020, 12, 1963 and preferential uptake of the vitamin by tissues. Changes in climate, such as drought, are also likely to impact on the vitamin C status of the populations reliant on local foods in the aﬀected region [36]. This also leads to increased reliance on staple crops like cassava that lose their vitamin content prior to consumption [38]. 4.3. Race A number of studies have indicated that vitamin C status varies by race (Table 4; reviewed in [74]). The prevalence of vitamin C deﬁciency appeared to be highest amongst South Asians and was thought to be partly due to traditional cooking practices. It was also suggested that the high proportion of individuals with low vitamin C concentrations in South Asian populations might contribute to their higher rates of cardiovascular disease. Only a few studies have assessed diﬀerent ethnic groups within the same study [15,26,30,73]. In 1980, Koh et al. [73] assessed vitamin C status in a cohort of black and white participants in Mississippi, USA. Black males and females had signiﬁcantly lower vitamin C status than white males and females (up to 10 µmol/L diﬀerence for females). The more recent US NHANES IV survey also found signiﬁcantly lower vitamin C status in black females compared with white females [15]. In London, the vitamin C status of black and South Asians was signiﬁcantly lower than white participants (up to 15 µmol/L lower for South Asian females) [30]. Similarly, in Singapore, Malays and Asian Indians were found to have signiﬁcantly lower vitamin C status than Chinese (up to 12 µmol/L for females) [26]. Studies in Uganda, South Africa, and Nigeria all showed high rates of deﬁciency in predominately black Africans [3]. 4.2. Age Some studies carried out in people aged >60 years (e.g., the French POLA study and the British National Diet and Nutrition Survey) have shown lower vitamin C status than other studies carried out in the same countries with younger age groups (e.g., the French SU.VI.MAX study and the European EPIC-Norfolk study); however, it is not ideal to compare values between diﬀerent studies due to potential methodological diﬀerences. A number of studies have indicated that older age within the same study population is associated with an increased prevalence of vitamin C deﬁciency, particularly in men, and older men also tend to have a lower vitamin C intake than older women [20,24,28,71]. However, a counterpoint to this is that overall vitamin C status can be higher in older people [15,29,50], although not in all cases, as was found in men in Finland who had lower vitamin C status with older age [72]. Schleicher et al. found a U-shaped curve for vitamin C status over the age range of 6 to 60+ years [15]. It is possible that the lower body mass of children and elderly, due to age-related frailty, could contribute to their higher vitamin C status. However, there are, clearly, subpopulations within the aging population who have increased deﬁciency, likely due to lower intake and/or comorbidities. 4.1. Sex 4.1. Sex In high-income settings, females appear to have higher vitamin C status and a lower prevalence of deﬁciency than males [3]. According to McCall et al. [28], UK males have a four-fold odds ratio of deﬁciency compared with females (Table 4). The diﬀerence in vitamin C status between males and females is thought to be partly a result of a volumetric dilution eﬀect due to the higher fat-free mass of males [29]. There are also diﬀerences in dietary intakes between men and women, with women generally having comparable or higher intakes than men in high-income countries, although this diﬀerence is less apparent in some low-income settings [3]. It should also be noted that pregnancy and lactation typically lower women’s vitamin C status due to hemodilution and the needs of the developing fetus and growing infant (see below). Many countries have higher dietary recommendations for men (to take into account the diﬀerences in body mass) and for pregnant and lactating women [13]. Table 4. Eﬀect of demographic factors on vitamin C status. Factor Summary and Comments References Sex Males generally have lower vitamin C status, and a higher prevalence of deﬁciency, than females; this is partly a result of a volumetric dilution eﬀect due to the higher fat-free mass of males. This diﬀerence is less apparent in some low- and middle-income countries. [14,15,17,19,22,24,26,28–30,42,43, 49,50,53,67–70] Age Both children and elderly tend to have higher vitamin C status in high-income settings; this could partly be due to lower body weight. Elderly can have a higher prevalence of vitamin C deﬁciency in some settings; this could be due to lower intake and/or comorbidities. [14,15,20,24,28,29,41,50,71,72] Race In the US and UK, African-Caribbean and South Asian people had a lower status than Caucasians. In South Asia, Malays and Indians had a lower status than Chinese; this is thought to be partly due to diﬀerences in culinary practices. Diﬀerences are more apparent between women of diﬀerent races. [15,26,30,73,74] Table 4. Eﬀect of demographic factors on vitamin C status. 7 of 19 Nutrients 2020, 12, 1963 7 of 19 5.1. Socioeconomic Status/Deprivation Socioeconomic status aﬀects diet quality as foods of higher quality and higher nutritional value generally cost more [75]. Therefore, it is not surprising that numerous studies have shown that lower socioeconomic status and deprivation negatively impact on vitamin C status (Table 2). In a UK study, McCall et al. [28] reported that those who were most deprived (based on the Townsend deprivation index) had a two-fold odds ratio of vitamin C deﬁciency relative to those with plasma values above 28 µmol/L. This ﬁnding was supported by Mosdol et al. [19], who speciﬁcally focused on the low-income population within the UK, and showed a higher prevalence of deﬁciency and insuﬃciency than has been reported for the general UK population. Bates et al. [41] reported a three-fold diﬀerence in plasma vitamin C status for people of low socioeconomic status in the UK relative to those of higher status. A two-fold diﬀerence in vitamin C intake between the two socioeconomic groups likely contributed to the observed diﬀerences in vitamin C status. Other more recent surveys have conﬁrmed the association of low vitamin C status with low socioeconomic status [15,43]. The British National Diet and Nutrition Survey indicated a 2 µmol/L increase in vitamin C status in adults for every additional £10,000 of income [44]. Nutrients 2020, 12, 1963 8 of 19 5.2. Education and Social Class Similar ﬁndings have been reported for the level of education, with the lowest level of education associated with the lowest vitamin C status [14,17]. McCall et al. [28] reported that those with the lowest level of education in the UK had a 4.5-fold odds ratio of deﬁciency relative to a vitamin C status of >28 µmol/L. The same authors investigated social class and found that those having manual occupations had a three-fold odds ratio of deﬁciency compared with those having nonmanual occupations [28]. Similar ﬁndings were reported by Wreiden et al. [20], with two- to three-fold higher prevalence of vitamin C deﬁciency for females and males, respectively, with manual occupations. 5.3. Institutionalization One survey of elderly people assessed institutionalized individuals relative to free-living individuals [41]. The authors found signiﬁcantly lower vitamin C status (25 vs. 44 µmol/L) and a higher prevalence of deﬁciency (40% vs. 14%) in institutionalized elderly compared with free-living elderly, respectively. Much of this is likely due to a higher proportion of individuals consuming less than the recommended nutrient intake for vitamin C in institutional settings [41]. This will become a growing concern with an increasingly aging population. Other institutionalized individuals, e.g., priests, prisoners, and boarding school children, have also been found to have lower vitamin C status, and higher hypovitaminosis C and severe deﬁciency, compared to control groups [46–48]. 6.1. Body Weight and Body Mass Index (BMI) Bodyweight and the related BMI, waist circumference, or waist-to-hip ratio are well known to have a signiﬁcant association with vitamin C status (Table 5). Numerous studies have shown an inverse association between body weight or BMI and vitamin C status, with obese individuals having the lowest vitamin C status [14–16,29,30,67,76]. Schleicher et al. [15] reported up to a 15 µmol/L diﬀerence in the vitamin C status of obese women compared with women of healthy weight (i.e., 45 versus 60 µmol/L, respectively). Pearson et al. [43] found that individuals with hypovitaminosis C (i.e., <23 µmol/L) had signiﬁcantly higher weight, BMI, and waist circumference. Lower vitamin C status in those of the highest weight could be partly due to lower dietary intake of the vitamin [76,77]. In support of this, lower vitamin C status was found in those with the highest fat intake [23]. It has also been suggested that the observed diﬀerences in vitamin C status could be a result of volumetric dilution due to diﬀerences in fat-free mass [29]. This premise is supported by supplementation studies that have indicated that individuals with higher body weight do not replete as readily as those of normal body weight [78,79]. Based on these observations, Block et al. [78] proposed that recommended vitamin C intakes should be based on a dose per kg body weight or in terms of an ideal plasma concentration. This is a prudent recommendation considering the ongoing increase in body weight globally, with the prevalence of obesity exceeding 50% in some countries [80,81]. Obesity is a risk factor for numerous diseases, particularly cardiometabolic diseases, such as diabetes and cardiovascular disease, which are also associated with lower vitamin C status and a higher prevalence of deﬁciency [9]. Enhanced dietary fat and sugar, which are risk factors for cardiometabolic diseases, are also associated with decreased vitamin C intake and status [22,23]. Nutrients 2020, 12, 1963 9 of 19 Table 5. Health aspects that aﬀect vitamin C status. Table 5. Health aspects that aﬀect vitamin C status. Factor Summary and Comments References Bodyweight, BMI Individuals with higher body weight or BMI have lower vitamin C status; this is likely partly due to a volumetric dilution eﬀect. [14–16,24,29,30,43,67,76,77,82,83] Physical activity Physical activity level positively correlates with vitamin C status, with inactive individuals having a 3-fold odds ratio of deﬁciency; this is likely partly due to associated lifestyle factors such as diet and body weight. 6.2. Pregnancy and Lactation Pregnant women typically have lower vitamin C status compared with nonpregnant women [34]. This is most likely due to hemodilution, as well as active transfer of the vitamin to the developing fetus [95]. Women with complications of pregnancy can have even lower vitamin C status [34,96]. Supplementation of pregnant women with vitamin C can potentially decrease the risk of some pregnancy-related complications [97]. Studies in high-income countries, which included women with normal vitamin C status, have failed to show beneﬁt from supplementation [98]. However, studies in low-income settings with high rates of vitamin deﬁciency show potential beneﬁts, including decreased rates of low birth weight, decreased hospital admissions, and possible decreased rates of pre-eclampsia [99]. It is noteworthy that the recently discovered epigenetic regulatory activities of vitamin C could have important roles to play in fetal development [100]. An animal model has indicated that maternal vitamin C can regulate the reprogramming of DNA methylation and germline development [101]. It is likely that lactating women also have lower vitamin C status due to the transfer of vitamin C to the growing infant via breastmilk. Many global authorities have taken the enhanced requirements of pregnant and lactating women into consideration, with recommendations above their standard dietary recommendations of +10–20 mg/d for pregnant women and +20–60 mg/d for lactating women [13]. 6.1. Body Weight and Body Mass Index (BMI) [28,29] Pregnancy and lactation Pregnancy is associated with lower vitamin C status; this is partly due to hemodilution and active transfer of vitamin C to the developing fetus and growing infant via breastmilk. [34] Genetic variants Polymorphisms in the genes for the vitamin C transporter (SVCT1) and haptoglobin (Hp2-2) are associated with lower vitamin C status; the latter is thought to be due to enhanced oxidative stress. [6,45,84–87] Smoking Smokers have lower vitamin C status and a higher prevalence of deﬁciency than nonsmokers; this is partly due to enhanced oxidative stress. [14–16,19,20,24,28–30,43,45,50,53, 54,67,71,88–91] Disease states Various communicable and noncommunicable diseases are associated with lower vitamin C status; this is partly due to inﬂammatory processes and enhanced oxidative stress. [9,92–94] 6.4. Smoking Smoking is a well-known source of oxidants and in vivo oxidative stress [108]. Multiple studies have shown depleted vitamin C status and a higher prevalence of deﬁciency in smokers compared with nonsmokers (Table 5). McCall et al. [28] reported that current smokers had over a seven-fold odds ratio of deﬁciency compared with nonsmokers, and Wrieden et al. [20] showed two- to three-fold more deﬁciency in male and female smokers, respectively. Smokers tended to have dietary intakes that were lower in vitamin C, e.g., lower fruit and vegetable intake and higher fat intake [88,89,109,110]. However, when diﬀerences in dietary intake were taken into account, smokers still exhibited lower vitamin C status and higher requirements than nonsmokers [88,89,111,112]. Kallner et al. [113] demonstrated over a 40% increase in vitamin C turnover in smokers compared with nonsmokers. A few international regulatory authorities have taken the enhanced requirements of smokers into consideration with additional intake recommendations of 20 to 80 mg/d above their normal adult recommendations [13]. However, it is likely that these additional intakes are insuﬃcient to compensate for the enhanced requirements of smokers [90]. Furthermore, some countries continue to show an increasing trend in smoking rates, which could potentially impact their population’s vitamin C status and requirements [114]. Of note, smoking cessation results in an increase in vitamin C status [115]. 6.3. Genetic Variants Vitamin C status can potentially be inﬂuenced by genetic variants. The SLC23A1 gene encodes the sodium-dependent vitamin C transporter-1 (SVCT1), which is responsible for active uptake of dietary vitamin C through the intestinal epithelium and reuptake of ﬁltered vitamin C via the kidney tubules and is essential in vitamin C-requiring animals [4,6]. A number of single nucleotide variants have 10 of 19 Nutrients 2020, 12, 1963 been identiﬁed in the SLC23A1 gene, and modeling of in vitro data for four of these variants indicates 40% to 75% decreases in vitamin C uptake [6,102]. These variants are relatively common in those of African descent (6–16%), and the variant with the largest decrease in vitamin C transport has a high prevalence in African Americans [6]. Meta-analysis has indicated a strong association of another of these variants with decreased vitamin C status in ﬁve cohorts in the UK (frequency of ~3%) [45]. In the British Women’s Heart and Health Study, a further two variants, with relatively common frequencies of ~30%, were also found to be associated with vitamin C status [45]. Cahill et al. [84] reported that variants of SVCT1 can also modify the strength of the correlation between dietary vitamin C and serum vitamin C. Vitamin C status could be further inﬂuenced by genetic variants aﬀecting the metabolism of the vitamin [103]. A common variant of the hemoglobin-binding protein haptoglobin (Hp2-2) has a decreased ability to bind hemoglobin and results in increased oxidation of vitamin C in vitro [85]. Several studies have shown that this variant is associated with lower circulating vitamin C concentrations [85–87]. The Hp2-2 variant appeared to have a greater eﬀect on individuals with dietary vitamin C intakes <90 mg/day [84]. It is noteworthy that genetic variants can show marked geographical diﬀerences; e.g., Hp2-2 is present in ~36% Northwest Europeans, 51% Iranians, 55% Thais and Chinese, and 84% Indians (where it is thought to have originated) [104]. Other genetic variants, such as those of the detoxifying enzyme glutathione S-transferase, may also have associations with vitamin C status, particularly in individuals with lower intakes, thereby resulting in an increased risk of deﬁciency [105]. High-dose vitamin supplementation has been shown to ameliorate certain gene variant defects [106] and is hypothesized to occur with vitamin C-related variants [107]. Therefore, individuals with genetic variants aﬀecting vitamin C status may require higher dietary intakes. 7. Has Vitamin C Status Changed Over Time? Various factors have changed over time that could potentially aﬀect vitamin C status. These include population demographics and health status, e.g., obesity and smoking rates. Public health policies, such as recommended dietary intakes for vitamin C, have changed in many countries [13]. Therefore, have these temporal changes been reﬂected in changing vitamin C status over time? There have been relatively few studies that have assessed vitamin C status in the same populations or regions over time. The ﬁndings can also be complicated by changes in analytical methodologies over time. Two surveys have been carried out 19 years apart on elderly people in Britain: one in 1979 by the Department of Health and Social Security, and the National Diet and Nutrition Survey in 1998 [41]. There was an increase in vitamin C status from 29 to 44 µmol/L between the two surveys. However, it should be noted that participants in the ﬁrst survey were older and analytical methodologies may have varied between the two surveys. In contrast, the more recent National Diet and Nutrition Surveys (2008–2017) have shown a signiﬁcant 6% increase in vitamin C deﬁciency over the nine-year period in women aged 19–64 [44]. Schleicher et al. [15] carried out an indepth comparison of US vitamin C status in NHANES IV (2003–2004) relative to NHANES III that was undertaken 10–15 years earlier (i.e., 1988–1994) [124]. For those aged ≥20 years, overall vitamin C deﬁciency decreased by about 44% (from 15% to 8%) between the two surveys. Of note, there was an increase in the recommended dietary intake for vitamin C in the US in 2000, from 60 to 90 mg/d for men and to 75 mg/d for women [125]. However, it is unlikely that this increase in dietary recommendations fully accounts for the decrease in vitamin C deﬁciency over time as there were also some diﬀerences in methodology between the two surveys, with the more recent survey likely being more accurate [15]. Looking back even earlier to the NHANES II survey (carried out between 1976 and 1980), vitamin C status was reported to be higher for both men and women (i.e., ≥50 µmol/L for men and ≥60 µmol/L for women), with 5% deﬁciency reported, despite lower dietary intakes compared to the later NHANES III survey [126]. 6.5. Disease States Vitamin C status can be depleted by various disease states due to inﬂammatory processes and enhanced oxidative stress (reviewed in [9,92,93]). A number of studies of hospitalized patients showed a high prevalence of depleted plasma vitamin C status, and concentrations were inversely correlated with inﬂammatory markers [116–119]. A wide range of medical conditions are associated with vitamin C insuﬃciency; these include noncommunicable diseases such as cardiovascular diseases (e.g., strokes, coronary artery disease and hypertension), congestive heart failure, malignancy, chronic inﬂammatory states (e.g., rheumatoid arthritis), metabolic disorders (e.g., diabetes), and cataracts [9,92,93]. Acute infectious diseases leading to enhanced inﬂammation are also associated with depleted plasma vitamin C concentrations in plasma and immune cells, as are a range of chronic infections such as HIV, 11 of 19 Nutrients 2020, 12, 1963 Helicobacter pylori, and tuberculosis, which are prevalent in many LMICs [94,120]. It should also be noted that requirements for vitamin C during infections increase with the severity of the infection, requiring signiﬁcantly enhanced intakes to reach normal plasma status [94]. This is particularly pertinent to the current global coronavirus (SARS-CoV-2) pandemic, which causes severe pneumonia and sepsis, conditions known to be associated with signiﬁcantly depleted vitamin C status [121,122]. It should also be noted that individuals with marginal vitamin C status are at higher risk of developing vitamin C deﬁciency [123], and once depleted, higher than recommended intakes of the vitamin are required to fully replete them again [78,79]. 8. Conclusions and Future Directions This narrative review describes the ﬁndings of numerous studies which highlight the various factors that can impact on vitamin C status and prevalence of deﬁciency. The studies were of variable quality; ideally, multivariate analysis of the various factors highlighted in these studies would be performed to further examine the relationships discussed. Another limitation is the variable quality of the processes and methodologies used to assess vitamin C status in the studies; this has been addressed in detail in our review on global vitamin C status [3]. Global vitamin C status appears to be a cause for concern, and we believe this is primarily due to poor diet, particularly in LMICs, although also apparent in subgroups within high-income settings [3]. Vitamin C concentrations provide a useful biomarker for a healthy diet; unfortunately, accurate analysis is costly, technically challenging, and time-consuming [130]. Thus, there is a need for low cost, accurate, and commercially available methods to assess plasma concentrations. Equally, the current assessment of intake is also limited and fails to take into account that the vitamin may be almost entirely eliminated prior to consumption in some globally important crops, such as cassava [38]. Future amendments to nutrient reference tables should take into this aspect into account. Clearly, there is a global need for increased consumption of vitamin-C-rich fresh fruit and vegetables. From our earlier review, this appears to be most notable in LMIC’s; however, it is also a concern in other at-risk groups [3]. Further education is required globally in how to meet the daily vitamin C requirements with locally available crops year-round. Some staple foods such as cassava require prolonged cooking to remove toxins (in this case, cyanide); eﬀorts have been made to reduce this content in genetically modiﬁed cassava, which could allow higher vitamin C content from this vitamin-rich plant at the time of consumption [38]. Given the high rates of deﬁciency in some diets, cooking practices may also require modiﬁcation to help increase intake. A number of studies have assessed the potential for vitamin C supplements to reduce a range of noncommunicable diseases and infections [9,94]. However, there remains much controversy as to the eﬃcacy of supplementation, primarily due to badly designed studies that do not take into account the baseline vitamin C status of the participants [98]. 7. Has Vitamin C Status Changed Over Time? It should be noted, however, that vitamin C in the NHANES II samples was analyzed using the 2,4-dinitrophenylhydrazine method, which can be prone to interference by other components in plasma and thus potentially overestimate vitamin C concentrations [127]. Other time course data is available from studies carried out in Finland and neighboring Russia between 1992 and 2002 [17,51]. In North Karelia, Finland, the vitamin C status of men decreased from 54 to 27 µmol/L over 5 years, then rose slightly to 37 µmol/L over the next 5 year period [17]. Nyyssonen et al. reported a small increase in baseline vitamin C values in Finish men over 6 years [72]. In Pitkäranta, Russia, the vitamin C status of women decreased from 22 to 13 µmol/L over a 5-year period, while the vitamin C status of Russian men remained in the deﬁciency range over a 10-year period [17]. The blood samples from the more recent survey (2002) were from fasting participants; however, it is not clear whether they were fasting in the earlier surveys (1992 and 1997). Another set of studies was carried out in Java, Indonesia, between 2005 and 2011 [128,129]. Mean vitamin C status decreased in the subjects from 45 to 29 µmol/L over 6 years, although the percentage of deﬁciency decreased from 15% to 11% over this time. The decrease in mean vitamin C status could partly be explained by the earlier study assessing nonfasting plasma (as demonstrated by a higher percentage of vitamin C status >23 µmol/L, i.e., 72% versus 54%). However, there are possibly Nutrients 2020, 12, 1963 12 of 19 other environmental, lifestyle, or health-related issues that could be responsible for the changes in vitamin C status over time in Indonesia, Finland, and Russia. Not all studies have shown changes in vitamin C status over time. A survey carried out in Shanghai, China, over a ﬁve-year period (between 1995 and 2000) showed no change in vitamin C status over this time period, although there was a dip in status at the midpoint (from 43 to 34 µmol/L) [52]. Thus, there is currently no clear trend in vitamin C status over time. However, much of the vitamin C data is very dated, and thus may not accurately reﬂect the current situation, particularly in countries where there have been changes over time in speciﬁc factors that can aﬀect vitamin C status. Acknowledgments: A.C.C. is a recipient of a Sir Charles Hercus Health Research Fellowship from the Health Research Council of New Zealand. hor Contributions: A.C.C. and S.R. contributed equally to the review. All authors have read and agreed to published version of the manuscript. References 1. Nishikimi, M.; Yagi, K. Molecular basis for the deﬁciency in humans of gulonolactone oxidase, a key enzyme for ascorbic acid biosynthesis. Am. J. Clin. Nutr. 1991, 54, 1203S–1208S. [CrossRef] 2. Stone, I. Homo sapiens ascorbicus, a biochemically corrected robust human mutant. Med. Hypotheses 1979, 5, 711–721. [CrossRef] 3. Rowe, S.; Carr, A.C. Global Vitamin C Status and Prevalence of Deﬁciency: A Cause for Concern? Preprints, 2020; 202005.0394.v1. 4. Savini, I.; Rossi, A.; Pierro, C.; Avigliano, L.; Catani, M.V. SVCT1 and SVCT2: Key proteins for vitamin C uptake. Amino Acids 2008, 34, 347–355. [CrossRef] 5 H i D Di t ib ti f bi id t b lit d l i d i l A N Y A d S i 5. Hornig, D. 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Conclusions and Future Directions Therefore, knowledge of the high-risk groups for deﬁciency should be utilized for future studies as these groups are more likely to beneﬁt from supplementation. pp In the meantime, global and local policymakers should consider the local data available on deﬁciency in an attempt to modify dietary intake and other modiﬁable risk factors. More of these risk factors should be taken into account during the review and update of global recommended dietary intakes for the vitamin. Clinicians worldwide should also remain vigilant to detect and correct hypovitaminosis C and deﬁciency in the at-risk groups highlighted. Author Contributions: A.C.C. and S.R. contributed equally to the review. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: A.C.C. is a recipient of a Sir Charles Hercus Health Research Fellowship from the Research Council of New Zealand. 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https://openalex.org/W4308914821	https://journals.plos.org/globalpublichealth/article/file?id=10.1371/journal.pgph.0001217&type=printable	English	null	Impact of COVID-19 lockdown on physical activity behaviours of older adults who participated in a community-based exercise program prior to the lockdown	PLOS global public health	2,022	cc-by	8,623	PLOS GLOBAL PUBLIC HEALTH PLOS GLOBAL PUBLIC HEALTH RESEARCH ARTICLE Impact of COVID-19 lockdown on physical activity behaviours of older adults who participated in a community-based exercise program prior to the lockdown Kaoru NosakaID1,2☯, Caitlin Fox-Harding1,3, Kazunori Nosaka1,3☯ 1 School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, Australia, 2 School of Nursing and Midwifery, Edith Cowan University, Joondalup, Western Australia, Australia, 3 Exercise Medicine Research Institute, Edith Cowan University, Joondalup, Western Australia, Australia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. kaoru.nosaka@ecu.edu.au Abstract This study investigated the impact of the 2020 COVID-19 lockdown on community-dwelling older adults attending a community-based exercise program to seek strategies to keep them active during self-isolated situations. A two-phase mixed methods approach included a survey followed by in-person focus groups. Forty-eight participants, with 32 starting a com- munity-based exercise program before the lockdown and 16 joining the program after the lockdown, completed a questionnaire survey about physical activities before and during the lockdown. This was followed by three focus groups (26 participants in total) to identify fac- tors influencing physical activity behaviours found in the survey. The survey found that the COVID-19 lockdown had varied impact on exercise adherence of the older adults: 43% of the participants exercised less during the lockdown than pre-lockdown, but 26% exercised more. Interestingly, among the participants approximately 80% still achieved the recom- mended physical activity level by the WHO during the lockdown. The focus groups revealed that exercise behaviours before the lockdown directly affected the behaviours during the lockdown. Participants’ recognition of the support from trustworthy people also influenced their motivation to perform exercises in an isolated environment. Remote exercise pro- grams, such as digital and printed exercise materials, were found beneficial for the partici- pants only when they came from the people the older adults trusted through their previous experience (i.e., the program). A sense of belonging to the exercise group was also essen- tial for the participants to achieve self-managed exercise. It was concluded that older adults need connections to an exercise group and a trustworthy exercise instructor who could con- tinuously support them to be physically active in isolated situations such as lockdowns, in addition to exercise knowledge and a better understanding of the benefits of exercise. OPEN ACCESS Citation: Nosaka K, Fox-Harding C, Nosaka K (2022) Impact of COVID-19 lockdown on physical activity behaviours of older adults who participated in a community-based exercise program prior to the lockdown. PLOS Glob Public Health 2(11): e0001217. https://doi.org/10.1371/journal. pgph.0001217 Citation: Nosaka K, Fox-Harding C, Nosaka K (2022) Impact of COVID-19 lockdown on physical activity behaviours of older adults who participated in a community-based exercise program prior to the lockdown. PLOS Glob Public Health 2(11): e0001217. https://doi.org/10.1371/journal. pgph.0001217 Introduction and Innovation, Government of Western Australia (G1005221 - DJTSI COVID 19 Project): https:// www.wa.gov.au/organisation/department-of-jobs- tourism-science-andinnovation. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. and Innovation, Government of Western Australia (G1005221 - DJTSI COVID 19 Project): https:// www.wa.gov.au/organisation/department-of-jobs- tourism-science-andinnovation. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Normal had become abnormal, and the abnormal has become the new normal since the World Health Organization (WHO) first confirmed a new virus outbreak (COVID-19) in Jan- uary 2020 [1]. This phenomenon has changed people’s daily lives worldwide, and Australia is no exception. Physical restrictions, including lockdowns, have had significant impacts on physical activities in Australia, ‘a sporting nation’ [2] from the time of the first COVID-19 active case identified in late January 2020 [3]. Approximately 20% of Australian children (aged 0–14 years) and adults (aged 15 years and over) who had participated in organised sports before the COVID-19 pandemic gave up the sports entirely by March 2021 [3]. Even in the state of Western Australia (WA), one of the relatively COVID-free locations, 30% of adults aged over 18 years were less engaged in physical activities (exercises and sports) during the COVID-19 period than before [4]. This reduction had severe impacts on older adults, espe- cially on their physical and mental fitness [5]. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. According to the WHO’s physical activity guidelines [6], older adults should be engaged in a minimum of 30 minutes of moderate physical activity at least five days a week, preferably every day, to reduce the risk of developing chronic diseases, improve the immune system, mental health, and the quality of life. However, the 2017–2018 National Health Survey by the Australian Bureau of Statistics [7] reported that 69% of males and 75% of females aged 65 years and over did not achieve the recommended physical activity even before the pandemic. Thus, a considerable concern existed that the pandemic-related self-isolation/lockdowns would increase sedentary lifestyles in older adults, which would cause various negative impacts on their physical and mental health. In addition, this could lead to an increase in the number of health-related national expenditures [8]. Editor: Nnodimele Onuigbo Atulomah, Babcock University, NIGERIA Editor: Nnodimele Onuigbo Atulomah, Babcock University, NIGERIA Copyright: © 2022 Nosaka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Yes - all data are fully available without restriction. In addition, we have uploaded 4 Supporting information files: 1) Survey questions, 2) Focus group questions, 3) Coding framework for qualitative analysis including codes, frequency, code description, and representative direct quotations, and 4) Quantitative data in Excel format. Funding: KN received a grant for this research from the Department of Jobs, Tourism, Science 1 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Introduction A recent systematic literature review paper [9] found that lockdowns decreased physical activity or increased sedentary time among older adults; however, pre-lockdown habits affected those changes, and practical lifestyle interventions minimised the negative impacts caused by the COVID-19 pandemic. Lee and colleagues [10] also reported that a 2-month lockdown in Singapore increased depressive symptoms and decreased physical activity among older people, but more targeted interventions would mitigate the negative impacts of the future lockdown. There has been an increase in studies related to understanding older adults’ physical activity experiences and behaviours during the COVID-19 pandemic [e.g., 11–13]. For instance, public health guidance published in 2021 by the National Institute for Health and Care Excellence (NICE) in the UK suggests that digital and mobile health interventions could be an effective health tool for supporting community-dwelling older people physically active [14] or videos can be an effective tool for older adults to cope with stress and stay physi- cally active [15]. In addition, Christensen and colleagues [16] have emphasised the universal promotion of physical activity in response to the significant physical activity declines due to the COVID-19 pandemic across whole societies around the world, particularly in older adults, to stop the further increase in inactive lifestyles. Some theories exist concerning exercise behaviour, such as the health belief model [17], protection motivation theory [18], theory of reasoned action [19], theory of planned behaviour [20], or social cognitive theory [21]. Sirur and colleagues [22] advocated that theory-based exercise interventions were critical to achieving exercise adherence within a targeted popula- tion. However, they also insisted that one theory alone could not explain definite determinants to predict people’s physical activity behaviour. They stated that as the concepts of self-efficacy and outcome expectations were common factors across the prominent theories, it should be crucial to identify the mediators of the self-efficacy and outcome expectations, depending on the targeted population. For instance, Collado-Mateo and colleagues [23] identified 14 factors 2 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults to increase adherence to physical exercises in older adults, emphasising that exercise adherence was affected by multiple factors; thus, intervention should be controlled and modified by those factors. Ethics statements The Edith Cowan University Human Research Ethics Committee granted the study’s ethical approval (REMS No: 2020-01766-NOSAKA). The authors obtained written formal consent to participate in this study from individual participants. Introduction Under those circumstances, it is significantly vital to deepen our understanding of the background of the older adults’ exercise experiences and behaviours before, during, and after the COVID-19 pandemic. In order to seek strategies to keep older adults physically active during self-isolated situa- tions like lockdowns in the future, it is inevitable to investigate the actual impact of a COVID- 19 lockdown on the physical activity behaviours of older adults. Western Australia had the first one-month lockdown in April 2020, although the number of infected community cases was small. The authors have been running community-based exercise programs for older adults in WA’s metropolitan region, and the lockdown forced us to suspend the program. However, unlike the reported high 20% sports termination ratio after the COVID-19 pan- demic in Australia [3] most of our program participants returned to the program after the lockdown. This provided us with a significant opportunity to investigate the impact of the COVID-19 lockdowns on the physical activities of the older adults who had attended a com- munity-based exercise program before the lockdown and probe the rational background of their exercise behaviours. This also made us better understand what factors would mediate the self-efficacy and outcome expectations to exercise regularly for community-dwelling older adults in an isolated environment, including how pre-lockdown exercise experience and knowledge would affect older adults when they face the necessity of self-managed exercises. The findings from the present study are expected to help develop strategies for older adults to be physically active in the post-pandemic world where other lockdowns or similar isolated environments are possible. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Study design A two-phase mixed methods under an embedded design [24,25] was employed to examine exercise-related perceptions and experiences of older adults in WA in relation to the COVI-19 pandemic and lockdowns. In phase one, quantitative and open-ended data were collected using a self-administered questionnaire to find older adults’ exercise experiences and behav- iours before and during the WA’s first a-month lockdown in April 2020. In phase two, qualita- tive data were obtained through focus group interviews to probe the findings of phase 1 and clarify critical elements to keep older adults physically active in future lockdown or similar iso- lated situations. The present study focused on how the COVID-19 lockdown affected exercises and physical activities in older adults who had previously participated in a community-based exercise program to investigate whether the exercise program impacted their ability and exer- cise knowledge to exercise alone during the lockdown. Therefore, participants were recruited using a purposeful sampling method targeting older adults (60 years and over) who partici- pated in the program before March 2020 and came back to the program after the lockdown in July 2020. The program consists of 2.5-hour weekly session for eight weeks; one hour of physi- cal exercise and one hour of cognitive health session with a morning tea in-between. Four pro- grams per year have been run, and each program has attracted 20–40 participants. The lockdown in this study included travel and gathering restrictions: the older adults were PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 3 / 14 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults required to stay home except for shopping for essentials, medical needs, up to one hour of exercise, or work, always wear a mask outside and have no visitor [26]. The participants who were in the program before the WA’s first lockdown in April 2020 are described as ‘pre-mem- bers’, and those who joined the program after the lockdown as ‘post-members’ in this study. In the present study, the exercise experience during the lockdown was measured, based on frequency distributions of self-reported exercise experience: less exercise than before the lock- down, more exercise than before the lockdown, or no change. Exercise patterns were investi- gated using self-reported exercise time per week and exercise types performed during the lockdown (see S1 Text for the survey questions). Study design This followed the evaluation approach that the public research institutions, including the Australian Sports Commission [3] and the Epi- demiology Branch of the Western Australian Government [4]. In the modern era, no one had experienced a pandemic until the COVID-19; therefore, to the best of our knowledge, no pre- vious studies evaluated exercise experiences or exercise patterns in older adults under pan- demic-enforced lockdowns, thus, we think that this approach was appropriate. Open-ended responses were gathered in the survey to outline the rationale of the experience and patterns, and we probed the questions in the focus groups based on the survey (see S2 Text for the focus group semi-structural questions). Survey study Fifty participants in a community-based exercise program for older adults in a metropolitan area of WA were invited to the survey verbally, via email or by phone through the program director after the WA’s first lockdown (July 2020). Forty-eight participants answered the ques- tionnaire, and written informed consent was obtained from each participant (96% response rate). Among 48 respondents (32 females and 16 males), 32 were pre-members, and 16 were post-members. No significant (p>0.05) differences in gender component between groups, and age (pre-member group: 66–95, 75.5 ± 6.6 y, post-member group: 60–88, 71.4 ± 8.1 y) were evident. A questionnaire was developed based on the best practices for survey research [27]. The final questionnaire for pre-members included eight closed and seven open-ended questions, and six closed and six open-ended questions were given to the post-members (see S1 Text for the survey questions). Printed self-administered survey questionnaires were distributed to the program participants who consented to participate in the survey at the beginning of the two programs after the lockdown on the 15th and 16th of July 2020. The respondents completed and returned the questionnaires to the program director by the end of each program. Data were analysed using Excel spreadsheets for open-ended questions and IBM SPSS Sta- tistics 27 [28] for closed-ended questions. Coding analysis classified the open-ended data and found patterns. Descriptive statistics captured the respondents’ characteristics, exercise experi- ence, and exercise patterns. Focus groups Active program members (who started the program before the lockdown and completed the survey) were invited to focus groups by email and in person through the program director. As a result, 26 members (20 females and 6 males) participated in one of three focus groups, 22 were pre-members who completed the survey, and the rest of the four were post-members who started the program after the lockdown. A written informed consent was obtained from each participant. One academic, who had never been involved in the program but had exten- sive focus group experience, facilitated the focus groups, which helped the focus groups mini- mise bias from existing relationships between facilitators and participants and maximise 4 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults disclosure. One of the investigators took notes of the focus group date and time, participant numbers, questions asked by the facilitator, and key themes that emerged from the three dis- cussions. The interviews were held on the 17th, 18th, and 31st of March 2021 in the facility’s meeting room, where the exercise program runs. Focus groups 1 and 2 had seven participants each (Group1: 5 females and 2 males: mean age = 76.9, Group2: 5 females and 2 males: mean age = 78.3). There was one post-member in Focus group 1 and two in Focus group 2. All twelve participants in Focus group 3 were pre-members, including 10 females and two males (mean age = 74.6). Each interview lasted about one hour, adhering to the WA Government’s COVID- 19 rules (venue attendance registration, 2-square meter social distancing, and hand sanitiser application) and keeping all venue windows open. It should be noted that there was no positive case in the WA communities when the focus groups were conducted. The data collected was digitally recorded using semi-structured focus group interviews. The semi-structured questions were developed to prove the results in the quantitative phase, based on the findings of phase one (see S2 Text for the focus group questions). After focus group 1, the focus group notes taken by the investigator and her feedback were used to assess the suit- ability of the semi-structured questions. That process confirmed the questions’ suitability; therefore, the initial semi-structured questions were used throughout the three focus groups. Focus groups All researchers discussed and agreed on data saturation after the third focus group, referring to the focus group notes and feedback from the note-taker, and decided on the termination of other focus groups. The recorded data was transcribed verbatim by one investigator of this research with the assistance of the YouTube auto-captions. The notes taken were not directly included in the data analysed and reported in this manuscript; however, they were referred to in the data analyses. The transcribed data were analysed using Nvivo12 [29] in a causal expla- nation analysis [27,30–32]. Two investigators independently coded the data to categorise that into different events and behavioural/attitudinal patterns. The coded and categorised data were compared between the two investigators. A third investigator undertook a further review if a consensus was not reached. This procedure continued until all the investigators reached an agreement on the coding and categorisation, followed by the three investigators independently producing a causal map based on the coded/categorised data. They subsequently compared the maps and reanalysed them when their consensus was not made. The steps continued until the investigators agreed with the causal networks of the influences on exercise behaviours pre, during, and after the COVID-19 lockdown. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Survey study In other words, they applied their exercise experience and knowledge acquired through the program to their self-managed exercise during the lockdown. In addition to the program, printed materials showing the eccentric and chair exercises with pictures to be performed at home were sent to the participants after entering the lock- down. The materials seemed to encourage the pre-members to do the eccentric exercises as well as the chair exercises during the lockdown. The results suggested that the previous atten- dance in the community-based exercise program helped the participants perform various exer- cises in the lockdown. Encouragement factors to exercise. Thirty-three people provided open-ended answers about what encouraged them to exercise in the lockdown. Those included "self-determination" (27%), "social interaction" (23%), "resources" (23%), followed by "reminder" and "fun" (6% each). Notable differences between the pre-and post-members were that the pre-members’ descriptions categorised as “self-determination” described more precisely what exercise Table 1. Self-reported exercise experience during the first lockdown in April 2020 with Demographics of Questionnaire Respondents in Perth, Western Australia. Variable Total n (%) Pre-member n (%) Post-member n (%) Exercise amount Less than before lockdown 20 (42.6) 14 (45.2) 6 (37.5) More than before lockdown 12 (25.5) 8 (25.8) 4 (25.0) As much as before lockdown 15 (31.9) 9 (29.0) 6 (37.5) Exercise time (min/week) 30 1 (2.2) 1 (3.2) 0 (0.0) 31–60 2 (4.4) 1 (3.2) 1 (7.1) 60–149 6 (13.3) 4 (12.9) 2 (14.3) 150 36 (80.0) 25 (80.6) 11 (78.6) Numbers of different exercises 1 5 (11.1) 2 (6.5) 3 (11.1) 2 15 (33.3) 6 (19.4) 9 (64.3) 3 10 (22.2) 8 (25.8) 2 (14.3) 4 15 (33.4) 15 (48.3) 0 (0.0) Three main exercise types Exercise 1 Walking 42 (87.5) Walking 28 (87.5) Walking 14 (87.5) Exercise 2 Eccentric exercise 24 (50.0) Eccentric exercise 23 (71.9) Yoga/Tai-Chi/Stretching 3 (18.8) Exercise 3 Chair exercise 19 (39.6) Chair exercise 17 (53.1) Pilates 3 (18.8) https://doi.org/10.1371/journal.pgph.0001217.t001 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults Table 1. Self-reported exercise experience during the first lockdown in April 2020 with Demographics of Questionnaire Respondents in Perth, Western Australia. Survey study Variable Total n (%) Pre-member n (%) Post-member n (%) Exercise amount Less than before lockdown 20 (42.6) 14 (45.2) 6 (37.5) More than before lockdown 12 (25.5) 8 (25.8) 4 (25.0) As much as before lockdown 15 (31.9) 9 (29.0) 6 (37.5) Exercise time (min/week) 30 1 (2.2) 1 (3.2) 0 (0.0) 31–60 2 (4.4) 1 (3.2) 1 (7.1) 60–149 6 (13.3) 4 (12.9) 2 (14.3) 150 36 (80.0) 25 (80.6) 11 (78.6) Numbers of different exercises 1 5 (11.1) 2 (6.5) 3 (11.1) 2 15 (33.3) 6 (19.4) 9 (64.3) 3 10 (22.2) 8 (25.8) 2 (14.3) 4 15 (33.4) 15 (48.3) 0 (0.0) Three main exercise types Exercise 1 Walking 42 (87.5) Walking 28 (87.5) Walking 14 (87.5) Exercise 2 Eccentric exercise 24 (50.0) Eccentric exercise 23 (71.9) Yoga/Tai-Chi/Stretching 3 (18.8) Exercise 3 Chair exercise 19 (39.6) Chair exercise 17 (53.1) Pilates 3 (18.8) https://doi org/10 1371/journal pgph 0001217 t001 lockdown reduced their exercise amount compared to the period before the lockdown, about 80% still achieved the recommended physical activity amounts (i.e., 150 min/week). This achievement ratio was much higher than that the national average reported by the Australian Bureau of Statistics [7]. It appears that the participants of the program were motivated to exer- cise regularly. It is possible that they attended the program because they had been already motivated well, or the exercise program made them exercise regularly. The number of exercises performed during the lockdown showed a remarkable difference between the two groups. The pre-member participants reported that they performed more than four different exercises most during the lockdown; however, the post-member partici- pants indicated that they performed two different exercises most. Walking was the most com- mon for both groups (87.5%) during the lockdown, but high proportions of the pre-members also performed “eccentric exercise” (71.9%) and “chair exercise” (53.1%) that had been instructed in the program that they had attended before the pandemic. These showed that peo- ple in the pre-member group performed the exercises that they had experienced in the pro- gram before the lockdown. In other words, they applied their exercise experience and knowledge acquired through the program to their self-managed exercise during the lockdown. In addition to the program, printed materials showing the eccentric and chair exercises with pictures to be performed at home were sent to the participants after entering the lock- down. Survey study Exercise experience during the lockdown. Exercise experience of participants in the study following the lockdown phenomenon in April 2020 was assessed on a self-reported exer- cise experience, indicating either ‘less exercise than before the lockdown’, ‘more exercise than before the lockdown’, or ‘no change’. As shown in Table 1, 20 people (42.6%) exercised less, 12 people (25.5%) exercised more during the lockdown than the period before the lockdown, and 15 people (31.9%) indicated no change in their physical exercise before and during the lock- down period. There was no significant difference between the pre-members and post-mem- bers in their physical exercise experience during the lockdown (p = 0.923). These results indicated that the COVID-19 lockdown affected the exercise amount in approximately 68% of the participants either negatively or positively. Exercise patterns during the lockdown. Exercise patterns were assessed from the exercise amount during the lockdown, the number of exercises performed, and the performed exer- cises. As shown in Table 1, while 42.6% of the participants reported that the COVID-19 5 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults lockdown reduced their exercise amount compared to the period before the lockdown, about 80% still achieved the recommended physical activity amounts (i.e., 150 min/week). This achievement ratio was much higher than that the national average reported by the Australian Bureau of Statistics [7]. It appears that the participants of the program were motivated to exer- cise regularly. It is possible that they attended the program because they had been already motivated well, or the exercise program made them exercise regularly. The number of exercises performed during the lockdown showed a remarkable difference between the two groups. The pre-member participants reported that they performed more than four different exercises most during the lockdown; however, the post-member partici- pants indicated that they performed two different exercises most. Walking was the most com- mon for both groups (87.5%) during the lockdown, but high proportions of the pre-members also performed “eccentric exercise” (71.9%) and “chair exercise” (53.1%) that had been instructed in the program that they had attended before the pandemic. These showed that peo- ple in the pre-member group performed the exercises that they had experienced in the pro- gram before the lockdown. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Survey study The materials seemed to encourage the pre-members to do the eccentric exercises as well as the chair exercises during the lockdown. The results suggested that the previous atten- dance in the community-based exercise program helped the participants perform various exer- cises in the lockdown. Encouragement factors to exercise. Thirty-three people provided open-ended answers about what encouraged them to exercise in the lockdown. Those included "self-determination" (27%), "social interaction" (23%), "resources" (23%), followed by "reminder" and "fun" (6% each). Notable differences between the pre-and post-members were that the pre-members’ descriptions categorised as “self-determination” described more precisely what exercise 6 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults routine settings and how to stick to the routines to encourage exercise in isolations. On the contrary, the post-members only focused on general physical movements. The participants in both groups stated that exercising with a social interaction element, such as exercising with their family and friends, could encourage them to achieve regular exercise even in isolated situ- ations. The materials to explain exercises, and resources, including exercise guides sent from the program organisers (e.g., exercise videos and handouts), were also described as essential motivators for regular self-exercise in isolation. Pre-members also stated that reminders from the program helped them keep exercising during the lockdown. Remote exercise programs/materials. The program director emailed the links to three home-based exercise videos and printed guides as attachments to all the program participants immediately after the announcement of the first lockdown. The pre-members evaluated the materials using five answering options (1 = never used, 2 = slightly helpful, 3 = moderately helpful, 4 = very helpful, and 5 = extremely helpful). Sixteen respondents (53.3%) reported that the exercise guides were helpful to do exercises during the lockdown. The participants also provided descriptions of the main reasons for their judgements. The descriptions were coded and categorised. The main reasons for the helpfulness were ‘exercise prompts’ (76%) and ‘easi- ness to follow’ (18%). Some respondents put the printed guides on a refrigerator to be seen anytime they were in the kitchen to motivate them to exercise. ‘Easiness to follow’ was also a key element determining the helpfulness level of the remote exercise guides. Survey study The respondents found the remote exercise guides unhelpful to do exercise during the lockdown when they had ‘no motivation to exercise’ (47%) or ‘having a self-exercise menu’ (13%) and saw the guides ‘need a sense of companionship’ (13%). It is important to note that 27% of the respondents reported that they had unavailability to access the videos or printed guides due to unfamiliarity with technology or no home printers. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Focus groups Qualitative data analyses of focus group sessions identified 38 influential elements of exercise behaviours and psychological responses during pre-lockdown, lockdown, and post-lockdown and their causal relationships, as shown in Fig 1. S1 Table summarises the coding framework, including code names, frequency, descriptions, and citations of the 38 elements. Pre-and during lockdown exercise behaviours. As shown in Fig 1, tacit causal relation- ships were found between pre-lockdown exercise behaviours and general psychological experi- ence during the lockdown. Social connections built before the lockdown tacitly but positively affected the general psychological experience of the participants. They re-realised the impor- tance of social connection and found that even the connection they had not realised before the lockdown brightened up their mood. It was also found that exercise behaviours before the lock- down directly affected the behaviours during the lockdown. Social connections built before the lockdown and exercise-related knowledge obtained before the lockdown contributed to older adults’ independent exercise, working as exercise facilitators. Both the participants’ exercise habits and recognition of the exercise support from trustworthy people before the lockdown formed their commitment to exercise, and that commitment worked as one of the exercise facil- itators during the lockdown. In addition, the general psychological experience tacitly affected the exercise behaviours during the lockdown. When the participants felt certainty, they were able to foresee what would happen next, which encouraged them to engage in exercise. The par- ticipants’ new realisation of the exercise importance, new exercise opportunities, and additional time for exercise also tacitly led them to see the lockdown as an opportunity to exercise. On the contrary, the participants’ uncertainty tacitly caused anxiety–no enjoyment, which prevented them from exercising. Social frustration—annoyance by those who did not follow 7 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults Fig 1. Chronological causal network: The influences on exercise behaviours and psychological responses before, during, and after the COVID-19 lockdown. Fig 1. Chronological causal network: The influences on exercise behaviours and psychological responses before, during, and after the COVID-19 l kd network: The influences on exercise behaviours and psychological responses before, during, and after the COVID-19 Fig 1. Chronological causal network: The influences on exercise behaviours and psychological responses before, durin lockdown. Focus groups https://doi.org/10.1371/journal.pgph.0001217.g001 the COVID-19-related restrictions or those who complained about the restrictions also tacitly caused no enjoyment within the participants. As a result, they tended to be discouraged from exercising during the lockdown due to negative feelings towards the environment surrounding them. Remote programs/materials and companionships. When the participants found the remote programs with companionships, they seemed motivated to work out. The flexibility of the programs also played a moderately important role in that. However, when the participants felt no companionship with the remote programs or found doing the remote programs dis- comfort, they were not motivated to exercise. In addition, it also became clear that the limited technological competence of the participants hindered them from engaging in exercise. In other words, older adults feel as if they are rejected when they cannot access remote programs or materials due to their technological competence. This type of feeling could discourage them from exercising. The results reported here may sound too obvious; however, the factors men- tioned in this section require more attention to including them in a remote program or mate- rial when the program or material is developed. Older adults want to feel companionship from program/material contents and even from how they are delivered. Sense of community, resilience, and remote program/material quality. The partici- pants’ general sense of community, resilience towards lockdowns, or short lockdown period were also vital elements in facilitating exercise in the future. At the same time, a long lockdown period would hinder them from exercising. It was suggested that effective remote programs should be stimulative, flexible, and empathetic in the future. If the participants regard the 8 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults remote programs as exercise stimulation, it will be highly possible to exercise; however, if the participants consider the remote programs not suitable to their environment, time, or non-evi- dence-based, they will not be engaged in an exercise in the future. The participants also emphasised the importance of variations of the remote programs and materials. No matter how good the programs and materials are, they need to be advanced and refined continually to stimulate older adults. Effective remote programs and materials. Focus groups Apart from the analysis reported above, the- matic analysis was also conducted to identify the critical features of an effective remote mate- rial/program from the participants’ perspective. The features were found in three dimensions: preferred program features, delivery methods, and distribution frequency. The participants favoured a remote material/program that could extend their exercise commu- nity even during a lockdown. Several participants made copies of the exercise materials sent to them by the instructors during the lockdown and handed them to their family members, friends, and neighbours. The materials became a tool for older adults to connect or expand their community. Some dropped the copy off in their neighbours’ letterbox, and some others forwarded the electronic materials to their friends and families overseas. They also preferred a remote material/program with options, e.g., programs to conduct both outside and inside or small group programs outside in line with the required social distance and group gather- ing numbers. For them, even a remote material/program should contain a sense of commu- nity. They also considered the materials/programs something that can reinforce their willingness to exercise as one participant stated that “I think it is important to have things to look forward to”. The participants suggested three delivery methods: 1) electric, such as emails, online meet- ing applications, online on-demand, and mobile texts, 2) in-person delivery, such as university students dropping a hardcopy material in a letterbox and having a short conversation with the participants through the front door, 3) posted hardcopy materials. Interestingly, preferred delivery methods appear to depend on the participants’ lifestyles. Some worried about paper consumption and posting costs for hardcopy materials, and others disliked electric methods concerning internet fraud. However, all participants agreed that the materials/programs should be delivered via multiple delivery channels. The most preferred distribution frequency was when information was updated on the one hand; the second most preferred frequency was once a week on the other hand. One of the par- ticipants supported the remote material/program distribution once a week as the once-a-week material/program delivery means for her a social connection and fun of waiting to happen. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Discussion We investigated the impact of the COVID-19 lockdown on community-dwelling older adults who had attended a community-based exercise program before the lockdown to seek strategies to keep them active during other self-isolated situations. The study collected quantitative data on self-reported exercise experience, exercise amount (minutes per week), the number of exer- cises performed, and the exercise activities during the COVID-19 lockdown of the older adults and qualitative data through three focus groups to probe the quantitative findings and achieve the objective. The quantitative data analyses found that the COVID-19 lockdown reduced the exercise amount in the majority of the older adults, but many maintained the recommended amount (150 minutes per week). These findings suggested that the participants adhered to exercise at the recommended level despite decreasing their exercise amount. The focus group discussions explored the rationale for the adherence behaviour. Exercise knowledge, reminders, and 9 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults encouragement from someone trusted and expert, and a sense of social interaction are essen- tial for older adults to remain physically active and minimise sedentary behaviours. The pres- ent study also revealed that the older adults who had attended an organised exercise program before the lockdown performed more variety of exercises independently than those who had not. The focus group participants reported that what they learnt in the program helped them become confident in their exercise knowledge and better understood the negative impacts of a sedentary lifestyle on their physical and mental health, which encouraged their intention to keep exercising during the lockdown. Knowledge and confidence are self-efficacy, and under- standing is outcome expectations. As suggested by the prominent theoretical models men- tioned in the introduction, knowledge and better understanding increased the intention to exercise in the participants. However, as indicated by Sirur et al. [22] and other researchers [33,34], they were still insufficient to maintain older adults physically active; intentions should be transformed into behaviours when they work with other factors. The participants admitted that they needed cues to transfer their exercise intentions to actual performance, although they knew how to exercise and why they should keep exercising. The participants recognised the gap and saw that the exercise materials (e.g., handouts, videos) were one of the cues to help them into actual exercise behaviour. Discussion However, at the same time, the focus groups also revealed that older adults did not necessarily accept any materials, and they were incredibly selective and even suspicious of such material. When such materials do not meet their preferences and win their credibility, they will never use them. Furthermore, they do not accept anything if treated as ‘weak’ or ‘ignorant’ people; they always desire to deepen their knowledge and expand their skills like young people. Additionally, the participants emphasised that the reliability of the material resources and a sense of social connection in the materials were also critical factors for achieving material acceptance. They particularly paid attention to who prepared the materials. If someone they already trusted qualified them, they counted the information and were willing to use it in an isolated environment. It became clear that building such rapport with the resource after being in an isolated environment is challenging for older adults. Therefore, it is inevitable for them, or those who would support them in the remote environment, to have a trusted relationship with the resource providers/communicators beforehand. Community-based programs could be one of the best ways to achieve this. Timely access or information sharing in the sense of community from program organisers also plays a vital role in keeping older adults physically active in an isolated environment. Older adults are motivated to exercise when they feel that they are not emotionally isolated and know someone close to them is also in the same situation and try to keep exercising. Hystad and Carpiano [35] have also confirmed a strong positive relationship between a sense of community belonging and health behaviour in Canadians. Therefore, setting up the most accessible measures to deliver the information or contact older adults is crucial. The focus groups additionally revealed that older adults preferred multiple selections of material formats. Some were familiar with the internet and new devices; others stuck to tradi- tional (e.g., more familiar) forms, including printed materials and landlines. Although technol- ogies are advancing and digital information has become standard for the general population, it should not be granted that older adults are also a part of that. The Office of the eSafety Com- missioner, the Australian Government [36], reported a strong relationship between age and digital literacy; the older, the more digitally disengaged. Therefore, older adults still require various material formats and delivery methods to cover the wider older population. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Limitations of the study First, when generalising the findings of this study, modifications will be required, depending on cultures, regions, or countries older adults live in, as the study was based on the WA com- munity-dwelling older adults. In addition, WA lockdowns to date have been softer than those in other states and countries. Hence, there would be other vital factors for physical activity in older adults in a restricted environment that this study has missed. Second, the effectiveness of the proposed initiative is unclear for sedentary older adults as this study did not deal with motivations for sedentary older adults to start exercising. Additional study with the inactive population is required for applying the initiative to the larger population. Gender bias in the collected data could affect the findings of this study. Although the female orientation in the participants of this study is typical within community-based programs, more male data will need to be collected and analysed to achieve more active older adults generally. Finally, other approaches could be possible to investigate exercise experience, such as weighted aggregate scoring of outcome variables of exercise experience, although the reli- ability and validity of the scoring under isolated conditions like the COVID-19 lockdown require justification. Discussion The focus groups proposed email, digital attachment, link sharing, online meeting tools, landlines, and postal service for hard copies. It also became clear that the older adults confirmed connecting 10 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults to others by receiving the materials in an isolated environment. They do not necessarily have to receive such materials daily or weekly; however, they must know in advance that they will be updated or accessed at least once a month to ensure they will not be forgotten. Based on the findings mentioned above, we propose a new initiative strategy called “BE ACTIVE” to facilitate older adults to exercise independently even when their living environ- ments suddenly change, or they are forced to be in an isolated environment: the strategy is encouraging older adults to 1) belong to the reliable exercise community and build a good rela- tionship/friendship with other community members/supporters, 2) acquire exercise knowl- edge and skills from experts, 3) seek assistance from trustworthy people, 4) be informed and updated regularly, 5) ensure they connect with others constantly, 6) be confident in their abil- ity to perform an exercise, 7) inspire others to get into exercise, enjoy various types of exercise, and 8) build up resilience against an isolated environment. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Acknowledgments The authors thank all research participants involved in this study, the university for adminis- trative support, and the director of the community-based exercise program for recruitment support. Supporting information S1 Table. Coding framework: Pre-lockdown, during lockdown, and future. (DOCX) Project administration: Kaoru Nosaka, Caitlin Fox-Harding. Validation: Kaoru Nosaka, Caitlin Fox-Harding. Visualization: Kaoru Nosaka. Visualization: Kaoru Nosaka. Writing – original draft: Kaoru Nosaka. Writing – original draft: Kaoru Nosaka. Writing – review & editing: Kaoru Nosaka, Caitlin Fox-Harding, Kazunori Nosaka. Writing – review & editing: Kaoru Nosaka, Caitlin Fox-Harding, Kazunori Nosaka. PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 Conclusions This study found the COVID-19 lockdown affected exercise amount among older adults who attended a community-based exercise program. However, the present study identified essential factors that would make older adults stay physically active even when they were forced to be isolated where their exercise opportunities are restricted. Older adults need to know about exercise and the benefits of exercise, have companionship connections with a community- based exercise group, have trustworthy exercise instructors and programmers, and receive updated exercise-related information to stay physically active in an isolated environment. Future research should investigate the effectiveness of the essential factors and conditions identified in the present study for changing older adults’ physical activity behaviour. Suppose the effectiveness or other additional factors are confirmed, older adults will accept the new normal and stay active even when provided physical activity opportunities are suddenly restricted or they cannot attend a community-based exercise program. Physical activity behav- iours cannot be changed suddenly; thus, what older adults are currently doing determines their behaviours in situations that could restrict their physical activities, such as lockdowns. The convergence of the COVID-19 pandemic is approaching, and we should urgently promote exercise for older adults for a healthier future. The convergence of the COVID-19 pandemic is approaching, and we should urgently promote exercise for older adults for a healthier future. 11 / 14 PLOS Global Public Health \| https://doi.org/10.1371/journal.pgph.0001217 November 11, 2022 PLOS GLOBAL PUBLIC HEALTH Impact of COVID-19 lockdown on physical activity behaviours of older adults Author Contributions Conceptualization: Kaoru Nosaka, Kazunori Nosaka. Conceptualization: Kaoru Nosaka, Kazunori Nosaka. Data curation: Kaoru Nosaka, Caitlin Fox-Harding, Kazunori Nosaka. Formal analysis: Kaoru Nosaka, Caitlin Fox-Harding. Funding acquisition: Kazunori Nosaka. Investigation: Kaoru Nosaka, Caitlin Fox-Harding, Kazunori Nosaka. Methodology: Kaoru Nosaka. Project administration: Kaoru Nosaka, Caitlin Fox-Harding. Resources: Kazunori Nosaka. Supervision: Caitlin Fox-Harding, Kazunori Nosaka. Validation: Kaoru Nosaka, Caitlin Fox-Harding. Visualization: Kaoru Nosaka. Writing – original draft: Kaoru Nosaka. 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https://openalex.org/W3120139464	https://www.nature.com/articles/s41467-020-20562-w.pdf	English	null	Giant isotropic magneto-thermal conductivity of metallic spin liquid candidate Pr2Ir2O7 with quantum criticality	Nature communications	2,021	cc-by	8,592	1 State Key Laboratory of Surface Physics, Department of Physics, and Laboratory of Advanced Materials, Fudan University, Shanghai 200438, China. 2 School of Physics, and Wuhan National High Magnetic Field Center, Huazhong University of Science and Technology, Wuhan 430074, China. 3 Collaborative Innovation Center of Advanced Microstructures, Nanjing 210093, China. 4 Shanghai Research Center for Quantum Sciences, Shanghai 201315, China. ✉email: tianzhaoming@hust.edu.cn; shiyan_li@fudan.edu.cn ARTICLE Giant isotropic magneto-thermal conductivity of metallic spin liquid candidate Pr2Ir2O7 with quantum criticality J. Cheng1, Y. J. Yu1, B. L. Pan1, Q. Li1, L. M. Xu2, Z. M. Tian 2✉& S. Y. Li 1,3,4✉ J. M. Ni1, Y. Y. Huang1, E. J. Cheng1, Y. J. Yu1, B. L. Pan1, Q. Li1, L. M. Xu2, Z. M. Tian 2✉& S. Y. Li 1,3,4✉ J. M. Ni1, Y. Y. Huang1, E. J. Cheng1, Y. J. Yu1, B. L. Pan1, Q. Li1, L. M. Xu2, Z. M. Tian Spin liquids are exotic states with no spontaneous symmetry breaking down to zero- temperature because of the highly entangled and ﬂuctuating spins in frustrated systems. Exotic excitations like magnetic monopoles, visons, and photons may emerge from quantum spin ice states, a special kind of spin liquids in pyrochlore lattices. These materials usually are insulators, with an exception of the pyrochlore iridate Pr2Ir2O7, which was proposed as a metallic spin liquid located at a zero-ﬁeld quantum critical point. Here we report the ultralow- temperature thermal conductivity measurements on Pr2Ir2O7. The Wiedemann–Franz law is veriﬁed at high ﬁelds and inferred at zero ﬁeld, suggesting no breakdown of Landau quasi- particles at the quantum critical point, and the absence of mobile fermionic excitations. This result puts strong constraints on the description of the quantum criticality in Pr2Ir2O7. Unexpectedly, although the speciﬁc heats are anisotropic with respect to magnetic ﬁeld directions, the thermal conductivities display the giant but isotropic response. This indicates that quadrupolar interactions and quantum ﬂuctuations are important, which will help determine the true ground state of this material. 1 State Key Laboratory of Surface Physics, Department of Physics, and Laboratory of Advanced Materials, Fudan University, Shanghai 200438, China. 2 School of Physics, and Wuhan National High Magnetic Field Center, Huazhong University of Science and Technology, Wuhan 430074, China. 3 Collaborative Innovation Center of Advanced Microstructures, Nanjing 210093, China. 4 Shanghai Research Center for Quantum Sciences, Shanghai 201315, China. ✉email: tianzhaoming@hust.edu.cn; shiyan_li@fudan.edu.cn 1 NATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w S pin ice state on a frustrated pyrochlore lattice has attracted numerous interests in condensed matter physics, due to the emergent magnetic monopole excitations from the manifold of degenerate ground states1,2. By introducing quantum ﬂuctua- tions with Jeff = 1/2 moments, quantum spin ice (QSI) states can be stabilized, exhibiting quantum electrodynamics with extra excitations like photons and visons3. Giant isotropic magneto-thermal conductivity of metallic spin liquid candidate Pr2Ir2O7 with quantum criticality (In this paper, we adopt the naming convention that the magnetic monopoles refer to the spin-ﬂip excitation, while the visons refer to the sources of emergent electric ﬁelds3.) Yb2Ti2O7, Tb2Ti2O7, and Pr2Zr2O7 are such promising QSI candidates3. On the other hand, iridates with 5d electrons have also drawn much attention in recent years owing to the various quantum phases and transitions therein, which originate from the competition between spin-orbit cou- pling and electron–electron correlation4,5. When these two aspects meet in the pyrochlore iridate Pr2Ir2O7, complex phe- nomena and exotic phases emerge6–13. scaling law in the Grüneisen ratio measurement10. It was also theoretically investigated as a QCP between antiferromagnetic ordering and nodal non-Fermi liquid11. Apparently, multiple mechanisms govern the ground state of Pr2Ir2O7, which may induce rich phenomena beyond the spin-ice physics. For such an exotic metallic spin-liquid candidate with quantum criticality, although various efforts have been made, two main issues remain to be solved. First, how do the electrons behave at the QCP? In other words, will the electrons still be well-deﬁned Landau quasiparticles? Second, probably due to the large neutron absorption cross-section of the iridium ions and the very small size of its single crystals14, little information is known for possible exotic magnetic excitations in Pr2Ir2O7, from which knowledge about the role of multipolar interactions beyond the dipolar interactions and quantum ﬂuctuations can be obtained. Ultralow-temperature thermal conductivity measurement is an important technique to address the above two issues. For the former one, the veriﬁcation of the Wiedemann–Franz (WF) law κ/σT = π2kB2/3e2 = L0 can be viewed as an evidence of the sur- vival of Landau quasiparticles at the QCP. Anomalous reduction of the Lorenz ratio L(T)/L0 with L(T) = κ/σT has been observed in CeCoIn5 (ref. 15), YbRh2Si2 (under debate (refs. 16–18)), and YbAgGe (ref. 19), while in some other compounds such as CeNi2Ge2 (ref. 20) and Sr3Ru2O7 (ref. 21), the WF law is veriﬁed at the QCP. For the latter one, a sizable residual linear term of thermal conductivity indicates the presence of highly mobile gapless excitations in triangular organics EtMe3Sb[Pd(dmit)2]2 (ref. 22) (note that this result has been challenged by two recent reports23,24). Spinon thermal conductivity with a linear tem- perature dependence was also found in the ideal spin-1/2 anti- ferromagnetic Heisenberg chain copper benzoate25. No magnetic thermal conductivities were observed in other two QSL candi- dates κ-(BEDT-TTF)2Cu2(CN)3 and YbMgGaO4 (refs. 26,27). Giant isotropic magneto-thermal conductivity of metallic spin liquid candidate Pr2Ir2O7 with quantum criticality p g Pr2Ir2O7 is a metal with the antiferromagnetic RKKY interac- tion of about 20 K in non-Kramers Pr 4 f moments mediated by Ir 5d conduction electrons6. The Kondo effect leads to a partial screening of the Pr 4 f moments and gives a lower Weiss tem- perature of \|θW\| = 1.7 K (ref. 6). No long-range magnetic order was observed down to 70 mK evidenced by the magnetic sus- ceptibility measurement, indicating a possible metallic spin liquid ground state, or even a U(1) QSI state6,13. A huge and anisotropic anomalous Hall effect (AHE) was probed under magnetic ﬁelds7,9, which may be the result of the spin chirality effect on the Ir sites from the noncoplanar spin texture of Pr 4 f moments. The observation of AHE in the absence of uniform magnetization at zero ﬁeld further indicates a long-sought chiral spin liquid state in Pr2Ir2O7 (ref. 8). More interestingly, a zero-ﬁeld quantum critical point (QCP) was uncovered from the diverging behavior and Fig. 1 Charge transport results of Pr2Ir2O7. a Temperature dependence of the resistivity at zero ﬁeld for Pr2Ir2O7 single crystal. Inset: zoomed view of the resistivity minimum at 45 K due to the Kondo effect. The solid line is the ﬁt to Hamann equation between 3 K and 35 K. b The magnetoresistance at T = 0.34 K. The magnetic ﬁeld is applied along the [111] direction. Inset: ρ(T) below 1 K in µ0H = 0, 3, and 6 T. g In this paper, we report ultralow-temperature thermal con- ductivity measurements on single crystals of Pr2Ir2O7. The WF law is veriﬁed at high ﬁelds and inferred at zero ﬁeld, suggesting the normal behavior of electrons at the QCP and the absence of fermionic magnetic excitations. A giant magneto-thermal con- ductivity at ﬁnite temperature is found, which may result from the strong scattering of phonons by the transverse ﬂuctuations. The thermal conductivity is isotropic in different magnetic ﬁeld directions, which is contrary to speciﬁc heat. We shall discuss the implications of these results. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w Unexpectedly, a crossover from the positive MTCs at low temperatures to the negative MTCs at high temperatures is observed at θw energy scale (for details, see Supplementary Note 4). We will come back to discuss the origin of this giant MTC later. We would like to emphasize that besides the electron thermal conductivities, κ above 5 T is entirely due to phonons without magnetic scatterings. First, a metamagnetic transition at Bc ~ 2.3 T can be induced only when applying the ﬁeld along the [111] direction in the magnetization M(B) measurements8. This means that a sizeable fraction of the “2-in, 2-out” conﬁgurations are transformed into the “3-in, 1-out” conﬁgurations at the critical ﬁeld. Therefore, it is natural to expect that the scatterings between the local spins and phonons will be reduced as the magnetization approaches saturation. In fact, the thermal conductivity of 3 T is quite close to the value of 5 T and 7 T, which coincides with the critical ﬁeld observed in the magnetization measurements. Second, the thermal conductivities are ﬁeld-independent between 5 T and 7 T. This indicates that even if the magnetic moments are not fully static in our experiment temperature range, they do not scatter phonons since the ﬁeld has no effect on the thermal conductivities. This behavior strongly suggests that the high-ﬁeld thermal conductivities are due solely to phonons without magnetic scatterings. Note that there may remain scatterings from structure disorder like stacking faults and grain boundaries. Fig. 2 Heat transport results of Pr2Ir2O7. a The thermal conductivity of Pr2Ir2O7 single crystal at various magnetic ﬁelds along the [111] direction. At zero ﬁeld, the thermal conductivity is strongly suppressed above Ts ≈ 0.12 K. b The data of thermal conductivity in (a) after subtracting the data of 5 T. A collapse region is clearly shown below the temperature at which the arrows point. This suppression temperature is deﬁned as Ts. Inset: ﬁeld dependence of the suppression temperature Ts. The error bars reﬂect the uncertainty in determining the temperature above which the data deviate from 0. They are estimated as the possible suppression temperature range due to the discrete data points. c The magneto-thermal conductivity MTC = (κ(H)−κ(0 T))/κ(0 T) × 100% at various temperatures. Above 5 T, the thermal conductivity tends to saturate. T, the thermal conductivity data overlap with each other. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w The dashed line is the Wiedemann- Franz law expectation L0/ρ0(5 T) = 0.032 mW K−2 cm−1 for sample A at 5 T, which meets the extrapolated κ/T ≡a = 0.031 mW K−2 cm−1 very well. The overlap of the 0 and 5 T curves below Ts ≈0.12 K suggests that the Wiedemann-Franz law is also satisﬁed in zero ﬁeld. Fig. 3 Veriﬁcation of Wiedemann–Franz Law in Pr2Ir2O7. The thermal Fig. 3 Veriﬁcation of Wiedemann–Franz Law in Pr2Ir2O7. The thermal conductivity of two Pr2Ir2O7 single crystals at µ0H = 0 and 5 T along the [111] direction, respectively. Solid lines are the ﬁts of the thermal conductivity data to κ/T = a + bTα−1 at 5 T below 0.3 K. The dashed line is the Wiedemann- F l t ti L / (5 T) 0 032 W K 2 1 f l A t 5 T g 2 2 7 conductivity of two Pr2Ir2O7 single crystals at µ0H = 0 and 5 T along the [111] direction, respectively. Solid lines are the ﬁts of the thermal conductivity data to κ/T = a + bTα−1 at 5 T below 0.3 K. The dashed line is the Wiedemann- Franz law expectation L0/ρ0(5 T) = 0.032 mW K−2 cm−1 for sample A at 5 T, which meets the extrapolated κ/T ≡a = 0.031 mW K−2 cm−1 very well. The overlap of the 0 and 5 T curves below Ts ≈0.12 K suggests that the Wiedemann-Franz law is also satisﬁed in zero ﬁeld. g 2 2 7 conductivity of two Pr2Ir2O7 single crystals at µ0H = 0 and 5 T along the [111] direction, respectively. Solid lines are the ﬁts of the thermal conductivity data to κ/T = a + bTα−1 at 5 T below 0.3 K. The dashed line is the Wiedemann- Franz law expectation L0/ρ0(5 T) = 0.032 mW K−2 cm−1 for sample A at 5 T, which meets the extrapolated κ/T ≡a = 0.031 mW K−2 cm−1 very well. The overlap of the 0 and 5 T curves below Ts ≈0.12 K suggests that the Wiedemann-Franz law is also satisﬁed in zero ﬁeld. transport at 0.34 K, the MTC is as large as 100% at 0.25 K and even 650% at 0.7 K. For other QSL candidates such as κ-(BEDT- TTF)2Cu2(CN)3 (ref. 26) and YbMgGaO4 (ref. 27), there is also a positive MTC, but the magnitude is much smaller. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w 2 Heat transport results of Pr2Ir2O7. a The thermal conductivity of 2Ir2O7 single crystal at various magnetic ﬁelds along the [111] direction. zero ﬁeld, the thermal conductivity is strongly suppressed above Ts ≈ 2 K. b The data of thermal conductivity in (a) after subtracting the data 5 T. A collapse region is clearly shown below the temperature at which e arrows point. This suppression temperature is deﬁned as Ts. Inset: ﬁeld pendence of the suppression temperature Ts. The error bars reﬂect the certainty in determining the temperature above which the data deviate m 0. They are estimated as the possible suppression temperature range e to the discrete data points. c The magneto-thermal conductivity TC = (κ(H)−κ(0 T))/κ(0 T) × 100% at various temperatures. Above 5 T, e thermal conductivity tends to saturate. Fig. 3 Veriﬁcation of Wiedemann–Franz Law conductivity of two Pr2Ir2O7 single crystals at µ direction, respectively. Solid lines are the ﬁts of to κ/T = a + bTα−1 at 5 T below 0.3 K. The d Franz law expectation L0/ρ0(5 T) = 0.032 mW which meets the extrapolated κ/T ≡a = 0.031 overlap of the 0 and 5 T curves below Ts ≈0.1 Wiedemann-Franz law is also satisﬁed in zero Fig. 2 Heat transport results of Pr2Ir2O7. a The thermal conductivity o Pr2Ir2O7 single crystal at various magnetic ﬁelds along the [111] directio At zero ﬁeld, the thermal conductivity is strongly suppressed above Ts 0.12 K. b The data of thermal conductivity in (a) after subtracting the da of 5 T. A collapse region is clearly shown below the temperature at wh the arrows point. This suppression temperature is deﬁned as Ts. Inset: ﬁ dependence of the suppression temperature Ts. The error bars reﬂect t uncertainty in determining the temperature above which the data devia from 0. They are estimated as the possible suppression temperature ran due to the discrete data points. c The magneto-thermal conductivity MTC = (κ(H)−κ(0 T))/κ(0 T) × 100% at various temperatures. Above 5 the thermal conductivity tends to saturate. Fig. 3 Veriﬁcation of Wiedemann–Franz Law in Pr2Ir2O7. The thermal conductivity of two Pr2Ir2O7 single crystals at µ0H = 0 and 5 T along the [111] direction, respectively. Solid lines are the ﬁts of the thermal conductivity data to κ/T = a + bTα−1 at 5 T below 0.3 K. Results Ch Charge and heat transport. Figure 1a shows the temperature dependence of the resistivity ρ(T) at zero ﬁeld for the Pr2Ir2O7 single crystal. The upturn behavior and the lnT dependence below 45 K where the resistivity displays a minimum, and the well ﬁt to the Hamann’s equation are the evidences for Kondo effect, as shown in the inset of Fig. 1a. This is consistent with ref. 6. The magnetoresistance MR = (ρ(H) −ρ(0 T))/ρ(0 T) × 100% at T = 0.34 K is presented in Fig. 1b. It is quite small, less than 5% up to 9 T, indicating the little inﬂuence of magnetic ﬁeld on the charge transport. Note that no anisotropy of resistivity is reported with respect to the electric current direction6, while the magnetore- sistance is anisotropic in different magnetic ﬁeld directions9. In the inset of Fig. 1b, ρ(T) below 1 K in µ0H = 0, 3, and 6 T are plotted. Since all the curves are very ﬂat, we can safely extrapolate them to the zero-temperature limit and get the residual resistivity ρ0 = 776, 757, and 769 µΩ cm for µ0H = 0, 3, and 6 T, respectively. Fig. 1 Charge transport results of Pr2Ir2O7. a Temperature dependence of the resistivity at zero ﬁeld for Pr2Ir2O7 single crystal. Inset: zoomed view of the resistivity minimum at 45 K due to the Kondo effect. The solid line is the ﬁt to Hamann equation between 3 K and 35 K. b The magnetoresistance at T = 0.34 K. The magnetic ﬁeld is applied along the [111] direction. Inset: ρ(T) below 1 K in µ0H = 0, 3, and 6 T. y The thermal conductivities of Pr2Ir2O7 single crystal up to 7 T are shown in Fig. 2a. The magnetic ﬁelds were applied perpendicular to the (111) plane. At high ﬁelds like 5 T and 7 NATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunication 2 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w ARTICLE NATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w The error bars reﬂect the above which the data deviate suppression temperature range neto-thermal conductivity rious temperatures. Above 5 T, Fig. 3 Veriﬁcation of Wiedemann–Franz Law in Pr2Ir2O7. The thermal conductivity of two Pr2Ir2O7 single crystals at µ0H = 0 and 5 T along the [111 direction, respectively. Solid lines are the ﬁts of the thermal conductivity data to κ/T = a + bTα−1 at 5 T below 0.3 K. The dashed line is the Wiedemann Franz law expectation L0/ρ0(5 T) = 0.032 mW K−2 cm−1 for sample A at 5 T which meets the extrapolated κ/T ≡a = 0.031 mW K−2 cm−1 very well. The overlap of the 0 and 5 T curves below Ts ≈0.12 K suggests that the Wiedemann-Franz law is also satisﬁed in zero ﬁeld. the thermal conductivity data overlap with each other. With creasing the ﬁeld, while κ/T data still overlap with the high-ﬁeld rves below a certain temperature Ts, they are suppressed more nd more strongly above Ts. In Fig. 2b, the collapse region of κ/T h d l Th b d h transport at 0.34 K, the MTC is as large a 650% at 0.7 K. For other QSL candi TTF)2Cu2(CN)3 (ref. 26) and YbMgGaO positive MTC, but the magnitude is muc crossover from the positive MTCs at negative MTCs at high temperatures is o (for details, see Supplementary Note 4 discuss the origin of this giant MTC late We would like to emphasize that bes conductivities, κ above 5 T is entirely magnetic scatterings. First, a metamagne can be induced only when applying t direction in the magnetization M(B) me that a sizeable fraction of the “2-in, 2 transformed into the “3-in, 1-out” con ﬁeld. Therefore, it is natural to expect th the local spins and phonons will be redu approaches saturation. In fact, the therm quite close to the value of 5 T and 7 T, critical ﬁeld observed in the magnetizatio the thermal conductivities are ﬁeld-inde 7 T. This indicates that even if the magne static in our experiment temperature r phonons since the ﬁeld has no effect on t This behavior strongly suggests that conductivities are due solely to pho scatterings. Note that there may remain disorder like stacking faults and grain b Veriﬁcation of Wiedemann–Franz La thermal conductivity data below 0.3 K f the WF law in Pr2Ir2O7. At ultra-low d ll b ﬁ d g. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w rlap with each other. With overlap with the high-ﬁeld , they are suppressed more b, the collapse region of κ/T 2b denote the suppression transport at 0.34 K, the MTC is as large as 100% at 0.25 K and even 650% at 0.7 K. For other QSL candidates such as κ-(BEDT TTF)2Cu2(CN)3 (ref. 26) and YbMgGaO4 (ref. 27), there is also a positive MTC, but the magnitude is much smaller. Unexpectedly, a crossover from the positive MTCs at low temperatures to the negative MTCs at high temperatures is observed at θw energy scale (for details, see Supplementary Note 4). We will come back to discuss the origin of this giant MTC later. We would like to emphasize that besides the electron therma conductivities, κ above 5 T is entirely due to phonons withou magnetic scatterings. First, a metamagnetic transition at Bc ~ 2.3 T can be induced only when applying the ﬁeld along the [111 direction in the magnetization M(B) measurements8. This mean that a sizeable fraction of the “2-in, 2-out” conﬁgurations are transformed into the “3-in, 1-out” conﬁgurations at the critica ﬁeld. Therefore, it is natural to expect that the scatterings between the local spins and phonons will be reduced as the magnetization approaches saturation. In fact, the thermal conductivity of 3 T i quite close to the value of 5 T and 7 T, which coincides with the critical ﬁeld observed in the magnetization measurements. Second the thermal conductivities are ﬁeld-independent between 5 T and 7 T. This indicates that even if the magnetic moments are not fully static in our experiment temperature range, they do not scatter phonons since the ﬁeld has no effect on the thermal conductivities This behavior strongly suggests that the high-ﬁeld therma conductivities are due solely to phonons without magnetic scatterings. Note that there may remain scatterings from structure disorder like stacking faults and grain boundaries. Veriﬁcation of Wiedemann–Franz Law. In Fig. 3, we ﬁt the thermal conductivity data below 0.3 K for µ0H = 5 T to examine the WF law in Pr2Ir2O7. At ultra-low temperatures, therma conductivity usually can be ﬁtted to κ/T = a + bTα−1 where aT a The thermal conductivity of ﬁelds along the [111] direction. rongly suppressed above Ts ≈ n (a) after subtracting the data elow the temperature at which ture is deﬁned as Ts. Inset: ﬁeld e Ts. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w Fig. 4 The giant isotopic magneto-thermal conductivity and anisotropic magneto-speciﬁc heat in Pr2Ir2O7. a The thermal conductivities of Pr2Ir2O7 single crystal in magnetic ﬁelds H // [111] and H ⊥Q (empty symbols) are compared with those in H ⊥[111] and H ⊥Q (ﬁlled symbols), where Q is the heat current. b They are also compared with those in H ⊥[111] and H // Q (half-ﬁlled symbols). The schematic illustrations of these three geometries are also shown. The overlap of these curves in all three ﬁeld directions show that the MTCs are isotropic. c The speciﬁc heats of Pr2Ir2O7 in magnetic ﬁelds H // [111] (empty symbols) are also compared with those in H ⊥[111] (ﬁlled symbols). Contrary to the magneto-thermal conductivities, anisotropy of magneto-speciﬁc heats is clearly seen in these two ﬁeld directions. plotted in Fig. 3. The ﬁtting gives κ0/T = 0.033 ± 0.006 mW K−2 cm−1 and α = 2.62 ± 0.12. Since it has ρ0(5 T) = 755 µΩ cm, thus L0/ρ0(5 T) = 0.032 mW K−2 cm−1, the WF law is also veriﬁed in sample B. The veriﬁcation of WF law above µ0H = 5 T is rea- sonable, because the thermal conductivity above µ0H = 5 T below 0.8 K is purely contributed from normal electrons and phonons, without other exotic excitations or magnetic scatterings. g g Since the thermal conductivity data at low ﬁelds collapse on the high-ﬁeld data below Ts (see Fig. 2b) and the MR is less than 2% for µ0H ≤5 T (see Fig. 1b), it would be inferred that the WF law is obeyed at all the applied ﬁelds, even at zero ﬁeld. This result is of great help to characterize the quantum criticality in Pr2Ir2O7. Many efforts have been made to describe the QCP phenomena30, among which two formalisms are highlighted: The Hertz-Millis formalism31,32 and the Kondo breakdown formalism33. In the former one, the critical ﬂuctuations are centered at a small part of the Fermi surface, called hot spots, leaving the majority unaffected and the electrons retaining as Landau quasiparticles. The WF law will be satisﬁed in this type of QCP due to the integrity of the electrons. In the latter one, the hot spots cover the whole Fermi surface and the critical ﬂuctuations reconstruct the Fermi surface abruptly. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w The WF law will be violated in this type of QCP due to the breakdown of quasiparticles. The veriﬁcation of the WF law in Pr2Ir2O7 at its QCP unambiguously excludes the possibility of the breakdown of Landau quasiparticles, and is incompatible with the Kondo breakdown formalism. However, this does not immediately indicate a Hertz–Millis type quantum criticality in Pr2Ir2O7, because the scaling exponents in the magnetic Grüneisen ratio experiment differ from the expectations within the Hertz–Millis theory10. Therefore, new kinds of quantum criticality where the quasiparticle picture is applicable may be realized in Pr2Ir2O7, and the conﬁrmation of the WF law puts strong constraints on the description of such QCP. Absence of positive contributions to κ from magnetic excita- tions. Also, the veriﬁcation of WF law in Pr2Ir2O7 demonstrates that there is no additional contribution to the thermal con- ductivity from mobile fermionic magnetic excitations. Further- more, since the phonon thermal conductivity in high ﬁelds deﬁnes the upper boundary of κ in Pr2Ir2O7, there is also no positive contribution to κ from other bosonic magnetic excita- tions. For pyrochlore Pr2Ir2O7, one scenario to describe its pos- sible spin liquid state is the QSI (refs. 3,13). Three topological excitations, including photon, vison, and magnetic monopole, may emerge from the ground state3. The gapless photons have a rather narrow bandwidth, about 1/1000 of the nearest-neighbor coupling Jzz (ref. 34). Since Jzz of Pr2Ir2O7 is only 1.4 K (ref. 8), the photons are deﬁnitely beyond the accessible temperature regime of our experiment. The gap of visons is about J3⊥/J2zz, in which J⊥ is the transverse exchange coupling between local moments3. Taking J⊥/Jzz = 0.3, a typical value for QSI materials34–36, the vison gap is estimated as 40 mK for Pr2Ir2O7, which is also likely beyond the experiment temperature range. For magnetic mono- poles, however, with the gap comparable to Jzz, the thermally excited magnetic monopoles should be detectable in our tem- perature range. But we do not observe their positive contributions here. One possibility is that the velocity and/or mean free path of these excitations may be too small so that their contribution to κ is negligible comparing to that of phonons. Another possibility is that the ground state of Pr2Ir2O7 is not a QSI, thus the above- mentioned magnetic excitations do not exist. Fig. 4 The giant isotopic magneto-thermal conductivity and anisotropic magneto-speciﬁc heat in Pr2Ir2O7. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w With decreasing the ﬁeld, while κ/T data still overlap with the high-ﬁeld curves below a certain temperature Ts, they are suppressed more and more strongly above Ts. In Fig. 2b, the collapse region of κ/T is shown in detail. The arrows in Fig. 2b denote the suppression temperatures Ts at corresponding ﬁelds. (κ(H)-κ(5 T))/T start to deviate from 0 above Ts. At zero ﬁeld, Ts is about 0.12 K. Similar behavior is also observed in another sample B (for details, see Supplementary Note 3). The ﬁeld-dependence of Ts is plotted in the inset of Fig. 2b. Veriﬁcation of Wiedemann–Franz Law. In Fig. 3, we ﬁt the thermal conductivity data below 0.3 K for µ0H = 5 T to examine the WF law in Pr2Ir2O7. At ultra-low temperatures, thermal conductivity usually can be ﬁtted to κ/T = a + bTα−1, where aT represents electrons and other fermionic quasiparticles such as spinons, while bTα represents phonons and other bosonic qua- siparticles such as magnons28,29. For phonons, the power α is typically between 2 and 3, due to the specular reﬂections at the sample surfaces28,29. The ﬁtting gives κ0/T ≡a = 0.031 ± 0.008 mW K−2 cm−1 and α = 2.41 ± 0.13 for sample A. From Fig. 1b, ρ0(5 T) = 764 µΩ cm is estimated, giving the WF law expectation L0/ρ0(5 T) = 0.032 mW K−2 cm−1 with L0 = 2.45 × 10 −8 W Ω K−2. Therefore, the WF law is veriﬁed nicely. In order to conﬁrm this result, the data of another sample B are also The magneto-thermal conductivity MTC = Δκ(H)/κ(0 T) = (κ(H) −κ(0 T))/κ(0 T) × 100% at various temperatures is plotted in Fig. 2c. MTC tends to saturate above 5 T below 0.8 K, when the thermal conductivity curves start to overlap with each other. In contrast to the magnetoresistance of less than 5% in charge 3 NATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunication NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w The possible observation of the quadrupole moment scatterings in Pr2Ir2O7 suggests the presence of quantum ﬂuctuations, which is a characteristic of QSI state. Indeed, due to the relatively small magnitude of the Pr3+ moments which reduces the dipolar interactions, quadrupolar interactions are expected to be important in Pr-based pyrochlore compounds, and quantum ﬂuctuations therein can melt classical spin ice states46. This has been experimentally evidenced both in Pr2Sn2O7 and Pr2Zr2O7 (refs. 47,48). To our knowledge, the roles of quadrupole moments and quantum ﬂuctuations in Pr2Ir2O7 have not been explored experimentally. Our results present the evidence of quantum effect in this spin liquid candidate, and put its 4 f moments in line with those in other Pr-based QSI candidates. Now that the well-deﬁned magnetic excitations such as magnetic monopoles neither contribute to κ nor suppress κ in Pr2Ir2O7, the strong scattering of phonons at low ﬁelds may be associated with the transverse quantum ﬂuctuations. Pr3+ can be represented by a 1/2-pseudospin. The z component along the local <111> direction carries a magnetic dipole moment, while the transverse component of the pseudospin corresponds to a quadrupole moment. Transverse ﬂuctuations of moments away from the quantization axis, characteristics of the quantum spin ice, can induce quantum dynamics, and may play an important role in this system. Intuitively, the transverse ﬂuctuations are weakened with lowering the temperature and increasing the ﬁeld. For Pr2Ir2O7, a bifurcation of the ﬁeld-cooled and zero-ﬁeld cooled magnetic susceptibility curves at about 0.12 K suggests that a partial fraction of spins freezes6,8, which coincides with Ts of zero ﬁeld in our thermal conductivity measurements. Therefore, this scenario may explain the temperature and ﬁeld dependence of thermal conductivity in Pr2Ir2O7. In each ﬁeld, the ﬂuctuations are very slow below Ts, due to the partially frozen spins, so that they do not scatter phonons. The ﬁeld will further weaken the phonon scatterings, thus the Ts increases with increasing ﬁeld. Such a simple scenario was used to interpret the thermal conductivity of YbMgGaO4 (ref. 27). One may consider whether it can also apply to the heat transport behavior of other QSI candidates such as Pr2Zr2O7 (ref. 36), as recently pointed out by Rau and Gingras in ref. 42. Nevertheless, unlike the anisotropic ﬂuctuations found in other spin ice materials, the ﬂuctuations in Pr2Ir2O7 should be isotropic in response to the applied ﬁeld. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w However, it has been well established that different magnetic structures may emerge in different ﬁeld directions in the dipole spin-ice formalism37. For example, threefold degenerate kagome- ice states can be stabilized for H // [111], while ﬁelds along [110] can align the spins on two independent chains. These different magnetic structures induce highly anisotropic speciﬁc heat in Dy2Ti2O7 (refs. 38,39). Especially, the different ﬁeld directions allow the Dy moments to develop phase transitions by decoupling some of the Ising moments40,41. For Pr2Ir2O7, anisotropic speciﬁc heats at μ0H = 2 T and 5 T in two ﬁeld directions are also observed, as shown in Fig. 4c. The low-temperature upturn is attributed to the Schottky anomaly from Pr nuclei. Since speciﬁc heats of phonons and electrons are isotropic in response to magnetic ﬁelds, the anisotropic part must be contributed from the spin system, likely the dipole moments as in Dy2Ti2O7. As a result, the scattering rate between phonons and monopoles Γp-m should be anisotropic, if one only considers the classical spin ice scenario originating from the local dipole moments. This leads to an anisotropic phonon thermal conductivity κp = 1/3Cpv2p/(Γp-m + Γother), where Cp, vp, Γother, are the phonon speciﬁc heat, phonon velocity and scattering rate from other mechanism like defects, respectively. Therefore, the isotropic MTC in Pr2Ir2O7 indicates that the scattering of phonons is unlikely from magnetic monopoles, the quasiparticles emerging from spin-ice physics due to ﬂips of dipole moments. Pr2Ir2O7 indicates that it is not ﬂuctuations from Ising dipole moments but ﬂuctuations from transverse part, likely quadrupole moments, that scatter phonons strongly above Ts. This implica- tion is important since the transverse interactions in addition to the classical Ising interactions is a key ingredient to drive the classical spin ice state into the U(1) QSI state3. For a simplest example, when introducing a transverse coupling J⊥into a clas- sical spin ice model, the resulting Hamiltonian H ¼ X <i;j> Jzzτz i τz j J? τþ i τ j þ τ i τþ j h i ð1Þ ð1Þ can result in quantum ﬂuctuations and capture the universal properties of U(1) QSI states, where pseudospin-1/2 component τz is along the local <111> direction carrying a magnetic dipole moment, and τx,y represent the quadrupole moments giving τ± = τx ± iτy (ref. 3). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w The magnetic excitation is another characteristic besides the quantum ﬂuctuation that determines whether a material lies in the QSI ground state. It has been claimed that the thermally excited magnetic monopoles contribute to the thermal conductivity in the QSI candidate Yb2Ti2O7, with the mean free path of such monopoles as long as 100 nm (ref. 45). More exotically, a two-gap behavior and an enhancement below 200 mK are observed in the thermal conductivity of another QSI candidate Pr2Zr2O7, which are suggested as the signatures of the three emergent excitations predicted in QSI materials36. However, here in Pr2Ir2O7, neither positive contributions nor negative contributions to κ from well- deﬁned magnetic excitations are detected. This may be a deviation from the QSI scenario despite the presence of quantum ﬂuctua- tions. However, we should note that although no contributions from magnetic excitations are detected in another QSI candidate Tb2Ti2O7 by the longitudinal heat transport study44, large thermal Hall effect is observed and indicates the existence of possible neutral spin excitations49. Therefore, thermal Hall measurements would be intriguing as a tool to determine the true ground state and emergent excitations in Pr2Ir2O7. The above discussion is restricted in the dipole spin ice or QSI categories. However, due to the unique metallic nature of Pr2Ir2O7, which is distinct from any other insulating spin ice candidates, the interactions between Pr 4 f moments and Ir 5d itinerant electrons do complicate the microscopic description and induce further correlations besides the spin–ice correlations6–10. When considering the effect of conduction d electrons, another possible origin of giant isotropic MTC may be the Kondo cou- pling between conduction electrons and local moments. The complex f-d coupling between Pr moments and Ir electrons may well give rise to a magneto-thermal response which is itself iso- tropic. The Kondo coupling is also suggested as the ingredient to produce the divergence in the magnetic Grüneisen ratio measurements50,51, which is also independent of ﬁeld directions. Interestingly, a scaling behavior of phonon thermal conductivities is observed, similar to the magnetic Grüneisen ratio10 (for details, see Supplementary Note 2). To check whether the f-d interactions NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w a The thermal conductivities of Pr2Ir2O7 single crystal in magnetic ﬁelds H // [111] and H ⊥Q (empty symbols) are compared with those in H ⊥[111] and H ⊥Q (ﬁlled symbols), where Q is the heat current. b They are also compared with those in H ⊥[111] and H // Q (half-ﬁlled symbols). The schematic illustrations of these three geometries are also shown. The overlap of these curves in all three ﬁeld directions show that the MTCs are isotropic. c The speciﬁc heats of Pr2Ir2O7 in magnetic ﬁelds H // [111] (empty symbols) are also compared with those in H ⊥[111] (ﬁlled symbols). Contrary to the magneto-thermal conductivities, anisotropy of magneto-speciﬁc heats is clearly seen in these two ﬁeld directions. suppression of κ at low ﬁelds apparently comes from the scat- tering of phonons by the spin system through the spin-lattice coupling, either by well-deﬁned excitations like magnetic mono- poles, which accompany ﬂips of the magnetic dipole moments pointing along the local <111> axes, or by transverse quantum ﬂuctuations, possibly including the quadrupole moments of Pr3+. Since the Ir 5d spins are Pauli paramagnetic and polarized due to the Kondo coupling6,7, the Ir moments are not responsible for the scattering process. In order to examine these possibilities, we measure the thermal conductivity in other two different ﬁeld directions and compare to the [111] direction, as shown in Fig. 4a and b. One can see that the curves in all three ﬁeld directions overlap with each other. It shows that the MTC is isotropic, i.e., insensitive to the direction of ﬁeld. Giant isotropic MTC and its origin. Now let us discuss the origin of the giant MTC in Pr2Ir2O7. In Fig. 2a, starting from the phonon thermal conductivity in high ﬁelds, the strong ATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunications 4 ARTICLE Methods S l Sample preparation. We prepare the high-quality single crystals of Pr2Ir2O7 using the KF ﬂux methods, as described in ref. 14. A polycrystalline sample of Pr2Ir2O7 was ﬁrst prepared by the solid-state reaction using stoichiometric starting materials Pr2O3(99.9%) and IrO2(99.9%). Then, single crystals were grown by combing the polycrystalline Pr2Ir2O7 with potassium ﬂuoride (KF) ﬂux in a ratio of 1:200. After being heated at 1100 °C for 36 h, the as-grown sample was slowly cooled down to 800 °C at a rate of 1 °C/h. The single crystals were obtained from the ﬂux by dissolving the ﬂux in water. The x-ray diffraction (XRD) measurement was per- formed on a typical Pr2Ir2O7 sample by using an x-ray diffractometer (D8 Advance, Bruker), and determined the largest surface to be the (111) plane (see Supplementary Note 1). 15. Tanatar, M. A., Paglione, J., Petrovic, C. & Taillefer, L. Anisotropic violation of the Wiedemann-Franz law at a quantum critical point. Science 316, 1320–1322 (2007). 16. Pfau, H. et al. Thermal and electrical transport across a magnetic quantum critical point. Nature 484, 493–497 (2012). p ( ) 17. Machida, Y. et al. Veriﬁcation of the Wiedemann-Franz law in YbRh2Si2 at a quantum critical point. Phys. Rev. Lett. 110, 236402 (2013). quantum critical point. Phys. Rev. Lett. 110, 236402 (2013) 18. Reid, J.-Ph et al. Wiedemann-Franz law and nonvanishing temperature scale across the ﬁeld-tuned quantum critical point of YbRh2Si2. Phys. Rev. B 89, 045130 (2014). 19. Dong, J. K., Tokiwa, Y., Bud’ko, S. L., Canﬁeld, P. C. & Gegenwart, P. Anomalous reduction of the Lorenz ratio at the quantum critical point in YbAgGe. Phys. Rev. Lett. 110, 176402 (2013). Speciﬁc heat measurements. The low-temperature speciﬁc heat was measured by the relaxation method in a physical property measurement system (PPMS, Quantum Design) equipped with a dilution refrigerator. 20. Kambe, S., Suderow, H., Fukuhara, T., Flouquet, J. & Takimoto, T. Spin- ﬂuctuation mediated thermal conductivity around the magnetic instability of CeNi2Ge2. J. Low. Temp. Phys. 117, 101–112 (1999). CeNi2Ge2. J. Low. Temp. Phys. 117, 101–112 (1999). 21. Ronning, F. et al. Thermal conductivity in the vicinity of the quantum critical end point in Sr3Ru2O7. Phys. Rev. Lett. 97, 067005 (2006). Charge and heat transport measurements. Discussion A i l As a topical system, thermal conductivities have been measured in various spin ice materials36,43–45. Highly anisotropic MTCs were discovered both in Dy2Ti2O7 and in another QSI candidate with non-Kramers doublets Tb2Ti2O7 (refs. 43,44). The aniso- tropic MTC in Dy2Ti2O7 was considered as a consequence of the mobility of magnetic monopole excitations in spin ice43. The anisotropic MTC with respect to H // [111] and H ⊥[111] in Tb2Ti2O7 was interpreted as a result of the scattering with pho- nons by anisotropic ﬂuctuating spins44. These behaviors are quite contrary to the isotropic MTC in Pr2Ir2O7. The isotropic MTC in 5 NATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w result in the isotropic MTC, it is meaningful to measure the ﬁeld orientation dependence of κ in Pr2Zr2O7, where both quantum ﬂuctuations and three exotic magnetic excitations were claimed to be observed without the contamination of conduction electrons36,48. Our results raise the question how interactions between the local moment system of a spin-ice state and con- duction electrons of a non-Fermi liquid state affect the heat transport properties and the ground state of Pr2Ir2O7. 2. Castelnovo, C., Moessner, R. & Sondhi, S. L. Magnetic monopoles in spin ice. Nature 451, 42–45 (2008). 3. Gingras, M. J. P. & McClarty, P. A. Quantum spin ice: a search for gapless quantum spin liquids in pyrochlore magnets. Rep. Prog. Phys. 77, 056501 (2014). 4. 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A giant isotropic magneto-thermal conductivity is found at ﬁnite temperatures, indicating that the quadrupolar interactions and quantum ﬂuctuations may play important roles. Methods S l One Pr2Ir2O7 single crystal (sample A) for electric and thermal conductivity measurements was cut and polished into a rectangular shape of dimensions 0.69 × 0.38 mm2 in the (111) plane, with a thickness of 0.20 mm. Another Pr2Ir2O7 single crystal (Sample B) with the dimension of 0.72 × 0.62 × 0.20 mm3 was also measured to check the reproduci- bility of the transport results (for details, see Supplementary Note 3). If not spe- ciﬁed, the data in the main text were taken on Sample A. The electric resistivities were measured using four-contact geometry in a 3He cryostat. The thermal con- ductivities at sub-Kelvin temperatures were measured in a dilution refrigerator, using a standard four-wire steady-state method with two RuO2 chip thermometers, calibrated in situ against a reference RuO2 thermometer. 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Nature 464, 199–208 (2010). NATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunic 6 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-20562-w 35. Ross, K. A., Savary, L., Gaulin, B. D. & Balents, L. Quantum excitations in quantum spin ice. Phys. Rev. X 1, 021002 (2011). 11421404), the NSAF (Grant No.: U1630248), the Ministry of Science and Technology of China (Grant No.: 2016YFA0300503), the Shanghai Municipal Science and Technology Major Project (Grant No. 2019SHZDZX01), and the fundamental research funds for central universities (2018KFYYXJJ038). q p y 36. Tokiwa, Y. et al. Discovery of emergent photon and monopoles in a quantum spin liquid. J. Phys. Soc. Jpn. 87, 064702 (2018). p q y p 37. Fukazawa, H., Melko, R. G., Higashinaka, R., Maeno, Y. & Gingras, M. J. P. Magnetic anisotropy of the spin-ice compound Dy2Ti2O7. Phys. Rev. B 65, 054410 (2002). p q y p 37. Fukazawa, H., Melko, R. 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Lett. 105, 047201 (2010). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 47. Zhou, H. D. et al. Dynamic spin ice: Pr2Sn2O7. Phys. Rev. Lett. 101, 227204 (2008). 48. Kimura, K. et al. Quantum ﬂuctuations in spin-ice-like Pr2Zr2O7. Nat. Commun. 4, 1934 (2013). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 49. Hirschberger, M., Krizan, J. W., Cava, R. J. & Ong, N. P. Large thermal Hall conductivity of neutral spin excitations in a frustrated quantum magnet. Science 348, 106 (2015). 50. Vojta, M. Frustration and quantum criticality. Rep. Prog. Phys. 81, 064501 (2018). 51. Oh, H., Lee, S., Kim, Y. B. & Moon, E.-G. Multiscale quantum criticality driven by Kondo lattice coupling in pyrochlore systems. Phys. Rev. Lett. 122, 167201 (2019). Author contributions S.Y.L. conceived the idea and designed the experiments. J.M.N. performed the speciﬁc heat and transport measurements with help from Y.Y.H., E.J.C., Y.J.Y., B.L.P., and Q.L. L. M.X. and Z.M.T synthesized the single crystal samples. J.M.N. and S.Y.L. wrote the manuscript with comments from all authors. 38. Hiroi, Z., Matsuhira, K., Takagi, S., Tayama, T. & Sakakibara, T. Speciﬁc heat of Kagome ice in the pyrochlore oxide Dy2Ti2O7. J. Phys. Soc. Jpn 72, 411 (2003). 39. Hiroi, Z., Matsuhira, K. & Ogata, M. Ferromagnetic ising spin chains emerging from the spin ice under magnetic ﬁeld. J. Phys. Soc. Jpn 72, 3045 (2003). p g The authors declare no competing interests. 41. Ruff, J. P. C., Melko, R. G. & Gingras, M. J. P. Finite-temperature transitions in dipolar spin ice in a large magnetic ﬁeld. Phys. Rev. Lett. 95, 097205 (2005). Competing interests g y p 40. Ramirez, A. P., Hayashi, A., Cava, R. J., Siddharthan, R. & Shastry, B. S. Zero- point entropy in ‘spin ice’. Nature 399, 333 (1999). Additional information 42. Rau, J. G. & Gingras, M. J. P. Frustrated quantum rare earth pyrochlores. Annu. Rev. Condens. Matter Phys. 10, 357–386 (2019). Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-20562-w. Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-20562-w. y 43. Kolland, G., Valldor, M., Hiertz, M., Frielingsdorf, J. & Lorenz, T. Anisotropic heat transport via monopoles in the spin-ice compound Dy2Ti2O7. Phys. Rev. B 88, 054406 (2013). Correspondence and requests for materials should be addressed to Z.M.T. or S.Y.L. © The Author(s) 2021 Acknowledgements We thank E.-G. Moon, Z.Y. Meng, Y. Wan, and Y. Zhou for helpful discussions. This work is supported by the Natural Science Foundation of China (Grant No. 12034004 and 7 NATURE COMMUNICATIONS \| (2021) 12:307 \| https://doi.org/10.1038/s41467-020-20562-w \| www.nature.com/naturecommunications
https://openalex.org/W2809007063	https://europepmc.org/articles/pmc6079211?pdf=render	English	null	Factorized Computation: What the Neocortex Can Tell Us About the Future of Computing	Frontiers in computational neuroscience	2,018	cc-by	3,114	Factorized Computation: What the Neocortex Can Tell Us About the Future of Computing Keywords: factorized computation, relational networks, uniﬁed brain theory, non-sequential, parallelization OPINION published: 31 July 2018 doi: 10.3389/fncom.2018.00054 OPINION published: 31 July 2018 doi: 10.3389/fncom.2018.00054 1Some formalisms express the entire problem as a product or conjunction of many terms, and in this case the terms are factors in a multiplicative sense. Other formalisms are not based on multiplication, but we still speak of the relations as factors that inﬂuence the desired computation, and we speak of a computation as having been factorized when it has been broken down into the relations that deﬁne it. 1. INTRODUCTION In ancient Greece our brains were presumed to be mainly important for cooling our bodies. When humanity started to understand that our brains are important for thinking, the way it would be explained was with water pump systems as this was one of the most sophisticated models at the time. In the nineteenth century, when we started to utilize electricity it became apparent that our brains also use electrical signals. Then, in the twentieth century, we deﬁned algorithms, improved electrical engineering and invented the computer. Those inventions prevail as some of the most common comparisons of how our brains might work. When taking a step back and comparing what we know from electrophysiology, anatomy, psychology, and medicine to current computational models of the neocortex, it becomes apparent that our traditional deﬁnition of an algorithm and of what it means to “compute” needs to be adjusted to be more applicable to the neocortex. More speciﬁcally, the traditional conversion from “input” to “output” is not as well deﬁned when considering brain areas representing diﬀerent aspects of the same scene. Consider for example reading this paper: while the input is quite clearly visual, it is not obvious what the desired output is besides maybe turning to the next page, but this should not be the goal in itself. Instead, the more interesting aspect is the change of state in diﬀerent areas of the brain and the corresponding changes in states of neurons. There are many types of models that have the interaction of modules as the central aspect. Among those are: Edited by: Si Wu, Peking University, China Reviewed by: Masami Tatsuno, University of Lethbridge, Canada *Correspondence: Matthew Cook cook@ini.ethz.ch Received: 11 January 2018 Accepted: 19 June 2018 Published: 31 July 2018 Citation: Diehl PU, Martel J, Buhmann J and Cook M (2018) Factorized Computation: What the Neocortex Can Tell Us About the Future of Computing. Front. Comput. Neurosci. 12:54. doi: 10.3389/fncom.2018.00054 Reviewed by: Masami Tatsuno, University of Lethbridge, Canada • Belief propagation • Dynamic ﬁelds • Relational networks • Interaction of brain areas in many psychological models • Basically all models describing ongoing interactions between neurons, e.g., STDP • Basically all models describing ongoing interactions between neurons, e.g., STDP They all use what we will refer to as “Factorized Computation.” Factorized Computation describes a common framework for distributed processing mechanisms and systems. The term Factorized Computation refers to how a problem gets factorized (decomposed) into smaller sub-problems such that many nodes are working together1. Typically, problems are broken down into a large set of small relations, whose composition represents the problem to be addressed. Citation: 1Some formalisms express the entire problem as a product or conjunction of many terms, and in this case the terms are factors in a multiplicative sense. Other formalisms are not based on multiplication, but we still speak of the relations as factors that inﬂuence the desired computation, and we speak of a computation as having been factorized when it has been broken down into the relations that deﬁne it. July 2018 \| Volume 12 \| Article 54 Frontiers in Computational Neuroscience \| www.frontiersin.org 1 Factorized Computation Diehl et al. of the individual that we see a diﬃculty with parallelization. Similarly, the multiple cores of a CPU or GPU can also all work simultaneously, barely aware of each other. It is only at the single core level that operations take on a purely sequential nature. This is not a coincidence, since after all, the theoretical understanding underlying a core’s operation (and indeed, underlying much of theoretical computer science) is historically based on modeling what an individual person does. This decomposition diﬀers from the conventional approach of breaking a problem down into a sequence of subproblems, in that in Factorized Computation the subproblems are interdependent, and they are solved jointly. There is no order for solving the subproblems, just like we don’t solve equations one by one–they must be solved together. Just as functions are deﬁned in terms of other functions or procedures (down to built-in ones), and are chained into sequences in order to transform the input into the output, relations can be deﬁned in terms of other relations (down to built-in ones), and can be arranged automatically into networks of variable nodes and relation nodes, with a connection wherever a variable is involved in a relation. This individual-thread approach to computing is largely a result of how we have trained ourselves to think and reason about computation. The idea of “computing” meaning the “execution of a program,” and a “program” as a “sequence of operations,” is very deeply ingrained in us, to the point that we are quick to see any computational process as the execution of a program in some sense. 1.1. The “Individual-Thread” Approach The ﬁrst computing architectures ever made implemented an idea that seems very natural to us: A task can be performed by dividing it into several sub-tasks to be carried out in a sequential order. As humans, this idea is natural to us since this is how we typically approach tasks in the world around us. We do one sub-task at a time, whether for physical tasks or paper-and-pencil tasks. Even when speaking about thought processes, we refer to “a train of thoughts,” reﬂecting our view that thinking involves a sequence of thoughts. Instructions are often arranged for clarity in numbered steps, making their sequential nature explicit. We may be instructed to skip to another point in the sequence, but we are practically never instructed to carry out two or more such sequences simultaneously. Citation: If there are other frameworks that would be better suited for understanding a given computation, we often don’t even notice them, because we are so quick to understand distributed computations in terms of the framework we are used to: a large number of parallel, individual threads, each following a sequence of instructions. In this article our goal is to outline how ﬁnding and using a unifying framework for the aforementioned models can beneﬁt our understanding of the neocortex, and how the process of understanding the neocortex can inspire new models and build a foundation for the next generation of hardware. Frontiers in Computational Neuroscience \| www.frontiersin.org 2. A RECURRING THEME: FACTORIZED COMPUTATION Many of the formalisms that have been proposed for speciﬁc types of parallel computations share an underlying theme in how they are structured and many separate developments in diﬀerent ﬁelds have repeatedly led to a similar style of distributed calculation, which we refer to collectively as Factorized Computation. Speciﬁc instances include Bayesian Brain theories (Knill and Pouget, 2004), dynamic ﬁelds (Wilimzig and Schöner, 2005), and relational networks (Diehl and Cook, 2016). Also, neuroanatomy tells us that connections between areas are almost always reciprocal/bidirectional (not on a per- neuron level but on a per-area level) (Felleman and Van Essen, 1991). This is in stark contrast to traditional feed-forward processing models and since reciprocal connections double the required resources, we would expect there to be a clear need for these connections in brain models. 2.1. A Simple Example As an example, a node in a network might represent the relation A + B = C. This node would be connected to other nodes involving these variables; for example, it might have a neighbor representing C + D = E −F, and these two nodes would send each other information about the value of C. If the A + B = C node hears from its neighbors that A is around 2, B is around 3, and C is around 4, then it might in turn tell its neighbors that A is probably a little less than 2, B is probably a little less than 3, and C is probably a little more than 4. Or, with a diﬀerent formalism for how to interpret values, if it hears that A = 2 and B ≥3, it might tell its neighbors that C ≥5. Other formalisms might send samples from distributions representing the current posterior for the variable, or send the set of remaining possible values for the variable as inconsistent values get ruled out, or simply send the current best estimate of the variable’s value. There are many such formalisms (Hinton and Sejnowski, 1986; Rumelhart and McClelland, 1987; Wegener, 1987; Shapiro, 1989; Dechter, 2003; Diehl and Cook, 2016), but the intuition behind creating the network is the same in each case: The variables are all the quantities you think about or compute when thinking about the problem, and the relations encode the way in which these variables are related to each other. Another direction where Factorized Computation is well- suited to applications is in understanding and re-engineering vision systems, where it is possible to use a large relational network to decompose sensory input into multiple physically- meaningful modalities. For example, from a retina or a dynamic vision sensor which reports only changes in light intensity without absolute brightness information, a network can infer the optic ﬂow, motion, and even the missing brightness information (Martel et al., 2015). Such networks are in principle capable of integrating input from more types of sensors, such as vestibular system (accelerometers), auditory system and so on. The Factorized Computation framework provides a very natural and general method for the problem of sensor fusion, where the more sensors you include, even of completely diﬀerent types, the more accurate the entire system will be. Down? (Viterbi, 1967), and 20 years later it was developed yet again to reason about causal relationships in the ﬁeld of artiﬁcial intelligence (Pearl, 1988). After another decade, in retrospect (Kschischang et al., 2001) it became clear that a lot of eﬀort could have been saved if the general form of these algorithms had been explicitly understood, rather than being worked out repeatedly and independently for what now appear to be various special cases of a general theme. model those quantities and their relationships, instead of modeling the sequence of steps you would take if doing the computation yourself. This is like writing down the equations that constrain the answer, without specifying how to solve the equations. This approach is almost the opposite of the standard parallelization of the “embarrassingly parallel” parts of a problem. The parts of a Factorized Computation are not independent, but are continuously communicating with each other in order to approach a solution. 3.2. Factorized Computation: A Direction There are many existing examples of systems within neuroscience which can be understood as Factorized Computation, such as belief propagation (Knill and Pouget, 2004), dynamic ﬁelds (Wilimzig and Schöner, 2005), relational networks (Diehl and Cook, 2016), or the interaction of brain areas in many psychological models. As we try to gain a more complete and uniﬁed understanding of this framework, a number of directions already present themselves. One direction that has the potential to greatly increase the range of applicability of Factorized Computation is improving the automated learning of latent variables and the relations between them. This direction includes how relations in deep hidden layers can be trained, as well as how other encodings such as temporal sequences can be usefully learned and used. We could even ask how the network of variables and relations could itself arise from a homogeneous sheet of computing elements, much as the cortical sheet of the brain self-organizes into areas that specialize in speciﬁc tasks. 2.1. A Simple Example Even distinct algorithms could be combined, providing “algorithm fusion” for example to combine multiple methods of computing optic ﬂow. Constructing and optimizing such brain- like frameworks and systems will allow us to better understand how our brains function and why they developed the way they did. As we can see, the calculation done at a single node is extremely simple. It is not a powerful equation solver—it is more like a neuron, capable of only a ﬁxed operation. Just like neurons, the power of these systems arises from having many nodes working together. Down? High level routines (in code) or procedures (for people) are often speciﬁed as a sequence of lower level routines, which themselves also use the individual-thread approach, i.e., they call a sequence of even lower level routines, and so on. Is this, then, what computation is, at heart? Is this the general method used by objects that compute? Factorized Computation is closely related to relational processing, an umbrella term covering many speciﬁc styles of setting up computations as some sort of relational network. Such a network consists of a set of variables, together with a set of relations between those variables, such as clauses or equations. The relations in the network behave as active elements rather than being used or inspected by a separate entity tasked with satisfying them. Each relation pays attention to the variables it relates, communicating with any other relations involving the same variables. A relation listens to its neighbors’ opinions about its variables, and by considering these opinions in the light of its own mathematical relation, it forms or updates its own opinion about its variables, which it then shares with its neighbors (an example is given below). Even though it might seem, as seen from the outside, that thinking is a single threaded-activity, or that the internal workings of a CPU are single-threaded, in fact these systems are strongly parallel on the inside. Looking inside our brain, we see it has activity going on simultaneously in many diﬀerent areas, collectively contributing to achieving one task, but with each area working asynchronously under its own control. Similarly, a modern processor has many activities going on simultaneously within a busy core, to aid in the processing of the main thread. So we see that below the level of the processor (whether CPU core or human), operations are highly parallelized. At higher levels, operations are again highly parallel. For example the employees and departments of an organization all work simultaneously, whether working together on shared goals or separately on distinct goals. So it is mainly at the level For Factorized Computation, the approach to “programming” is to examine the concepts with which we understand the quantity or behavior to be computed, and to directly Frontiers in Computational Neuroscience \| www.frontiersin.org July 2018 \| Volume 12 \| Article 54 2 Factorized Computation Diehl et al. 3.1. Where Can We Go From Here? 3.1. Where Can We Go From Here? While it is nice to observe that many existing algorithms are examples of Factorized Computation, and that there are surely many more algorithms of this form waiting to be discovered, is there anything in particular that really needs to be done, or should we just sit back and enjoy the show? For comparison, if we look at the history of one particular form of Factorized Computation, namely belief propagation in factor graphs, we see that this formalism was developed multiple times over many years by many researchers. First developed in periodic forms by statistical physicists (Bethe, 1935), it was developed again 30 years later in various linear forms for decoding noisy signals in the ﬁeld of communications Frontiers in Computational Neuroscience \| www.frontiersin.org REFERENCES Rumelhart, D. E., and McClelland, J. L. (1987). Parallel Distributed Processing, Vol. 1. Cambridge, MA: MIT Press. Bethe, H. A. (1935). Statistical theory of superlattices. Proc. R. Soc. Lond. A Math. Phys. Eng. Sci. 150, 552–575. Shapiro, E. (1989). The family of concurrent logic programming languages. ACM Comput. Surv. 21, 413–510. y g Dechter, R. (2003). Constraint Processing. San Francisco, CA: Morgan Kaufmann. y g Dechter, R. (2003). Constraint Processing. San Francisco, CA: Morg Viterbi, A. J. (1967). Error bounds for convolutional codes and an asymptotically optimum decoding algorithm. IEEE Trans. Inform. Theor. 13, 260–269. Diehl, P. U., and Cook, M. (2016). Learning and inferring relations in cortical networks. arXiv:1608.08267. Felleman, D. J., and Van Essen, D. C. (1991). Distributed hierarchical processing in the primate cerebral cortex. Cereb. Cortex 1, 1–47. Wegener, I. (1987). The Complexity of Boolean Functions. New York, NY: John Wiley & Sons, Inc. the primate cerebral cortex. Cereb. Cortex 1, 1–47. Hinton, G. E., and Sejnowski, T. J. (1986). “Learning and relearning in Boltzmann machines,” in Parallel Distributed Processing: Explorations in the Microstructure of Cognition, Vol. 1, eds D. E. Rumelhart and J. L. McClelland (Cambridge, MA: MIT Press), 282–317. Wilimzig, C., and Schöner, G. (2005). “The emergence of stimulus-response associations from neural activation ﬁelds: dynamic ﬁeld theory,” in Proceedings of the 27th Annual Conference of the Cognitive Science Society (Stresa). Knill, D. C., and Pouget, A. (2004). The Bayesian brain: the role of uncertainty in neural coding and computation. Trends Neurosci. 27, 712–719. doi: 10.1016/j.tins.2004.10.007 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Kschischang, F. R., Frey, B. J., and Loeliger, H.-A. (2001). Factor graphs and the sum-product algorithm. IEEE Trans. Inform. Theor. 47, 498–519. doi: 10.1109/18.910572 Copyright © 2018 Diehl, Martel, Buhmann and Cook. 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. Martel, J. N. P., Chau, M., Dudek, P., and Cook, M. (2015). AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. July 2018 \| Volume 12 \| Article 54 Frontiers in Computational Neuroscience \| www.frontiersin.org 3 Factorized Computation Diehl et al. Frontiers in Computational Neuroscience \| www.frontiersin.org July 2018 \| Volume 12 \| Article 54 REFERENCES “Toward joint approximate inference of visual quantities on cellular processor arrays,” in 2015 IEEE International Symposium on Circuits and Systems (ISCAS) (Lisbon), 2061–2064. Pearl, J. (1988). 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https://openalex.org/W4391756894	https://www.nature.com/articles/s41599-024-02767-2.pdf	English	null	Mobile money, medical cost anxiety and welfare of individuals within the reproductive age in Malaysia	Humanities & social sciences communications	2,024	cc-by	9,130	1 Institute of Energy Policy and Research (IEPRe), Universiti Tenega Nasional (UNITEN), Kajang, Malaysia. 2 Laboratoire d’Economie Publique, Faculté des Sciences Economiques et de Gestion, Université d’Abomey-Calavi, Abomey-Calavi, Benin. 3 UNESCO-TWAS, Trieste, Italy. 4 Shaanxi University of Technology, Hanzhong, China. 5 Department of Global Health Economics and Policy, University of Kragujevac, Kragujevac, Serbia. ✉email: romik247@gmail.com HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:260 \| https://doi.org/10.1057/s41599-024-02767-2 Introduction T This is because mobile money is often more accessible and affordable than traditional banking services (Apeti, 2023). As a result, households that were before disqualiﬁed from the formal ﬁnancial sector can now access a range of ﬁnancial services, which can help to increase their overall welfare (Jakovljevic et al., 2023). The rapid development of mobile phone technologies has played a crucial role in mobile money emergence because it is an inno- vative and convenient ﬁnancial service that enables users to con- duct transactions through their mobile phones (Apeti, 2023; Kikulwe et al., 2014). In Malaysia, mobile money has become increasingly popular in recent years, particularly among rural populations (Demirgüç-Kunt et al., 2018). Indeed, according to the global Findex survey during 2014 and 2017, only 3% and 11% of people aged 15 years old and above have mobile money accounts (Demirgüç-Kunt et al., 2018). Also, in Malaysia in 2014 and 2017, only 3% in 2014 and 10% in 2017 of people aged 15 years old and above respectively paid utility bills using a mobile phone (Demir- güç-Kunt et al., 2018). This is because mobile money provides a suitable way for households to transfer money, pay bills, and access ﬁnancial services without having to visit a physical bank branch (Apeti, 2023; Munyegera and Matsumoto, 2016). For agriculture households, this has signiﬁcant implications for their overall wel- fare because mobile money adoption improves welfare (Islam et al., 2022; Kilombele et al., 2023; Tabetando et al., 2022). By reducing medical cost anxiety and improving access to ﬁnancial services, mobile money can help households manage their ﬁnances more effectively and improve their livelihoods. As such, mobile money is expected to continue to play a key role in the development of rural communities in Malaysia. The objective of this paper is to analyse the effects of mobile money and medical cost anxiety on household welfare in Malaysia. On the other hand, the paper tends to answer the following research questions: i. What is the impact of mobile money on household welfare? d d h d l ﬂ i. What is the impact of mobile money on household welfare? ii. How does anxiety associated with medical costs inﬂuence household welfare? Medical cost anxiety is a common problem among households in developing countries such as Malaysia, particularly those in rural areas (Houeninvo et al., 2023; Pradhan and Mukherjee, 2018). Indeed, Houeninvo et al. Introduction T allocations (Vuković et al., 2012) and remains a bottleneck inefﬁ- ciency in so many low- and middle-income countries’ health sys- tems (Ranabhat et al., 2020). Mobile money can help alleviate medical cost anxiety by providing households with a convenient way to save money and access ﬁnancial services (Sakyi-Nyarko et al., 2022). For example, households can use mobile money to set up savings accounts, which can be used to pay for medical expenses. T he ﬁnancial sector plays a crucial role in household welfare as it provides access to business occasions, investment, savings, consumption smoothing, and insurance against sudden events (Apeti, 2023; Demirgüç-Kunt et al., 2008; Demirguc-Kunt and Klapper, 2012; Munyegera and Matsumoto, 2016; Okafor et al., 2023). However, in emerging nations, a huge portion of the population lacks access to basic ﬁnancial services (Micah et al., 2021; Munyegera and Matsumoto, 2016). Deﬁ- ciency of access to basic ﬁnancial services limits the rural poor capacity to invest and participate in both formal and informal insurance procedures aimed at smoothing consumption and limiting poverty (Local Burden of Disease HIV Collaborators, 2021). These socioeconomic inequalities are reﬂected in health- care affordability as well, with the most prominent examples among the leading emerging BRICS (Jakovljevic, 2014) markets across the Global South nations (Jakovljevic et al., 2021). Most rapidly developing large emerging BRICS economies being the drivers of global real GDP growth (Jakovljevic et al., 2016), continue to shape the medical spending landscape worldwide. Mobile money can also be used to access insurance products, which can provide households with ﬁnancial protection in the event of a medical emergency. Thus, according to Nghiem and Connelly (2017), health care is a necessity, not a luxury good as some other studies in this ﬁeld found. So, there is a need for government to put in place health insurance accessible to their population, especially the poorest (Wu et al., 2023). In addition to reducing medical cost anxiety, mobile money can also improve the overall welfare of households in Malaysia. This is because mobile money can improve access to ﬁnancial services, which can help households manage their ﬁnances more effectively. For example, households can use mobile money to pay bills, transfer money, and access credit, which can help them to invest in their farms and improve their livelihoods. Furthermore, mobile money can also help to enhance ﬁnancial inclusion among agri- cultural households in Malaysia. Introduction T (2023) found in Benin that out-of-pocket health spending directs households to make additional spending on health care that in ﬁne crowds out expenses in other essential goods such as education items. This is because healthcare costs can be expensive, and households may not have access to adequate insurance or savings to cover these costs (Houeninvo et al., 2023; Nghiem and Connelly, 2017; Nasir et al., 2022; Sui et al., 2021). f iii. How does mobile money moderate the impact of anxiety associated with medical costs on household welfare? This study focused on Malaysia. Focusing the study on Malaysia offers a compelling rationale due to the country’s unique position as a hub of mobile money innovation and its diverse healthcare landscape. One convincing reason to concentrate on Malaysia is the country’s robust adoption of mobile money ser- vices and its progressive strides in healthcare accessibility and affordability. Also, Malaysia’s diverse population and multi- faceted healthcare system create an opportunity to analyse the interplay between mobile money, medical cost anxiety, and var- ious welfare indicators within and outside the reproductive age group, allowing for nuanced insights that could inform not only local policy but potentially broader regional strategies. The rest of the paper is organised as follows: next to the introduction section, we have the literature review section. Then, the third section is devoted to the methodology and the ﬁnal section concludes and provides some policy recommendations. The estimate by Dieleman et al. (2017) recommends that worldwide health expenditure may perhaps increase from US$8 trillion in 2018 to $18 trillion in 2040 with a forecast of about 9% of the global GDP to be assigned to health in 2040, giving to the Institute for Health Metrics and Evaluation (IHME) (Zhou et al., 2020). As a result, households can wait to seek medical treatment or forego it wholly, which can lead to serious health con- sequences. A typical consequence of this side effect of the unaf- fordability of medical care is the so-called boomerang effect (Turabian, 2019). Namely, the patient comes back later with a far more severe, neglected disease stage when he or she is in acute need of expensive hospital admission and intensive care unit care (Tyrovolas et al., 2018). Mobile money, medical cost anxiety and welfare of individuals within the reproductive age in Malaysia This study examines the association between mobile money, medical cost anxiety, and the welfare of households in Malaysia. The study made use of the data sourced from the Global ﬁnancial survey conducted by the World Bank. The study applied the logit model and test of mean difference, probit and instrumental variable regressions to check for robustness. Findings show that mobile money has a positive and signiﬁcant impact on the welfare of households in Malaysia. Mobile money enables households to access ﬁnancial services easily and conveniently, which in turn leads to better ﬁnancial management and increased household welfare. The study also ﬁnds that medical cost anxiety hurts household welfare. Households that are more anxious about medical costs tend to experience lower levels of welfare. The results of this study have signiﬁcant implications for policymakers and ﬁnancial service providers in Malaysia. To improve the welfare of households, policymakers should promote mobile money use and encourage ﬁnancial inclusion. Financial service providers should also develop products and services tailored to the needs of households, especially those that are more vulnerable to medical cost anxiety. Additionally, policymakers should consider implementing policies that address rising healthcare costs and alleviate medical cost anxiety among households in Malaysia. 1 ARTICLE Literature review Several studies around the world have examined the mobile money impact on household welfare (Apeti, 2023; Islam et al., 2022; Kikulwe et al., 2014; Kilombele et al., 2023; Munyegera and Matsumoto, 2016; Riley, 2018; Suri and Jack, 2016; Tabetando At this time in most contemporary societies such life-saving care is not being denied but instead, it is provided at a much higher cost compared to initial screening or prevention (Sahoo et al., 2023). This cost burden is covered by public or state-owned health budgetary HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:260 \| https://doi.org/10.1057/s41599-024-02767-2 2 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 indicators by nation, region, and income group. Data is also included sum by gender, income (adults living in the richest 60% and poorest 40% of households), labour force involvement (adults in or out of the workforce), age (young and older adults), and rural and urban residence. This study is based in Malaysia which is located in Southeast Asia. et al., 2022). For example, Suri and Jack (2016) analysed in Kenya the long-run impacts of mobile money and found a positive inﬂuence on households by taking out of poverty, 2% of Kenyans. The author’s ﬁndings are explained by ﬂuctuations in ﬁnancial behaviour–growing ﬁnancial resilience and savings–and efﬁcient labour sharing after a change from agriculture to business. g g g Basing their studies on Ugandan rural households, Munyegera and Matsumoto (2016) found that mobile money adoption improves households’ welfare via its capacity to ease remittances. Apeti (2023) found in developing countries that nations with mobile money demonstrate smaller consumption instability. Riley (2018) found in Tanzania that next to a village-level precipitation shock, it’s only mobile money users can avoid a decrease in their consumption. For the author the remittances received by households allow them to smooth consumption. Kikulwe et al. (2014) analysed in Kenya the impacts of mobile money tech- nology on smallholder farm households’ welfare and showed that mobile money use improves household income via remittances received from relatives and friends. Econometric approach. In this current study which analyses the link between mobile money, medical cost anxiety, and the welfare of households in Malaysia, the dependent variable is welfare. Literature review Generally, to capture welfare, the literature used per capita con- sumption expenditure (Chakrabarty and Mukherjee, 2022), and income per capita (Nguyen and Nguyen, 2019; Tabetando et al., 2022) but in this study, welfare is captured by income quintile, measured as 1 = households within the richest fourth 20% and ﬁfth 20%; and 0 = household within the poorest 20%, second 20% and middle 20%. This gives a binary character to the dependent variable and leads us to run a logit regression approach to make inferences about welfare status in Malaysian households. Due to its wide use to measure the determinants of poverty status in developing countries (Islam et al., 2022; Nguyen and Nguyen, 2019), this paper used logit models to analyse the between mobile money, medical cost anxiety, and the welfare of households in Malaysia. The logit model is speciﬁed as: Kilombele et al. (2023) showed in Tanzania that mobile money use increases maize productivity and in turn reduces poverty likelihood because in farmers who use mobile money services maize productivity is increased by about 124 kg/acre, indicating a reduction of their poverty likelihood by nearly 25%. Tabetando et al. (2022) found in Uganda that mobile money adoption improves farm welfare by 13% through the increase of the probability of using chemical fertiliser on maize plots by 11%. Islam et al. (2022) found in Bangladesh that every 1 billion Taka (approximately US$ 11.76 million) rise in mobile money trans- actions through the bKash system in 2015 is linked with a 0.71% drop in the poverty rate. ln Pj ð1 PjÞ " # ¼ δ0 þ ∑ n i¼1 δiXij þ μj ð1Þ ð1Þ Where, Pj is the probability that the jth household is within the richest fourth 20% and ﬁfth 20%, δ0 and δi are the parameters to be estimated, μj is the random error, and Xij are the explanatory variables deﬁned in Table 1. The empirical model used to investigate the association between mobile money, medical cost anxiety, and the welfare of households in Malaysia is derived from Munyegera and Matsumoto (2016), Kilombele et al. (2023) and Sakyi-Nyarko et al. (2022) studies and is described as follows: p p y Medical cost anxiety which refers to the fear of not being able to afford medical treatment or healthcare services is a common issue among households in Malaysia due to rising healthcare costs. Literature review Indeed, according to statistics from the Global Findex survey undertaken in 2021, only 19% of the Malaysian population aged 15 years and above are not worried at all about not being able to pay for medical costs in case of a serious illness or accident while 40% are very worried and 40% are somewhat worried. These statistics showed that medical cost anxiety is a signiﬁcant issue that affects the ﬁnancial well-being and quality of life of households in Malaysia. In this situation, mobile money has the potential to reduce medical cost anxiety and improve the welfare of households in Malaysia because according to the global Findex survey, the percentage of the Malaysian population aged 15 years and above who have mobile money accounts experienced an increase, moving from 3% in 2014 to 11% in 2017 and 28% in 2021. EðWFijXiÞ ¼ α0 þ α1ANXi þ α2MMi þ α3LOCi þ α4EMPi þ α5EDUCi þ εi ð2Þ ð2Þ Where E(WFi\|Xi) denote the expected state of welfare of the household i conditioned on X variables such as cost of medical services (ANXi), and mobile money (MM), among others. The interest variables are ANXi and MMi are which interest variables and refer to ﬁnancially worried: bills associated with medical costs and access and usage of mobile devices of the household i, respectively. LOCi, EMPi and EDUCi are the control variables and represent the location, employment status and educational level of household i, respectively. α0; ¼ ; α5 are the parameters to be estimated and εi is the error term. All estimation variables used, and their measurements are summarised in Table 1. To capture the interaction or moderate the impact of mobile money on anxiety associated with medical costs, we present Eq. (3) EðWFijXiÞ ¼ α0 þ α1ANXi þ α2MMi þ α3LOCi þ α4EMPi þ α5EDUCi Indeed, mobile money improves welfare by providing a means of payment for medical bills that is affordable and accessible, mobile money services can help alleviate ﬁnancial stress and anxiety among households. Policymakers in Malaysia should consider promoting mobile money services to improve the ﬁnancial well- being of households and reduce medical cost anxiety. By doing so, they can ensure that all households in Malaysia have access to affordable and accessible healthcare services. Results and discussion S i i f Summary statistics of variables. Table 2 reports the descriptive statistics of all the variables used for aggregate, within the reproductive age and not within the reproductive age. For the aggregate, the mean of the full sample for the households within the richest fourth 20% and ﬁfth 20% (WF) is 0.448. To consider the gender dimension, we computed the descriptive statistics for all the variables for males and females. The mean of males and females within the richest fourth 20% and ﬁfth 20% is 0.434 and Literature review This study comple- ments the extant literature by showing that mobile money can improve the welfare of individuals within the reproductive1 through the provision of a means of payment for medical bills that is affordable and accessible and that reduces medical cost anxiety. ð3Þ Methodology d About 0.643 of the aggregate are in the workforce with 0.524 for the male sample and 0.784 for the female sample. Concerning the educational level of the respondents, 0.834 of the aggregate are educated with 0.815 for the male sample and 0.856 for the female sample. Logit regression results. Table 3 reported the estimated results with the logit model. The probability of the Chi2 shows that the models’ speciﬁcation (aggregate, within the reproductive age and not within the reproductive age) as soon models speciﬁcation for the total, male and female are globally signiﬁcant at 1%. The outcome showed that anxiety associated with medical costs decreases the welfare of Malaysian households for the aggregate, within the reproductive age and not within the reproductive age as soon as for the total, male–female samples. For aggregate, a rise of one unit of anxiety associated with medical costs worsens the welfare of Malaysian households of 0.748, 0.697 and 0.796 for the total, male and female, respectively. Similarly, within the reproductive age, an increase of one unit of anxiety associated with medical cost lessens Malaysia households’ welfare by 0.852 (total), 0.755 (male) and 0.987 (female) while not within the reproductive age, only the ﬁndings of the female are signiﬁcant, meaning that one unit increase of anxiety associated with medical cost decrease female, not within the reproductive age welfare of 0.377. Within the reproductive age, the mean of the full sample for the households within the richest fourth 20% and ﬁfth 20% (WF) is 0.459. To consider the gender dimension, we computed the descriptive statistics for all the variables for males and females. The mean of males and females within the richest fourth 20% and ﬁfth 20% is 0.449 and 0.471, respectively. For anxiety associated with medical costs, 0.753 (total) of the households are very worried and somewhat worried while 0.747 and 0.761 of males and females are respectively very worried and somewhat worried. Within the reproductive age, the percentage of households who have access to and usage of mobile devices in payment of purchase including those associated with medical bills is 0.288. This percentage is 0.301 for males while it’s about 0.273 for females. For the full sample, about 0.761 of the households are urban residents. About 0.769 males are urban residents while 0.751 females are urban residents. Methodology d Data and sources. Data used in this study was sourced from the Global Financial Index 2021 which provides approximately 300 indicators on topics such as account ownership, payments, saving, credit, and ﬁnancial resilience. Findex data is described for all HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:260 \| https://doi.org/10.1057/s41599-024-02767-2 3 ARTICLE Table 1 Variables description and measurement. Variables Deﬁnition Measurement WF The welfare of the household Income Quintile of the household; 1 = households within the richest Fourth 20% and ﬁfth 20%; and 0 = households within the Poorest 20%, Second 20% and Middle 20% ANX Anxiety associated with medical cost Financially worried: bills associated with medical cost. 1 = Very worried and somewhat worried, and 0 = Not worried at all) LOC Location of the household 1 = urban residents, and 0 = rural residents EMP The employment status of the respondents 1 = in the workforce, 0 = out of the workforce EDUC The educational level of the respondents Educated and non-education 0 = completed secondary school or less (non-educated) 1 = completed secondary, tertiary education or more (educated) MM Mobile money Access and usage of mobile device, eg phone, etc in payment of purchases including those associated with medical bills. Source: Authors using the Global Financial Index (2021). Note: reproductive age = 15–49 years (WHO, 2021). Table 1 Variables description and measurement. males while it’s about 0.099 for females. For the full sample, about 0.700 of the households are urban residents. About 0.726 males are urban residents while 0.671 of females are urban residents. About 0.432 of the aggregate are in the workforce with 0.269 for the male sample and 0.621 for the female sample. Concerning the educational level, about 0.666 of the aggregate are educated with 0.618 for the male sample and 0.720 for the female sample. 0.465, respectively. For anxiety associated with medical costs, 0.71 (total) of the households are very worried and somewhat worried while 0.708 and 0.712 of males and females are respectively very worried and somewhat worried. For the aggregate, the percentage of households who have access to and usage of mobile devices in payment of purchase including those associated with medical bills is 0.225. This percentage is 0.236 for males while it is 0.212 for females. For the full sample, about 0.74 of the households are urban residents. About 0.755 males are urban residents while 0.723 females are urban residents. Methodology d Table 3 Logit regression. Variable Aggregate Within the reproductive age Not within the reproductive age Total Male Female Total Male Female Total Male Female ANX −0.748* (0.000) −0.697* (0.001) −0.796* (0.000) −0.852* (0.000) −0.755* (0.004) −0.987* (0.001) −0.510 (0.043) −0.551 (0.114) −0.377* (0.000) MM 0.577* (0.000) 0.799* (0.000) 0.341 (0.170) 0.622* (0.001) 0.855* (0.001) 0.361 (0.198) 0.731 (0.072) 0.797 (0.144) 0.698* (0.000) LOC 0.460 (0 00 ) 0.704* (0 002) 0.214 (0 36 ) 0.335 (0 09 ) 0.614 (0 033) 0.089 (0 60) 0.676 (0 0 6) 0.862 (0 028) 0.368* (0 000) Variable Aggregate Within the reproductive age Not within the reproductive age 1 2 3 5 6 7 8 9 10 Total Male Female Total Male Female Total Male Female Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] WF 0.448 (0.498) 0.434 (0.496) 0.465 (0.499) 0.459 (0.499) 0.449 (0.498) 0.471 (0.500) 0.427 (0.495) 0.403 (0.492) 0.453 (0.499) ANX 0.71 (0.453) 0.708 (0.455) 0.712 (0.453) 0.753 (0.431) 0.747 (0.435) 0.761 (0.427) 0.628 (0.484) 0.634 (0.483) 0.621 (0.487) MM 0.225 (0.418) 0.236 (0.425) 0.212 (0.409) 0.288 (0.453) 0.301 (0.459) 0.273 (0.446) 0.107 (0.309) 0.113 (0.317) 0.099 (0.300) LOC 0.74 (0.439) 0.755 (0.431) 0.723 (0.448) 0.761 (0.427) 0.769 (0.421) 0.751 (0.433) 0.700 (0.459) 0.726 (0.447) 0.671 (0.471) EMP 0.643 (0.479) 0.524 (0.499) 0.784 (0.412) 0.755 (0.430) 0.657 (0.475) 0.872 (0.335) 0.432 (0.496) 0.269 (0.445) 0.621 (0.487) EDUC 0.834 (0.372) 0.815 (0.388) 0.856 (0.352) 0.923 (0.266) 0.919 (0.274) 0.929 (0.257) 0.666 (0.472) 0.618 (0.487) 0.720 (0.450) Source: Authors’ computation. Methodology d About 0.755 of the aggregate are in the workforce with 0.657 for the male sample and 0.872 for the female sample. Concerning the educational level of the respondents, about 0.923 of the aggregate are educated with 0.919 for the male sample and 0.929 for the female sample. The main hypothesis in this paper is that mobile money can moderate the impact of anxiety orchestrated by medical costs by improving access to ﬁnancial services helping households manage their ﬁnances more effectively and improving their livelihoods. From Table 3, medical cost anxiety is signiﬁcantly and negatively related to welfare. Without the interaction value of medical cost anxiety and mobile money, medical cost anxiety decreases welfare by 0.748 (total), 0.697 (male) and 0.796 (female) for the aggregate, 0.852 (total), 0.755 (male) and 0.987 (female) within the reproductive age and ﬁnally by 0.377 (female) not within the reproductive age. This implies that anxiety about medical costs is harmful to individuals’ welfare in Malaysia and so needs mobile money access and usage to move this negative effect to a positive one. p p Not within the reproductive age, the mean of the full sample for the households within the richest fourth 20% and ﬁfth 20% (WF) is 0.427. To consider the gender dimension, we computed the descriptive statistics for all the variables for males and females. The mean of males and females within the richest fourth 20% and ﬁfth 20% is 0.403 and 0.453, respectively. For anxiety associated with medical costs, 0.628 (Total) of the households are very worried and somewhat worried while 0.634 and 0.621 of males and females are respectively very worried and somewhat worried. Not within the reproductive age, the percentage of households who have access to and usage of mobile devices in payment of purchase including those associated with medical bills is 0.107. This percentage is 0.113 for The outcomes showed that the interaction value of medical cost anxiety and mobile money is positively associated with welfare. Indeed, one unit increase in interaction value enhances the total sample and female sample for an aggregate of 0.587 and 0.736, respectively. Similarly, one unit increase in interaction value improves the full sample and female male sample within the reproductive age welfare of 0.554 and 0.714, respectively. Methodology d The implication of the outcome is that mobile money access and usage HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:260 \| https://doi.org/10.1057/s41599-024-02767-2 4 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 Table 3 Logit regression. Variable Aggregate Within the reproductive age Not within the reproductive age Total Male Female Total Male Female Total Male Female ANX −0.748* (0.000) −0.697* (0.001) −0.796* (0.000) −0.852* (0.000) −0.755* (0.004) −0.987* (0.001) −0.510 (0.043) −0.551 (0.114) −0.377* (0.000) MM 0.577* (0.000) 0.799* (0.000) 0.341 (0.170) 0.622* (0.001) 0.855* (0.001) 0.361 (0.198) 0.731 (0.072) 0.797 (0.144) 0.698* (0.000) LOC 0.460 (0.004) 0.704* (0.002) 0.214 (0.364) 0.335 (0.097) 0.614 (0.033) 0.089 (0.760) 0.676 (0.016) 0.862 (0.028) 0.368* (0.000) EMP 0.361 (0.014) 0.322 (0.098) 0.367 (0.148) 0.407 (0.040) 0.383 (0.120) 0.335 (0.375) 0.530 (0.033) 0.569 (0.134) 0.531* (0.000) EDU 1.257* (0.000) 1.043* (0.000) 1.592* (0.000) 1.201* (0.003) 1.153 (0.041) 1.279 (0.030) 1.399* (0.000) 1.167* (0.002) 1.884* (0.000) interaction 0.587* (0.001) 0.405 (0.123) 0.736* (0.001) 0.554* (0.004) 0.355 (0.224) 0.714* (0.005) 0.680 (0.113) 0.632 (0.343) 0.736 (0.190) Constant −1.473* (0.000) −1.562* (0.000) −1.497* (0.000) −1.395* (0.002) −1.716* (0.007) −1.023 (0.142) −1.769* (0.000) −1.709* (0.000) −2.062* (0.000) Log-likelihood −626.109 −333.525 −289.931 −416.803 −221.432 −193.192 −204.919 −109.959 −93.967 Pseudo R2 0.089 0.1007 0.0835 0.0747 0.0960 0.0593 0.1345 0.1233 0.1527 Prob > chi2 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 Source: Authors’ computation. Note: p-values are in (), * means signiﬁcance at 1%. Variable Aggregate Within the reproductive age Not within the reproductive age 1 2 3 5 6 7 8 9 10 Total Male Female Total Male Female Total Male Female Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] WF 0.448 (0.498) 0.434 (0.496) 0.465 (0.499) 0.459 (0.499) 0.449 (0.498) 0.471 (0.500) 0.427 (0.495) 0.403 (0.492) 0.453 (0.499) ANX 0.71 (0.453) 0.708 (0.455) 0.712 (0.453) 0.753 (0.431) 0.747 (0.435) 0.761 (0.427) 0.628 (0.484) 0.634 (0.483) 0.621 (0.487) MM 0.225 (0.418) 0.236 (0.425) 0.212 (0.409) 0.288 (0.453) 0.301 (0.459) 0.273 (0.446) 0.107 (0.309) 0.113 (0.317) 0.099 (0.300) LOC 0.74 (0.439) 0.755 (0.431) 0.723 (0.448) 0.761 (0.427) 0.769 (0.421) 0.751 (0.433) 0.700 (0.459) 0.726 (0.447) 0.671 (0.471) EMP 0.643 (0.479) 0.524 (0.499) 0.784 (0.412) 0.755 (0.430) 0.657 (0.475) 0.872 (0.335) 0.432 (0.496) 0.269 (0.445) 0.621 (0.487) EDUC 0.834 (0.372) 0.815 (0.388) 0.856 (0.352) 0.923 (0.266) 0.919 (0.274) 0.929 (0.257) 0.666 (0.472) 0.618 (0.487) 0.720 (0.450) Source: Authors’ computation. ARTICLE The outcomes are also reinforced by Sakyi- Nyarko et al. (2022) who showed that being on a higher ﬁnancial inclusion raises the likelihood of never going without medicine or medical treatment when needed by approximately 8%. y pp y For the control variables inserted in the model, the outcomes showed that males located in urban areas for the aggregate increased welfare by 0.704 while the welfare of females not within the reproductive age improved by 0.368. This ﬁnding is similar to Nguyen and Nguyen (2019) and Sakyi-Nyarko et al. (2022) who found that location improves individual welfare. The outcome also showed that for females in the workforce not within the reproductive age welfare is improved by 0.531, indicating employment plays a crucial role in welfare improvement by improving wages to individuals to satisfy their needs. The outcomes are consistent with Tran (2015) and Nguyen and Tran (2018) who found that wage employment improves household welfare in Vietnam. Finally, the results found that educational level improves Malaysian households’ welfare. Indeed, for the aggregate, one unit increase in educational level increases welfare by 1.257 (Total), 1.043 (Male) and 1.592 (Female). One unit increase in full sample educational level within the reproductive age increases welfare by 1.201 while this increases full sample, male sample and female sample not within the reproductive age welfare by 1.399, 1.167 and 1.884, respectively. The implication is that educational level allows individuals to access job opportunities, which can help them to satisfy their needs and in turn improve their welfare. This outcome is similar to Kilombele et al. (2023) who found that educational level decreases poverty, improving welfare and Nguyen and Nguyen (2019) and Sakyi-Nyarko et al. (2022) who found that educational level improves welfare in Ghana and Vietnam, respectively. p y The result of the interaction shows that access to mobile money signiﬁcantly moderates the impact of medical costs on welfare by 0.587 (for all households) and 0.736 (female), while it has no signiﬁcant moderation when it comes to male-headed house- holds. This is also the same for the households within the reproductive age. On the other hand, for those not within the reproductive age, mobile money has no moderate power on medical cost anxiety. ARTICLE (T-stat) ANX 0.858 [0.727] 0.131* (5.215) 0.861 [9.699] 0.161* (4.28) 0.856 [0.753] 0.1034 (3.075) 0.915 [0.76] 0.155* (5.556) 0.910 [0.735] 0.175*** (4.092) 0.918 (0.781) 0.137 [3.734] 0.758 [0.662] 0.096* (1.98) 0.772 [0.63] 0.142* (1.99) 0.747 [0.693] 0.054* (0.813) MM 0.150 [0.317] 0.225* (6.395) 0.155 [0.276] −0.1218* (−3.213) 0.1465 [0.353] −0.206* (−5.77) 0.206 [0.383] 0.287* (−5.05) 0.216 [0.335] −0.119 (−2.314) 0.198 [0.425] −0.22* [4.75] 0.050 [0.182] −0.13* −4.025 0.050 [0.182] −0.13 (−4.02) 0.054 [0.20] −0.145 (−3.150) LOC 0.682 [0.81] −0.127* (−4.61) 0.677 [0.775] 0.042 (−2.323) 0.687 [0.842] −0.1552 (−0.227) 0.714 (0.816) −0.10* (−3.087) 0.726 [0.778] −0.052 (−1.041) 0.704 [0.85] −0.15* (−3.29) 0.628 [0.797] −0.17* (−3.44) 0.591 [0.767] −0.176 (−2.396) 0.657 [0.826] −0.169 (−2.565) EMP 0.583 [0.716] −0.133* (−4.409) 0.743 [0.830] −0.088 (−2.293) 0.456 [0.612] −0.1567* (−3.658) 0.711 0.806 −0.095 (−2.844) 0.847 [0.90] −.0528 (−1.361) 0.918 [0.78] 0.137* (3.73) 0.357 [0.534] −0.17* (−3.334) 0.556 [0.698] −0.141 (−1.85) 0.198 [0.373] −0.388* (−4.0) EDUC 1.876 [2.26] −0.384* (−9.49) 1.869 [2.263] −0.393* (−6.939) 0.736 [0.919] −0.183* (−5.587) 0.602 0.725 −0.122 (−2.445) 2.039 [2.383] −0.343 (−7.263) 2.006 2.3571 −0.35* (−5.05) 0.356 [0.53] −0.18*** (−3.33) 0.556 [0.698] −0.141* (−1.85) 0.198 [.373] −0.175 (-2.67) Note: CG means control group, TG means treatment group and their values are contained in square brackets []. The treatment group are the focus category. For example, anxiety associated with medication cost (ANX) = 1 (treatment group) if the households are worried about this, and 0 (control group) if otherwise. Location (LOC) applies 1 = urban residents (treatment group), and 0 = rural residents (control group). Employment status of the respondents, 1 = in the workforce (treatment group), 0 = out of the workforce (control group); education (EDUC) 1 if educated (treatment group) and 0 if non-education (control group), mobile money (MM), 1 if used mobile money (treatment group) and 0 if did not use mobile money (control group). Diff represents the mean difference (CG-TG). T-stat is in brackets (), , * and * means that the absolute values are signiﬁcant at 1%, 5% and 10%, respectively. Source: Authors’ computation. The ﬁndings are consistent with Munyegera and Matsumoto (2016) and Kilombele et al. (2023) who found in Uganda and Tanzania that mobile money improves welfare. Similarly, Wang et al. (2022) study in rural China showed that mobile money usage signiﬁcantly increased farmers’ welfare. This is similar to Jakovljevic et al. (2023). Similar outcomes are found by Peprah et al. (2020) in Ghana. ARTICLE ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 Note: CG means control group, TG means treatment group and their values are contained in square brackets []. The treatment group are the focus category. For example, anxiety associated with medication cost (ANX) = 1 (treatment group) if the households are worried about this, and 0 (control group) if otherwise. Location (LOC) applies 1 = urban residents (treatment group), and 0 = rural residents (control group). Employment status of the respondents, 1 = in the workforce (treatment group), 0 = out of the workforce (control group); education (EDUC) 1 if educated (treatment group) and 0 if non-education (control group), mobile money (MM), 1 if used mobile money (treatment group) and 0 if did not use mobile money (control group). Diff represents the mean difference (CG-TG). T-stat is in brackets (), *, and * means that the absolute values are signiﬁcant at 1%, 5% and 10%, respectively. Source: Authors’ computation. help to moderate the impact of anxiety orchestrated by medical costs and improve Malaysian households’ welfare. Variable Total sample Reproductive age group (15–49 years) Non-reproductive age group (50 years +) Total Male Female Total Male Female Total Male Female 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 CT [TG] Diff. (T-stat) CG [TG] Diff. (T-stat) CG [TG] Diff. (T-stat) CT [TG] Diff. (T-stat) CG [TG] Diff. (T-stat) CG [TG] Diff. (T-stat) CT [TG] Diff. (T-stat) CG [TG] Diff. (T-stat) CG [TG] Diff. ARTICLE This result suggests that for people who are not in the reproductive age range, the use of mobile money does not seem to signiﬁcantly affect how worried they are about the costs of medical care. In other words, the presence or absence of mobile money usage does not appear to be strongly linked to their level of anxiety about covering medical expenses. Methodology d Variable Aggregate Within the reproductive age Not within the reproductive age Total Male Female Total Male Female Total Male Female ANX −0.748* (0.000) −0.697* (0.001) −0.796* (0.000) −0.852* (0.000) −0.755* (0.004) −0.987* (0.001) −0.510 (0.043) −0.551 (0.114) −0.377* (0.000) MM 0.577* (0.000) 0.799* (0.000) 0.341 (0.170) 0.622* (0.001) 0.855* (0.001) 0.361 (0.198) 0.731 (0.072) 0.797 (0.144) 0.698* (0.000) LOC 0.460 (0.004) 0.704* (0.002) 0.214 (0.364) 0.335 (0.097) 0.614 (0.033) 0.089 (0.760) 0.676 (0.016) 0.862 (0.028) 0.368* (0.000) EMP 0.361 (0.014) 0.322 (0.098) 0.367 (0.148) 0.407 (0.040) 0.383 (0.120) 0.335 (0.375) 0.530 (0.033) 0.569 (0.134) 0.531* (0.000) EDU 1.257* (0.000) 1.043* (0.000) 1.592* (0.000) 1.201* (0.003) 1.153 (0.041) 1.279 (0.030) 1.399* (0.000) 1.167* (0.002) 1.884* (0.000) interaction 0.587* (0.001) 0.405 (0.123) 0.736* (0.001) 0.554* (0.004) 0.355 (0.224) 0.714* (0.005) 0.680 (0.113) 0.632 (0.343) 0.736 (0.190) Constant −1.473* (0.000) −1.562* (0.000) −1.497* (0.000) −1.395* (0.002) −1.716* (0.007) −1.023 (0.142) −1.769* (0.000) −1.709* (0.000) −2.062* (0.000) Log-likelihood −626.109 −333.525 −289.931 −416.803 −221.432 −193.192 −204.919 −109.959 −93.967 Pseudo R2 0.089 0.1007 0.0835 0.0747 0.0960 0.0593 0.1345 0.1233 0.1527 Prob > chi2 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 Source: Authors’ computation. Note: p-values are in (), * means signiﬁcance at 1%. Variable Aggregate Within the reproductive age Not within the reproductive age 1 2 3 5 6 7 8 9 10 Total Male Female Total Male Female Total Male Female Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] Mean [SD] WF 0.448 (0.498) 0.434 (0.496) 0.465 (0.499) 0.459 (0.499) 0.449 (0.498) 0.471 (0.500) 0.427 (0.495) 0.403 (0.492) 0.453 (0.499) ANX 0.71 (0.453) 0.708 (0.455) 0.712 (0.453) 0.753 (0.431) 0.747 (0.435) 0.761 (0.427) 0.628 (0.484) 0.634 (0.483) 0.621 (0.487) MM 0.225 (0.418) 0.236 (0.425) 0.212 (0.409) 0.288 (0.453) 0.301 (0.459) 0.273 (0.446) 0.107 (0.309) 0.113 (0.317) 0.099 (0.300) LOC 0.74 (0.439) 0.755 (0.431) 0.723 (0.448) 0.761 (0.427) 0.769 (0.421) 0.751 (0.433) 0.700 (0.459) 0.726 (0.447) 0.671 (0.471) EMP 0.643 (0.479) 0.524 (0.499) 0.784 (0.412) 0.755 (0.430) 0.657 (0.475) 0.872 (0.335) 0.432 (0.496) 0.269 (0.445) 0.621 (0.487) EDUC 0.834 (0.372) 0.815 (0.388) 0.856 (0.352) 0.923 (0.266) 0.919 (0.274) 0.929 (0.257) 0.666 (0.472) 0.618 (0.487) 0.720 (0.450) S A h ’ i 5 Within the reproductive age p g g p The outcomes using probit regression (Table 5) and instru- mental variable regression (Table 6) revealed that medical cost anxiety reduces Malaysian households’ welfare not only for total, males and females in the aggregate but also for total, males and females within the reproductive age and for total in not within the reproductive age. As found in Table 3, the outcome also indicated that the association of mobile money with medical cost anxiety (interaction value) help to moderate the impact of anxiety orchestrated by medical cost and improve Malaysian households’ welfare. Similarly, as found in Table 3, the control variables such as location, employment status and education level, inserted in the probit and instrumental variable regression improve Malay- sian households’ welfare. All the outcomes found for probit and instrumental variable regressions are consistent with the out- comes found using logit regression. This indicates that outcomes are robust. Table 4 Test of mean difference. This observation could be important for policymakers, healthcare providers, and ﬁnancial institutions, as it indicates that the impact of mobile money on medical cost anxiety may not be uniform across different age groups. Understanding these dynamics can help tailor interventions and support mechanisms to better address the speciﬁc needs and concerns of different demographic segments. Robustness: mean difference, probit and instrumental variable regression. To check for the robustness of our logit outcomes reported in Table 3, we apply the test of mean difference, probit regression and instrumental variable regression. Table 4 reported HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:260 \| https://doi.org/10.1057/s41599-024-02767-2 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:260 \| https://doi.org/10.1057/s41599-024-02767 6 ARTICLE ENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 UNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 Variable Aggregate Within the reproductive age Not within the reproductive age Total Male Female Total Male Female Total male Female 1 2 3 4 5 6 7 8 9 ANX −0.517 (0.000) −0.585* (0.000) −0.427* (0.004) −0.638* (0.000) −0.698* (0.001) −0.569*** (0.006) −0.303* (0.059) −0.384 (0.105) −0.2075 (0.352) MM 0.302* (0.004) 0.1965 (0.209) 0.423* (0.003) 0.2959 (0.012) 0.1703 (0.337) 0.4229 (0.008) 0.471* (0.056) 0.4697 (0.210) 0.482 (0.147) LOC 0.3061* (0.002) 0.1819 (0.205) 0.432* (0.002) 0.223* (0.073) 0.0983 (0.586) 0.3696 (0.034) 0.454 (0.006) 0.2865 (0.248) 0.5653 (0.015) EMP 0.1955 (0.030) 0.2053 (0.182) 0.1650 (0.169) 0.2117* (0.081) 0.2885 (0.222) 0.1593 (0.001) 0.314 (0.035) 0.244 (0.273) 0.3636 (0.112) EDU 0.485* (0.000) 0.6045* (0.000) 0.3915 (0.000) 0.4995 (0.000) 0.5810* (0.000) 0.4163* (0.001) 0.5388* (0.000) 0.7031* (0.000) 0.451* (0.005) interaction 0.367* (0.001) 0.253 (0.123) 0.4602 (0.001) 0.346* (0.004) 0.2225 (0.223) 0.446* (0.005) 0.4254 (0.111) 0.3949 (0.340) 0.4605 (0.189) Constant −1.140* (0.000) −1.205* (0.000) −1.148 * (0.000) −1.077 (0.000) −1.127* (0.009) −1.086* (0.002) −1.449* (0.000) −1.5451 (0.000) −1.458 (0.000) Log-likelihood −617.854 −282.438 −332.780 −405.296 −184.85 −218.75 −207.370 −95.2196 −111.189 Pseudo R2 0.1016 0.1072 0.1027 0.1003 0.0999 0.1069 0.1241 0.1414 0.1135 Prob > chi2 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 Note: p-values are in (), *, and * means that the absolute values are signiﬁcant at 1%, 5% and 10%. Source: Authors’ computation. Table 4 Test of mean difference. the outcomes of the test of mean difference and revealed the mean difference between the households who are worried about medical cost anxiety and those who are not worried about medical cost anxiety signiﬁcantly differ for total and males both in the total sample and the reproductive age group and positive and signiﬁcant for total, male and female in the non-reproductive age group. It implies that those who are pricked by medical cost anxiety have lower welfare outcomes compared to those who are not worried. The mean difference between the households who used mobile money and those who did not use mobile money is positive and signiﬁcant for total and negative and signiﬁcant for males and females both in the total sample and the reproductive age group and negative and signiﬁcant for total, male and female in the non-reproductive age group. The mean difference between the households who live in urban areas and those who live in rural areas is negative and signiﬁcant for total and positive and signiﬁcant for males in the total sample, both negative and signiﬁcant for total and female in the reproductive age group and for total, male and female in the non-reproductive age group. The mean difference between the households in the workforce and those out of the workforce is negative and signiﬁcant both for total, male and female in the total sample, negative and signiﬁcant for total and positive and signiﬁcant for female in the reproductive age group and ﬁnally, negative, and signiﬁcant bot for total and female in the non-reproductive age group. The mean difference between educated respondents and non-educated respondents is negative and signiﬁcant both for total, males and females in the total sample, the reproductive age group and the non-reproductive age group. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 Variable Aggregate Within the reproductive age Not within the reproductive age Total Male Female Total Male Female Total male Female 1 2 3 4 5 6 7 8 9 ANX −0.1846* (0.000) −0.248* (0.001) −0.198 (0.006) −0.225* (0.000) −0.248* (0.001) −0.1985 (0.006) −0.1071* (0.052) −0.1335 (0.100) −0.0748 (0.330) MM 0.1137* (0.002) 0.064 (0.319) 0.1597 (0.006) 0.1111 (0.009) 0.064 (0.319) 0.1597 (0.006) 0.168 (0.045) 0.1621 (0.199) 0.180 (0.120) LOC 0.1083* (0.002) 0.0340 (0.600) 0.1324** (0.028) 0.081* (0.063) 0.0340 (0.600) 0.132 (0.028) 0.1528 (0.006) 0.0959 (0.253) 0.1851 (0.016) EMP 0.069** (0.02) 0.098 (0.236) 0.0587 (0.273) 0.074* (0.085) 0.0986 (0.236) 0.058 (0.273) 0.110 (0.034) 0.090 (0.240) 0.1218 (0.137) EDU 0.1684* (0.000) 0.201* (0.000) 0.1422* (0.001) 0.1723* (0.000) 0.2010* (0.000) 0.142* (0.001) 0.187* (0.000) 0.2454* (0.000) 0.1546 (0.005) interaction 0.1457* (0.001) 0.1008 (0.122) 0.1819* (0.001) 0.1376* (0.004) 0.0885 (0.224) 0.1766 (0.005) 0.1684 (0.108) 0.156 (0.339) 0.1818 (0.184) Constant 0.0869 (0.169) 0.0810 (0.416) 0.0947 (0.243) 0.4296* (0.000) 0.1110 (0.456) 0.1152 (0.345) 0.984 (0.141) −0.0386 (0.782) 0.0047 (0.969) R2 0.2148 0.1294 0.1580 0.1294 0.1286 0.1382 0.1580 0.1803 0.1431 Prob > F 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000*** variable regressions to check for robustness. The results found that medical cost anxiety decreases individuals’ welfare while its association with mobile money has a positive and sig- niﬁcant inﬂuence on households’ welfare. Furthermore, the result of the interaction shows that access to mobile money signiﬁcantly moderates the impact of medical costs on welfare by 0.587 (for all households) and 0.736 (for females), while it has no signiﬁcant moderation when it comes to male- headed households. This is also the same for the households within the reproductive age. On the other hand, for those not within the reproductive age, mobile money has no moderate power on medical cost anxiety. This result suggests that for people who are not in the reproductive age range, the use of mobile money does not seem to signiﬁcantly affect how worried they are about the costs of medical care. In other words, the presence or absence of mobile money usage does not appear to be strongly linked to their level of anxiety about covering medical expenses. The ﬁndings from this study have signiﬁcant implications for policymakers and ﬁnancial service providers in Malaysia. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 To promote the welfare of households, policymakers should promote the use of mobile money and encourage ﬁnancial inclusion Financial service providers should also develop products and services tailored to the needs of households, especially those that are more vulnerable to medical cost anxiety. Furthermore, policymakers should consider imple- menting policies that address rising healthcare costs and alleviate medical cost anxiety among households in Malaysia. The implementation of these policies will allow to achieve- ment Sustainable Development Goal (SDG 3) which concerns good health and well-being. This study is not without limitations. One of the limitations is that the study focused on a single country and on one section (2021) of the global ﬁnancial survey, which is unable to account for long-term changes in mobile money adoption and medical cost anxiety impact on the welfare of households over time. As a recommendation, there is a need for long-term studies that track changes in mobile money adoption and medical cost anxiety over time can provide insights into the dynamic nature of this rela- tionship. Longitudinal data would help in capturing changes and trends that might not be observable in a single cross-sectional study. Data availability Th d l The datasets analysed during the current study are available from the corresponding author upon reasonable request. Received: 26 September 2023; Accepted: 26 January 2024; Note 1 According to the World Health Organization (WHO), reproductive age is explained as the stage between 15 and 49 years for females, during which they are considered to be at risk of pregnancy. For males, reproductive age is generally considered to extend from the onset of puberty to old age, as sperm production can continue throughout their lives (WHO, 2021). Conclusion d l Medical cost anxiety refers to the fear of not being able to afford medical treatment or healthcare services, which is a common issue among households in Malaysia due to rising healthcare costs. This situation reduces individuals’ welfare in Malaysia. Thus, to cope with this, mobile money which is a ﬁnancial service that allows users to achieve several transac- tions, including sending and receiving money, paying bills, and purchasing goods and services, using their mobile phones is essential to reduce medical cost anxiety and in turn improves individuals’ welfare. This study examines the association between mobile money, medical cost anxiety, and the welfare of households in Malaysia. 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J Dev Econ 135(6):43–58. https://doi.org/10.1016/j.jdeveco.2018.06.015 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2024) 11:260 \| https://doi.org/10.1057/s41599-024-02767-2 9 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-024-02767-2 ARTICLE 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. 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https://openalex.org/W2071165491	https://europepmc.org/articles/pmc2706241?pdf=render	English	null	Carbon offsets, reversal risk and US climate policy	Carbon balance and management	2,009	cc-by	4,767	Published: 15 June 2009 Carbon Balance and Management 2009, 4:3 doi:10.1186/1750-0680-4-3 This article is available from: http://www.cbmjournal.com/content/4/1/3 © 2009 Mignone et al; licensee BioMed Central Ltd. g This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: One controversial issue in the larger cap-and-trade debate is the proper use and certification of carbon offsets related to changes in land management. Advocates of an expanded offset supply claim that inclusion of such activities would expand the scope of the program and lower overall compliance costs, while opponents claim that it would weaken the environmental integrity of the program by crediting activities that yield either nonexistent or merely temporary carbon sequestration benefits. Our study starts from the premise that offsets are neither perfect mitigation instruments nor useless "hot air." Results: We show that offsets provide a useful cost containment function, even when there is some threat of reversal, by injecting additional "when-flexibility" into the system. This allows market participants to shift their reduction requirements to periods of lower cost, thereby facilitating attainment of the least-cost time path without jeopardizing the cumulative environmental integrity of the system. By accounting for market conditions in conjunction with reversal risk, we develop a simple offset valuation methodology, taking into account the two most important factors that typically lead offsets to be overvalued or undervalued. Conclusion: The result of this paper is a quantitative "model rule" that could be included in future legislation or used as a basis for active management by a future "carbon fed" or other regulatory authority with jurisdiction over the US carbon market to actively manage allowance prices. Methodology Carbon offsets, reversal risk and US climate policy Bryan K Mignone1,2, Matthew D Hurteau3, Yihsu Chen4 and Brent Sohngen5,6 Address: 1The Brookings Institution, 1775 Massachusetts Avenue, NW, Washington, DC 20036, USA, 2Center for Applied Macroeconomic Analysis, Australian National University, Canberra ACT 0200, Australia, 3Western Regional Center of the National Institute for Climatic Change Research, Northern Arizona University, Box 6077, Flagstaff, AZ 86011, USA, 4School of Engineering, University of California, Merced, 5200 North Lake Road, Merced, CA 95343, USA, 5Agricultural, Environmental and Development Economics, The Ohio State University, 2120 Fyffe Road, Columbus, OH 43210-1067, USA and 6Resources for the Future, 1616 P Street NW, Washington, DC 20036, USA Email: Bryan K Mignone - bmignone@brookings.edu; Matthew D Hurteau - Matthew.Hurteau@nau.edu; Yihsu Chen - Yihsu.Chen@ucmerced.edu; Brent Sohngen - sohngen.1@osu.edu * Corresponding author Received: 19 March 2009 Accepted: 15 June 2009 Published: 15 June 2009 Page 1 of 6 (page number not for citation purposes) Carbon Balance and Management Open Access Background ff For example, the northeast Regional Greenhouse Gas Initia- tive (RGGI) discounts offset credits at 10% [8] and early versions of the Waxman-Markey bill discounted offset credits at 20% [6]. The Voluntary Carbon Standard requires that a proportion of the offset credits be depos- ited in a buffer pool, with that proportion determined by a categorical risk assessment [9]. However, the wisdom of including large quantities of mit- igation from outside the energy sector (and from the land- use sector, in particular) has been widely questioned by those concerned with a wide range of potential problems. Reversals of either kind (on-site or off-site) are not prob- lematic unless they fail to be properly internalized by the underlying crediting framework. In that case, permits may be awarded in excess of the net integrated emissions reductions specified by the policy. In a world in which the trajectory of carbon loss could be reasonably well antici- pated, this outcome could be avoided in one of two ways. First, project developers or offset buyers could individu- ally retain liability for future carbon loss, in which case those entities would be responsible for purchasing addi- tional permits ex post, whenever sequestered carbon was shown (through monitoring) to have escaped. A number of mechanisms to place liability on the buyer of offsets have been proposed. One is to create "rental con- tracts" for temporary sequestration [12]. Another is to issue so-called temporary sequestration credits (tCERs) under which holders of such credits must make up the car- bon content of these credits in other ways after the term of the temporary credit expires [13]. Finally, credits for risky assets might be issued in the usual manner, but with the regulator imposing an additional condition that those holding such assets acquire private insurance just as vehi- cle owners are required to demonstrate proof of insurance before registration. Of course, this requirement is feasible only if there is an existing insurance market for offsets. For an example of how this might be implemented, see the model rule developed as part of the northeast Regional Greenhouse Gas Initiative [8]. While this approach internalizes some of the risks associ- ated with such carbon assets, the discount factors used in practice are largely qualitative and fail to account for the economic value of such instruments. Background ff disturbance and forest health [2-5], the land-use contribu- tion to total emissions will almost certainly remain quan- titatively significant [2]. Consequently, architects of US cap-and-trade policies have sought to include instruments to reduce such emissions by issuing carbon offsets for projects that avoid expected emissions or deliberately sequester additional carbon [6]. Offsets are economically g The efficiency of the natural carbon sink is in decline, with land-use change contributing approximately 16% of annual carbon emissions from 2000–2006 [1]. While the future trajectory of land-use emissions will depend on a variety of uncertain factors, ranging from future patterns of rural development to the impacts of climate change on Page 1 of 6 (page number not for citation purposes) http://www.cbmjournal.com/content/4/1/3 Carbon Balance and Management 2009, 4:3 climate system itself. An offset is permanent, in an opera- tional sense, if the emissions reduction does not reappear as a source in a later compliance period, although the proper length of this horizon is debatable. Offset credits from land-use projects are particularly important to eval- uate in this context, because carbon stored in forests or other terrestrial systems could escape back into the atmos- phere for a variety of reasons, ranging from natural distur- bances (e.g., fire) to inadequate protection from human interference (e.g., logging) [11]. As discussed above, future losses of carbon off-site due to economic leakage could also jeopardize the effective permanency of stored carbon. and politically attractive because, by expanding the scope of the program to include lower cost options, they increase the number of compliance opportunities in the market, exert downward pressure on carbon prices and minimize the overall social cost of abatement [7]. However, the wisdom of including large quantities of mit- igation from outside the energy sector (and from the land- use sector, in particular) has been widely questioned by those concerned with a wide range of potential problems. These include potential future losses of carbon on-site that could result from natural disturbance, poor manage- ment or other factors (collectively referred to as "non-per- manence") and potential losses of carbon off-site due to activity shifting or market price adjustments that drive up carbon losses elsewhere (typically called "leakage"). Exist- ing regulations governing offset project development have attempted to account for the possibilities of non-perma- nence and leakage by applying a discount factor to per- mits commensurate with their perceived reversal risk. Background ff As a result, they raise the costs of forest carbon without acknowledging any of the benefits that risky carbon offsets might provide by mitigating upside price shocks and reducing overall miti- gation costs. This paper shows how to quantitatively adjust the discount factor to account for these benefits. The resulting "model rule" could be used by regulators to dynamically adjust the supply of offsets in an emissions permit market to more finely manage carbon prices. Page 2 of 6 (page number not for citation purposes) Results ll While the failure to properly account for reversal risk leads to the possibility of overvaluation, a failure to acknowl- edge the economic efficiency benefits of (even risky) off- sets can lead to systematic undervaluation. As an example, consider an offset project in which all of the carbon ini- tially sequestered is lost in a future compliance period. Fully discounting carbon reversals, as above, would imply that no credits should be issued for such a project. How- ever, if credits were initially issued at a time when permit prices in the market were high, and credits for future car- bon loss were later surrendered at a time when prices were low, then this combined transaction would lower total abatement costs by allowing market actors to endog- enously shift abatement across time in pursuit of an eco- nomically efficient outcome. Tightening and loosening constraints on offsets in real time and in response to actual market conditions has been proposed previously in the context of a "carbon fed" [14], but markets may behave better in this case if clear, quan- titative rules are written up front. When it comes to risky carbon assets, the regulator's fundamental objective is to make sure that the crediting system correctly balances the tendency to overvalue offsets (by not sufficiently account- ing for reversal risk and the implied risk to the climate sys- tem) with the tendency to undervalue them (by not sufficiently accounting for the benefit of when-flexibility). Within the broad class of risky assets, we contend that the underlying economic and environmental goals would be best served by explicitly quantifying reversal risk along a continuum and transparently and dynamically adjusting valuations on a project-specific basis to reflect the nature of such risk in the context of the broader carbon market. The application of such a valuation rule by a central authority would increase when-flexibility (lower compli- ance costs) without violating the cumulative environmen- tal integrity of the system. When liability for future carbon losses is privately held, those trading offsets must decide for themselves when these types of transactions offer credible arbitrage oppor- tunities, given their own expectations about future prices. Under this type of regulation, firms would use a collection of compliance tools, consisting of both risky and non- risky assets, to navigate an efficient abatement path. Results ll Typically, broad concerns over environmental integrity take two distinct forms, reflected in requirements that off- sets be both "additional" and "permanent" [10]. The first criterion effectively requires that the activity under consid- eration yield emissions reductions that would not other- wise occur. Assignment of "additionality" therefore hinges on confidence in the relevant emissions baseline path, since this is the trajectory against which any reduc- tions will be measured and against which credits will be awarded. Alternatively, the regulator could discount credits ex ante (at the time of certification) to account for anticipated future carbon loss, thus effectively transferring liability to itself. In this case, the government would issue less than one tradable permit for every ton of carbon sequestered (assuming each permit represents the legal right to emit one ton), thereby acquiring a greater number of permits up front to sufficiently compensate for future carbon losses. A similar objective could also be pursued through the creation of a "buffer pool" [9] rather than through direct discounting. While allowing private entities the The second criterion requires that a project safely seques- ter carbon over the long time horizons demanded by the Page 2 of 6 (page number not for citation purposes) Page 2 of 6 (page number not for citation purposes) http://www.cbmjournal.com/content/4/1/3 http://www.cbmjournal.com/content/4/1/3 Carbon Balance and Management 2009, 4:3 In the above example, the regulator is essentially con- straining liquidity in the market when it believes that prices have risen to levels above those justified by funda- mentals. This mechanism would thus complement firm- level borrowing decisions (assuming such actions were allowed by the policy) and help to collapse speculative bubbles early. Of course, any mechanism designed to enhance when-flexibility, whether realized through firm- level borrowing decisions or through the actions of a cen- tral regulator, necessarily requires judgments to be made about future market conditions, and prices in particular. A limited discretionary mechanism, like the one proposed here, simply provides an additional check on the judg- ments made by firms and spreads the decision about whether to shift abatement across a more diverse set of actors in the system. opportunity to internalize risk is arguably more efficient than these approaches, some form of regulator discount- ing or "buffering" is likely to be needed in the short run before a robust offset insurance market develops and because private liability would be difficult to implement in the case of future off-site reversals. Page 3 of 6 (page number not for citation purposes) Results ll Page 3 of 6 (page number not for citation purposes) Page 3 of 6 (page number not for citation purposes) Carbon Balance and Management 2009, 4:3 http://www.cbmjournal.com/content/4/1/3 Applied discount factor (δ) in percent as a function of the share of sequestered carbon expected to be lost over the relevant time horizon (f) and the factor by which the spot price for permits exceeds the target price set by the regulator (S) or by which it exceeds the expected discounted future equilibrium price Figure 1 Applied discount factor (δ) in percent as a function of the share of sequestered carbon expected to be lost over the relevant time horizon (f) and the factor by which the spot price for permits exceeds the target price set by the regulator (S) or by which it exceeds the expected discounted future equilibrium price. The limits described in the main text are easy to identify on this figure. The no-reversal limit is found along the left edge of the figure; the discount fac- tor of 100% is independent of the value of S. The full reversal limit returns a low discount factor when S is small (lower right- hand corner) but a high discount factor when S is large (upper right-hand corner). Applied dis time horiz which it ex Figure 1 Applied discount factor (δ) in percent as a function of the share of sequestered carbon expected to be lost over the relevant time horizon (f) and the factor by which the spot price for permits exceeds the target price set by the regulator (S) or by which it exceeds the expected discounted future equilibrium price Figure 1 Applied discount factor (δ) in percent as a function of the share of sequestered carbon expected to be lost over the relevant time horizon (f) and the factor by which the spot price for permits exceeds the target price set by the regulator (S) or by which it exceeds the expected discounted future equilibrium price. The limits described in the main text are easy to identify on this figure. The no-reversal limit is found along the left edge of the figure; the discount fac- tor of 100% is independent of the value of S. The full reversal limit returns a low discount factor when S is small (lower right- hand corner) but a high discount factor when S is large (upper right-hand corner). Results ll In the alternative case in which credits are discounted up front, the regulator would need to determine an appropriate dis- count rate that incorporates both reversal risk and the ben- efits of increased when-flexibility. The regulator, however, would like to employ a discount rule that mimics, as much as possible, the incentives that individual actors would face were they themselves held liable for future car- bon losses. Consider the expected price path, Pe(t) of carbon permits, derived, for example, from model estimates of a prescribed emissions reduction path [15]. In effect, this price path rep- resents the "target price" that the regulator hopes to defend through the constrained use of offsets. Suppose offsets are allowed into the system in some period t = τ, but that a frac- tion f of the carbon initially sequestered is lost over the rel- evant time horizon. The parameter f in this model will inevitably vary by project or project type, but existing research provides ample guidance [16]. The immediate economic benefit (per ton of carbon) of allowing such cred- its into the market is the spot price, Ps(τ), because that reflects the marginal (per ton) cost of an avoided permit. The total economic cost of using the offset is the sum of the deployment cost of the offset itself, C(τ), and the present value of the future permit that must be purchased at the For example, suppose a project developer sequesters two tons of carbon in the present period when prices exceed the expected discounted long-run average price by a factor of two, knowing that all of the carbon stored initially will ultimately be lost within the relevant time horizon. In response to this submission, the regulating agency prints two permits, issues one to the project developer (effec- tively discounting at 50% from the developer's perspec- tive) and retains one for itself. The regulator immediately liquidates its own permit in the secondary market and uses the revenue to buy back and retire two permits in the future, once the price drops back to (or below) the long- run average value. Because the regulator removes two per- mits from circulation in a future period, the carbon loss is properly internalized and the cumulative integrity of the program is not violated. Page 4 of 6 (page number not for citation purposes) Results ll future target price to compensate for the eventual carbon loss. Assuming that Pe(t) rises at the long-term interest rate, the net present value of the cost is C(τ) + f·Pe(τ). The value of the offset V(τ) can be measured by one's will- ingness to pay for such an instrument, which in turn, can be found by solving for C(τ) when the total cost of using the instrument exactly balances the benefit of using it. This returns the maximum price at which offsets would be an attractive compliance vehicle: V(τ) = Ps(τ) - f·Pe(τ). Further dividing through by the spot price, the value of the offset relative to the spot price is δ = V(τ)/Ps(τ) = 1 - f·Pe(τ)/ Ps(τ). Since, from a cost containment perspective, we are most concerned with the case where Ps(τ) > Pe(τ), it is use- ful to define S as the relative price shock at t = τ, or equiv- alently, as the factor by which the spot price exceeds the target price, so that S = Ps(τ)/Pe(τ). This implies that δ = 1 - f/S. If δ is the relative value of the offset, it is also the fac- It is worth noting that the full present value of the carbon loss term (assuming the fraction f is lost in time T) can be written as f·Pe(τ)·erT·e-rT. The exponentially increasing term represents the steadily increasing forward price on carbon (as forecast by most model assessments), and the declining exponential term represents the discount factor (with the discount rate also assumed to be equal to the long-term interest rate). Because the exponential terms cancel, the longer expression simplifies to f·Pe(τ), which is what appears in the preceding paragraph. Page 4 of 6 (page number not for citation purposes) Page 4 of 6 (page number not for citation purposes) http://www.cbmjournal.com/content/4/1/3 http://www.cbmjournal.com/content/4/1/3 http://www.cbmjournal.com/content/4/1/3 Carbon Balance and Management 2009, 4:3 tor by which a given ton of emissions should be dis- counted at the time of crediting. tor by which a given ton of emissions should be dis- counted at the time of crediting. ties may use valuation tools like the ones above to deter- mine the optimal use of risky assets within their larger mitigation portfolio. Authors' contributions All authors contributed to the intellectual development of the methodology described here. BKM drafted the manu- script and all authors read and approved the final version. Results ll If risk is internalized publicly by dis- counting ex ante, which may be particularly necessary if reversals result from economic leakage off-site rather than physical losses on-site, then the regulator may apply our valuation tools to the project certification process itself. Details of the discounting algorithm could be included in legislation or left to a regulatory body to implement, in either case providing a methodology by which forest car- bon and other risky carbon assets could be properly val- ued and regulated within a future cap-and-trade system. Competing interests p g The authors declare that they have no competing interests. The authors declare that they have no competing interests Methods All methods are described in the main text of the manu- script. References Kurz WA, Dymond CC, Stinson G, Rampley GJ, Neilson ET, Carrol AL, Ebata T, Safranyik L: Mountain pine beetle and forest carbon feedback to climate change. Nature 2008, 452:987-990. g , 5. Weidinmyer C, Neff J: Estimates of CO2 from fires in the United States: Implications for carbon management. Carbon Balance and Management 2007, 2:10. Discussion The discount factor δ is plotted as a function of f and S in Figure 1. A few limiting cases are worth discussing explic- itly. First, consider the case f → 0, in which there is no reversal over the relevant time horizon. In that case, δ → 1, meaning that the offset credit should not be devalued at all at the time of crediting, regardless of market prices. That is, the credit should be treated like any other ton of emissions abatement from the energy sector. For many activities related to changes in land manage- ment, 0 <f < 1, but the exact risk-adjusted value of f may not be known precisely. For simplicity, consider the alter- native limit f → 1, which implies that all carbon originally sequestered is ultimately lost. Arguably, this is a good approximation when the relevant time horizon is very large. Under this limit, δ depends on the size of the rela- tive price shock S. When S is extremely large (e.g. Ps(τ) → ∞), then δ → 1. Intuitively, when the spot price is high rel- ative to the target price, even permits associated with full reversals would not be discounted much because the opportunity to shift abatement is valuable when spot prices are considerably higher than expected future prices. This situation is most likely to be encountered during the early periods of a new compliance regime, when technol- ogy substitutes, like carbon capture and storage (CCS), may not be widely available. In the opposite extreme, where there is no cost shock whatsoever so that S = 1 (i.e. Ps(τ) → Pe(τ)), then δ → 0 and no permits would be issued for offsets associated with full reversals, because there is no economic value in moving abatement to future periods when the spot price exactly equals the discounted expected future price. Acknowledgements The authors would like to acknowledge travel and workshop support from National Science Foundation collaborative grants EAR-0435728 (Whitman College, C. S. Weiler P.I.) and EAR-0435719 (University of Oregon, R. B. Mitchell P.I.). In addition, BKM acknowledges financial support from the Energy Foundation; MDH received financial support from the U.S. Depart- ment of Energy's Office of Science (BER) through the Western Regional Center of the National Institute for Climatic Change Research at Northern Arizona University; and BS appreciates financial support from the Ohio State University Climate, Water and Carbon Initiative. Finally, BKM thanks A. Morris for helpful comments on the manuscript. References 1. Canadell JG, Le Quere C, Raupach MR, Field CB, Bultenhuls ET, Ciais P, Conway TJ, Gillett NP, Houghton RA, Marland G: Contributions to accelerating atmospheric CO2 growth from economic activity, carbon intensity and efficiency of natural sinks. Proc Nat Acad Sci 2007, 104:18866-18870. 2. Levy PE, Friend AD, White A, Cannell MGR: The influence of land use change on global-scale fluxes of carbon from terrestrial ecosystems. Clim Change 2004, 67:185-209. 3. Westerling AL, Bryant BP: Climate change and wildfire in Cali- fornia. Clim Change 2008, 87(Supple 1):S231-S249. 4. Kurz WA, Dymond CC, Stinson G, Rampley GJ, Neilson ET, Carrol AL, Ebata T, Safranyik L: Mountain pine beetle and forest carbon feedback to climate change. Nature 2008, 452:987-990. 5. Weidinmyer C, Neff J: Estimates of CO2 from fires in the United States: Implications for carbon management. Carbon Balance and Management 2007, 2:10. 6. E.g., The American Clean Energy and Security Act of 2009 (H.R. 2454) [http://energycommerce.house.gov/Press_111/ 20090515/hr2454.pdf] 7. E.g., A call for action: Consensus principles and recommen- dations from the US Climate Action Partnership US Climate Action Partnership 2009 [http://www.us-cap.org/USCAPCallForAc tion.pdf]. 8. The Regional Greenhouse Gas Initiative Model Rule [http:// www.rggi.org/docs/model_rule_corrected_1_5_07.pdf] 9. The Voluntary Carbon Standard [http://www.v-c-s.org/afl.html] 1. Canadell JG, Le Quere C, Raupach MR, Field CB, Bultenhuls ET, Ciais P, Conway TJ, Gillett NP, Houghton RA, Marland G: Contributions to accelerating atmospheric CO2 growth from economic activity, carbon intensity and efficiency of natural sinks. Proc Nat Acad Sci 2007, 104:18866-18870. In applying this rule to cases where Ps(τ) < Pe(τ), we rec- ommend that S be set to 1. If the policy allows permits to be banked for future use, as most do, then prices that fall short of expectations probably do not indicate an ineffi- cient time path (such inefficient allocations should largely be arbitraged away through banking), but rather that abatement costs are simply lower than projected. In these circumstances, offset valuations should be based only on the extent to which they result in perfect sequestration, not on broader market conditions. , 2. Levy PE, Friend AD, White A, Cannell MGR: The influence of land use change on global-scale fluxes of carbon from terrestrial ecosystems. Clim Change 2004, 67:185-209. y g 3. Westerling AL, Bryant BP: Climate change and wildfire in Cali- fornia. Clim Change 2008, 87(Supple 1):S231-S249. g ( pp ) 4. Conclusion g 6. E.g., The American Clean Energy and Security Act of 2009 (H.R. 2454) [http://energycommerce.house.gov/Press_111/ 20090515/hr2454.pdf] The analytic framework described here provides practical guidance to policymakers charged with regulating the future carbon market. Our model is agnostic about the extent to which liability for reversal risk should reside in private or public hands, as long as it is internalized in some way. If internalized privately, then individual enti- 7. 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Clim Policy 2004, 4:65-74. instrument for carbon bears. Clim Policy 2004, 4:65-74 14. Cost Containment for the Carbon Market: A proposal Nicho- las Institute White paper 2007 [http://www.nicholas.duke.edu/institute/ carboncosts/carboncosts.pdf]. Duke University p ] y 15. U.S. Environmental Protection Agency Analyses of Climate Change Legislation [http://www.epa.gov/climatechange/econom ics/pdfs/HR2454_Analysis.pdf] 16. Sohngen B, Brown S: Measuring leakage from carbon projects in open economies: a stop timber harvesting project in Bolivia as a case study. Can J For Res 2004, 34:829-839. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 6 of 6 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge
https://openalex.org/W3138280130	https://bmcmededuc.biomedcentral.com/track/pdf/10.1186/s12909-020-02262-7	English	null	Assessing perceived organ donation competencies among pediatric intensivists in Saudi Arabia: A national survey	Research Square (Research Square)	2,020	cc-by	8,322	Kazzaz and Da’ar BMC Medical Education (2020) 20:358 https://doi.org/10.1186/s12909-020-02262-7 Kazzaz and Da’ar BMC Medical Education (2020) 20:358 https://doi.org/10.1186/s12909-020-02262-7 (2020) 20:358 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. Abstract Background: As increasing the number of organ donations presents a global challenge, Saudi Arabia is no different. Intensivists can play a major role in maximizing the organ donation process and minimize the challenges. The purpose of this study was to investigate Saudi pediatric intensivists’ comfort and importance levels of organ donation competencies. Methods: We conducted a cross-sectional survey whose sampling frame included 100 pediatric intensivists. The pediatrician intensivists were identified through an updated list provided by the Saudi Critical Care Society. We assessed 14 competencies categorized into four domains: the general donation, donation after brain death (DBD), neurological determination of death, and medicolegal, religious, and ethical domains. Then we investigated the association between these competencies and physicians’ characteristics. Results: With a response rate of 76%, we found that 40–60% of the surveyed pediatric intensivists rated their comfort in 6 out of 14 competencies as high or very high. There was a statistically significant gap in the intensivists’ rating of 10 competencies (i.e., high importance but low comfort levels). Ordinal regression showed that comfort levels with the general donation, neurological determination of death, and medicolegal, religious, and ethical domains were higher in intensivists who were frequently involved with DBD than those who had never been exposed. Conclusions: Pediatric intensivists expressed low comfort levels to organ donation competencies that are essential for maximizing donation rates. Adapting mandatory comprehensive donation education programs and dedicated physician specialists may be beneficial in critical care units aiming to increase donation rates. Keywords: Donation after brain death, Intensive care unit physician, Organ donation, Tissue donation, Saudi Arabia after brain death [DBD]) or after circulatory death (DCD). The primary source of organ donation is DBD [3]. Organ donation has legislative and regulatory implications that vary across countries. Worldwide organ donation rates are measured in donors per million of population (PMP). De- ceased donation rates vary among different countries, ran- ging from 0.2 PMP to 46.90 PMP [4]. Assessing comfort level of organ donation competencies among pediatric intensivists in Saudi Arabia: a national survey Yasser M. Kazzaz1,2,3* and Omar B. Da’ar3,4 Background End-stage organ failure is associated with high mortality, and organ transplantation is the current standard of care [1]. Organ transplantation would not be possible without the generosity of living and deceased organ donation [2]. Deceased donation is possible after brain death (donation In spite of the global efforts to improve organ dona- tion rates, the number of organ donors remains low [5]. Patients with end-organ failure face long waiting times on the waitlist, which sometimes ends with mortality * Correspondence: kazzazy@ngha.med.sa 1Department of Pediatrics, Ministry of National Guards - Health Affairs, Riyadh, Kingdom of Saudi Arabia 2College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia Full list of author information is available at the end of the article * Correspondence: kazzazy@ngha.med.sa 1Department of Pediatrics, Ministry of National Guards - Health Affairs, Riyadh, Kingdom of Saudi Arabia 2College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia Full list of author information is available at the end of the article Methods The important role intensivists play in the process of organ donation has been recognized by leading countries. Spain, for instance, has the world’s highest deceased organ donation rate (46.9 PMP), followed by Portugal in a distant second with 34 PMP (actual deceased donors) during the same period [15]. While Spain’s success in deceased organ dona- tion is multifactorial, the role of trained intensivists forms a cornerstone of that success. Intensivists both lead and iden- tify the disclosure process. All health professionals in Spain are trained in deceased organ donation [12, 16]. We conducted a nationwide prospective cross-sectional survey of pediatric intensivists working in teaching and nonteaching governmental pediatric ICUs across Saudi Arabia. We identified all pediatric intensivists across Saudi Arabia, through an updated list provided by the Saudi Critical Care Society. We administered the survey using web-based software. The invitation to participate in the study was extended through an email containing the link to the online survey to all pediatric intensivists. Non-responders received electronic reminders 4 weeks apart, up to three reminders. The study was approved by the Institutional Review Board of the King Abdullah International Medical Research Center, and informed consent was obtained from each participant prior to par- ticipation in the study. Pediatric intensivists play the same pivotal role in the identification and management of pediatrics organ do- nors [8]. The American Academy of Pediatrics recom- mends national strategies for the training of physicians involved in all aspects of organ transplantation—includ- ing psychological, social, and medical aspects [17]. Evi- dence suggests pediatric intensivists have less knowledge when compared to adult intensivists on matters of brain death, and only one-third (33%) of pediatric intensivists considered themselves with a high level of assurance in explaining brain death to family members [18]. We used a previously validated Canadian survey, and permission to use the survey was obtained from the au- thors [23]. The survey contained competencies in five domains—including general topics in organ and tissue donation, the neurological determination of death dona- tion, the circulatory determination of death donation, medicolegal and religious considerations, and transplant. Given that organ DCD is not performed in Saudi Arabia, we omitted this domain. Two religious consideration competencies were added to the neurological consider- ation and medicolegal domains. For each competency, participants were asked two questions using a Likert Organ transplantation spans over four decades in Saudi Arabia [19, 20]. Page 2 of 11 Page 2 of 11 Page 2 of 11 Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Kazzaz and Da’ar BMC Medical Education (2020) 20:358 The Saudi Center for Organ Transplantation (SCOT) has laid down clear policies for the diagnosis of death by brain function criteria and acquisition of the approval of religious scholars on DBD. While SCOT facilitates the process of organ donation through coordinators assigned for different hospitals, most intensivists are not aware of this role [20, 22]. Children who are potential donors comprise a significant proportion of all donors, with those under 10 years of age and those between 10 and 20 years of age accounting for 11 and 28.4% of all do- nors, respectively [21]. before receiving an organ. The situation is worse for the pediatric population; twice as many pediatric patients die while waiting for organ transplants when compared to adults [6]. The proportion of deceased organ donation in pediatrics is lower than in adults due to several rea- sons—including low mortality in the pediatric age group, medical unsuitability, contraindications, and missed op- portunities with potential donors [7]. This has led to limiting organ transplant surgeries in pediatrics [8, 9]. Reasons for poor donation rates are multifactorial and include medical contraindication, failure to identify po- tential donors, failure of donor organ maintenance, fail- ure to confirm brain death diagnosis, and refusal by relatives [10]. Brain death disclosure and DBD discus- sions occur mostly in the intensive care unit (ICU), ren- dering intensivists major stakeholders in this process [11]. Intensivists need to acquire essential competencies to achieve successful donations—such as identifying po- tential donors, guiding families through the process of brain death, and facilitating donor management [12, 13]. A lack of these competencies leads to missed donations. A retrospective study of all deaths in ICUs and emer- gency rooms in Alberta, Canada, identified 64 patients with a high probability of becoming brain death donors that were missed, which could have increased the organ donation rate by 7.5 PMP [14]. Thus, organ donation in Saudi Arabia could greatly benefit from involving intensivists in the process. How- ever, to formalize the organ donation process, intensi- vists’ competencies need to be identified and knowledge gaps and skill deficiencies assessed. This study, therefore, set out to identify intensivists’ competency comfort levels relating to the brain death process and organ do- nation. Unlike previous studies, which were general in terms of assessing the organ and tissue donation, the present study addresses physicians’ competencies with respect to several aspects, including the neurological de- termination of death and DBD, while exploring the med- ical, legal, and cultural/religious considerations of transplantation. Methods The country is the fourth-highest in living organ donations, with 27.7 PMP. However, de- ceased donation rates remained relatively low (3.3 PMP) in 2017 [15]. From 2010 to 2016, the annual number of reported potential DBD cases remained relatively un- changed between 570 and 710 [21]. Page 3 of 11 Page 3 of 11 Kazzaz and Da’ar BMC Medical Education (2020) 20:358 (65.8%), consent discussions (47.4%), ethical considerations (57.9%), and legal considerations (43.4%). Largely, respon- dents rated all competencies related to the general donation, DBD, and medicolegal, religious, and ethical domains as of high or very high importance to their practice (69.7–94.7%), and around 50% of the respondents rated the transplant do- main as of high or very high importance to their profession. McNemar’s test indicated a statistically significant difference between comfort and importance levels (low comfort and high importance) in 10 competencies. Those competencies included offering families opportunities of organ donation (p = 0.001); the referral process for SCOT (p < 0.001); an ex- planation that Islamic law permits organ donation (p = 0.001); the management of potential brain death donor (p = 0.013); consent discussions for organ and tissue donation (p = 0.002); ethical (p = 0.009), legal (p < 0.001), and religious considerations in a deceased donor (p = 0.023); recipient prioritization and organ allocation (p < 0.001); and organ transplant outcomes (p = 0.017). scale of five points: the first question concerned “com- fort with competency” and the second question, “the im- portance of this competency to your work.” IBM SPSS version 26 was used to analyze the data. The median and percentile (Q1–Q3) were used to describe the continuous variables. Frequencies and percentages were used to describe the categorical variables such as gender, the health sector, region among others. McNemar’s test was used to assess the differences between the importance of organ donation competencies and the perceived com- fort level of the same competencies. Ordinal logistic regression was used to model the asso- ciation between participant characteristics and ordinal response variables (comfort levels in the four domains of organ and tissue donation competencies). Each domain score was derived from the mean of the competencies of that domain. Whenever the proportional odds assump- tion was violated, categories were collapsed (very low, low, and average; high and very high), and the Fisher exact test was performed. Results Table 1 shows the respondents’ demographics and organ donation experiences. A total of 76 physicians out of the targeted 100 responded to the survey, representing a re- sponse rate of 76% across all Saudi health sectors. The median age of the responding physicians and their clin- ical experience was 42.5 years (IQR 38.25–49) and 12 years (IQR 8–8.75), respectively. All health-care sectors and regions were represented. Nearly half (46.1%) of the surveyed participants reported involvement in DBD two to five times per year, and 6.6% reported that they had never been involved in DBD. Responding physicians largely worked at academic centers (62.3%) and centers that have postgraduate fellowship critical care programs (57.1%). The proportion of participants from centers with a DBD program and centers with a transplant pro- gram were 67.5 and 39%, respectively. When asked about potential barriers to organ and tis- sue donation in Saudi Arabia, most of the respondents (85.3%) chose negative family beliefs as a major barrier. Three-fifths (60%) of the respondents agreed that dis- comfort in offering families opportunities of donation was a major barrier as well. In addition, more than 40% of the respondents agreed that a lack of staff designated to provide donation services and their competency levels were barriers (Fig. 1). When asked about their opinion on the benefit of a potential national education program on organ donation to intensivists, 60% of the respon- dents rated the program to be of great value, and an additional 30% rated it of moderate significance. Table 2 summarizes physicians’ responses to the questions regarding the perceived comfort levels with organ donation competencies and how important they viewed the same competencies in their practice. Overall, more than 40% of the respondents rated their comfort to the general donation, DBD, and medicolegal, religious, and ethical domains as high or very high (43.4–96.1%). However, less than 50% of the re- spondents rated their comfort to the transplant domain as high or very high (26.3–50%). It is important to note that only 40–60% of the respondents rated comfort to key com- petencies as high/very high. Those competencies include of- fering families opportunities of donation (52.5%), referral to SCOT (60.5%), an explanation that Islamic law permits DBD Methods Associations identified in the analyses were expressed as odds ratios and 95% confi- dence intervals. A p-value < 0.05 was considered statisti- cally significant. Tables 3, 4, 5 and 6 show the association between the phy- sicians’ characteristics (the health sector, region, frequency of involvement in DBD, practice at a center with a DBD pro- gram, and practice at a center with a transplant program) and comfort levels in the four domains of organ and tissue donation competencies. Ordinal regression showed statisti- cally significant differences in the comfort levels in three out of the four domains (the general organ donation, DBD, and medicolegal, religious and ethical domains); intensivists ex- posed more to DBD perceived higher comfort levels in those domains. Similar to the frequency of exposure, both the health sector and region of practice was associated with the general donation domain. Additionally, physicians working at a center with a DBD program appeared to be associated with the comfort levels of medicolegal, religious, and ethical com- petencies. Finally, working at a transplant center was associ- ated with more comfort with these competencies. Discussion Saudi Arabia has taken important steps in improving organ donation rates in the country. There are four organ donation initiatives in the National Transformation Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Page 4 of 11 Table 1 Characteristics of participants Variable Median Q1-Q3 Age (Years) 42.5 38.25–49 Experience (Years) 12 8–18.75 n % Gender Male 60 78.9% Female 16 21.1% Years of experience < 5 3 3.9% 5 to 9 26 34.2% 10 to 14 16 21.1% 15 to 19 14 18.4% > 20 17 22.4% Health Sector Ministry of Health 28 36.8% Ministry of National Guard-Health Affairs 17 22.4% Ministry of Defense and Aviation - Armed Forces Hospitals 14 18.4% King Faisal Specialist Hospital & Research Centre 5 6.6% Ministry of Higher Education 12 15.8% Region Central 47 61.8% Eastern 12 15.8% Northern 1 1.3% Southern 5 6.6% Western 11 14.5% Academic or Community Academic 48 63.2% Community 28 36.8% Critical Care Training Critical Care training 69 90.8% Without Critical Care training 7 9.2% PICU type All 21 27.6% Medical Only 10 13.2% Medical Surgical 40 52.6% Cardiac 5 6.6% Fellowship Centre Yes 44 57.9% No 32 42.1% Frequency of involvement in DBD Never 5 6.6% Once every few years 14 18.4% Once per year 15 19.7% 2–5 times per year 35 46.1% 6 or more times per year 7 9.2% Center Performing DBD Yes 52 68.4% No 22 28.9% I don’t know 2 2.6% Center Performs Solid Organ Transplant Yes 30 39.5% No 46 60.5% Table 1 Characteristics of participants Program. This is a program with several radical initiatives regarding the structure and function of the healthcare sys- tem to improve the quality of healthcare. The initiatives include a national awareness campaign for organ donation and transplant programs, an interactive virtual laboratory for training on organ donation and transplantation, and a national program for evaluating organ transplant centers and promoting organ donation programs in ICUs [24]. and transplant programs, an interactive virtual laboratory for training on organ donation and transplantation, and a national program for evaluating organ transplant centers and promoting organ donation programs in ICUs [24]. Discussion Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Page 5 of 11 Table 2 Comparison between organ donation competencies comfort and importance level Competency Comfort Importance p Very Low/ Low/ Average High/ Very High Very Low/ Low/ Average High/ Very High n % n % n % n % The benefits of organ and tissue donation 3 3.9% 73 96.1% 7 9.2% 69 90.8% 0.289 Offering families the opportunity for organ and tissue donation 36 47.4% 40 52.6% 21 27.6% 55 72.4% 0.001 The referral process for tissue donation to SCOT 30 39.5% 46 60.5% 12 15.8% 64 84.2% < 0.001 Identification of potential brain death donors 11 14.5% 65 85.5% 9 11.8% 67 88.2% 0.754 Diagnosis of brain death 8 10.5% 68 89.5% 4 5.3% 72 94.7% 0.289 Explaining brain death to family 10 13.2% 66 86.8% 5 6.60% 71 93.4% 0.125 Explaining that Islamic law permits organ donation 26 34.2% 50 65.8% 12 15.8% 64 84.2% 0.001 Management of a potential brain death donor until organ procurement 20 26.3% 56 73.7% 10 13.2% 66 86.8% 0.013 Consent discussions for organ and tissue donation 40 52.6% 36 47.4% 23 30.3% 53 69.7% 0.002 Ethical considerations in deceased donation 32 42.1% 44 57.9% 17 22.4% 59 77.6% 0.009 Legal considerations in deceased donation 43 56.6% 33 43.4% 17 22.4% 59 77.6% < 0.001 Religious considerations in deceased donation 31 40.8% 45 59.2% 19 25% 57 75% 0.023 Recipient prioritization and organ allocation 56 73.87% 20 26.3% 33 43.4% 43 56.6% 0.000 Organ transplant outcomes 38 50% 38 50% 28 36.8% 48 63.2% 0.017 2 Comparison between organ donation competencies comfort and importance level intensivist approach and skills in discussing organ dona- tion with families [28]. Respondents in our study ac- knowledged that discomfort in offering families donation opportunities is a barrier to organ donation. This finding of discomfort is consistent with the evidence in other studies, which show that only one-third (33%) of poten- tial donors are reported in the country [29]. On explor- ing factors associated with organ donation decisions, several studies point out that family agreement for dona- tion is predicted by healthcare providers’ comfort with answering families’ donation questions, their confidence, and education level [30, 31]. This has led to the World Brain Death Project to recommend education about counseling families during end-of-life care to healthcare providers managing brain death patients [32]. Discussion Second, the Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Page 6 of 11 Table 3 Participants’ characteristics and comfort level with general organ donation competencies Variable OR (95%CI) VL/L/Ave H/VH P Health Sector Ministry of Health 0.395 (0.086 to 1.822) 0.234 Ministry of National Guards - Health Affairs 0.413 (0.091 to 1.878) 0.252 Ministry of Defense and Aviation - Armed Forces Hospitals 0.095 (0.019 to 0.484) 0.005 King Faisal Specialist Hospital & Research Centre 0.284 (0.034 to 2.351) 0.243 Ministry of Higher Education 1 . Region West 0.457 (0.122 to 1.713) 0.245 South 0.1 (0.012 to 0.868) 0.037 East 1.705 (0.447 to 6.501) 0.434 Central 1 (. to.) . Frequency of involvement in donation after brain death 6 or more times per year 31.762 (2.102 to 479.92) 0.013 2–5 times per year 60.775 (5.834 to 633.13) 0.001 Once per year 12.86 (1.173 to 141.037) 0.037 Once every few years 34.993 (3.06 to 400.164) 0.004 Never 1 . Center with a DBD program* Yes 12 (22.2%) 42 (77.8%) 0.204 No 8 (36.4%) 14 (63.6%) Transplant center* Yes 10 (33.3%) 30 (66.7%) 0.262 No 10 (21.7%) 46 (78.3%) OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test. OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test. OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, ve with high and very high using Fisher exact test. that healthcare providers be trained in cultural sensitiv- ity and communication [32]. associated with the frequency of exposure to cases of DBD. The variation by region is consistent with the evidence in previous studies where donation rates were found to vary markedly depending on region, ranging between 0 PMP and 7.5 PMP [41]. This variation persisted between densely popu- lated areas with similar ICU facilities [41]. Discussion Since 1994 all Australian intensivists complete educational programs on organ donation and family conversation. A study conducted in 4 teaching hospitals in Australia, al- most all intensivists felt skilled at approaching families and attributed this to the educational program [33]. This study was developed in line with the National Trans- formation Program initiatives for improving organ dona- tion rates. This study was developed in line with the National Trans- formation Program initiatives for improving organ dona- tion rates. The main purpose of this study was to assess/measure pediatric intensivists’ perceived organ donation compe- tencies. The findings suggest low perceived comfort levels in several organ donation competencies, and com- fort levels in several organ donation domains were influ- enced by the participants’ frequency of exposure to DBD, in addition to the health sector and region of prac- tice. Although the participants viewed most of the com- petencies as important to their practice, they reported less comfort levels with the same competencies. A majority of the pediatric intensivists rated the im- portance of key competencies as high or very high but the comfort levels of the same competencies as lower. The low comfort levels in several competencies and per- ceived gaps in knowledge are in agreement with the current literature. In Saudi Arabia, one study reported that less than one-third of the surveyed intensivists knew the role of SCOT in the researchers’ knowledge and atti- tude study [22]. Another study in the country found that healthcare providers in ICUs possess inadequate know- ledge regarding organ donation [25]. Organ donation discussions require intensivists to be familiar with relevant laws and be aware of religious scholars’ perspectives. Family ethical or religious under- standing influences organ donation decisions and may lead to refusal of donation [34–36]. In our study, intensi- vists reported low comfort in legal, ethical, and religious considerations. Although the majority of Islamic Scholars acknowledge brain death as true death and per- mit organ donation [37], it has been shown that Muslim families frequently reject brain death and refuse organ donation [38]. World Brain Death Project recommends Respondents reported lower comfort rating in offering families donation opportunity and consent discussions. Studies from North America found that intensivists were not prepared to support organ donation decisions [26, 27]. Two issues seem to account for missing potential donors in critical care units. First, families are not approached by healthcare providers [27]. Discussion y The Saudi Commission for Health Specialties (SCFHS) is the governmental body responsible for developing, ap- proving, and supervising professional postgraduate med- ical education programs in the nation [39]. The SCFHS included brain death evaluation and certification, as well as principles of organ donation and transplantation, in the mandatory objectives of critical care fellowship [40]. Neither SCFHS nor SCOT does not direct formal teach- ing on principles and processes of organ donation. The low comfort rate in several competencies reflects that educational objectives alone are not sufficient. In addition, our study showed that the higher exposure to DBD was associated with a higher comfort level of dona- tion competencies, which suggests that pediatric intensi- vists do acquire their knowledge in donation from experience rather than education. The gap in the participants’ perceived competencies (i.e., high importance but lower comfort level) is a pointer that the intensive care community is conscious of the need for an educational curriculum. A majority of the participants rated a possible national education pro- gram on organ donation as of moderate to high value. This is in contrast to previous studies, where only one- fifth of the surveyed intensivists thought there was a need for intensivist training on organ donation in Saudi Arabia [22]. Yet another study found that only one-third of the participants were willing to participate in organ donation training [25]. The change in attitude toward needed education is encouraging, suggesting that a na- tional program will be well-received. Discussion The comfort level with organ donation varied among intensivists from different regions and health sectors and was Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Page 7 of 11 Table 4 Participants’ characteristics and comfort level with neurological determination of death competencies Variable OR (95%CI) VL/L/ Ave H/VH P Health Sector Ministry of Health 0.368 (0.086 to 1.575) 0.178 Ministry of National Guards - Health Affairs 0.572 (0.128 to 2.552) 0.464 Ministry of Defense and Aviation - Armed Forces Hospitals 0.21 (0.042 to 1.047) 0.057 King Faisal Specialist Hospital & Research Centre 1.573 (0.173 to 14.315) 0.688 Ministry of Higher Education 1 Region West 1 (9.1%) 10 (90.9%) 0.141 South 2 (40%) 3 (60%) East 0 (0%) 12 (100%) Central 6 (12.8%) 41 (87.2%) Frequency of involvement in donation after brain death 6 or more times per year 28.324 (2.467 to 325.24) 0.007 2–5 times per year 13.874 (1.92 to 100.268) 0.009 Once per year 9.151 (1.121 to 74.717) 0.039 Once every few years 4.573 (0.553 to 37.783) 0.158 Never 1 Center with a DBD program* Yes 6 (11.1%) 48 (88.9%) 0.408 No 4 (18.2%) 18 (81.8%) Transplant center* Yes 5 (16.7%) 25 (83.3%) 0.465 No 5 (10.9%) 41 (89.1%) * OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test tics and comfort level with neurological determination of death competencies Transplant center * OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very with high and very high using Fisher exact test OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very with high and very high using Fisher exact test Overall, our study shows that Saudi intensivists who participated in the survey had the same comfort levels in most of the competencies. Discussion However, in comparison, a higher number of Canadian intensivists rated four com- petencies as high or very high [23]—including offering families opportunities for organ donation, the referral process of organ donation, consent discussion, and eth- ical considerations. pediatric intensivists in organ donation was found to be relatively infrequent in our study, with 44.7% of intensi- vists reporting involvement in organ donation once per year or less [21]. Due to this low exposure, it is evident that there is no single solution for improving organ dona- tion rates, and a bundle of interventions should be consid- ered to reform the current approach of organ donation in critical care units. Successful countries in organ donation have identified key interventions that included the integra- tion of donation physician specialists and training in organ donation for critical care staff [42, 44]. The current approaches to the evaluation, reporting, identification, and referral of potential donors in Saudi Arabia somewhat lack consistency among regions and do not involve intensive care physicians. The importance of integrating intensive care physicians in leading the organ donation process has been recognized globally. Spain, the world’s leader in organ donation, has an inte- grated system built on three pillars: (a) a transplant co- ordinator, who is mainly a critical care physician reporting to the hospital medical director with a part- time dedication to transplant activities; (b) training on organ donation through different types of courses for transplant coordinators and intensive care physicians; and (c) a quality assurance program for the deceased do- nation process [42, 43]. Dedicated donation physician specialists were intro- duced initially in Spain, then replicated by other coun- tries (Croatia, Portugal, United Kingdom, Australia, the United States, and Canada) [44–46]. Donation physician specialist is a hospital-based staff, mostly intensivist with expertise in organ donation, who is responsible for pro- gram administration, education, and training for critical care staff [45]. Medical education and training as well as the afore- mentioned donation physician specialist is an essential key intervention. Findings from the study identified defi- ciencies in key competencies that can tailor a medical education curriculum. Discussion In Spain, all intensive care physi- cians are mandated to complete training in the Although in Saudi Arabia, pediatric donors comprise up to 40% of all potential donors, the involvement of Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Page 8 of 11 Table 5 Participants’ characteristics and comfort level with medicolegal considerations, religious and ethics competencies Variable OR (95%CI) VL/L/Ave H/VH P Health Sector Ministry of Health 0.465 (0.112 to 1.927) 0.291 Ministry of National Guards - Health Affairs 1.197 (0.29 to 4.944) 0.804 Ministry of Defense and Aviation - Armed Forces Hospitals 0.616 (0.141 to 2.687) 0.519 King Faisal Specialist Hospital & Research Centre 2.258 (0.298 to 17.088) 0.43 Ministry of Higher Education 1 . Region West 1.552 (0.423 to 5.699) 0.508 South 6.344 (0.792 to 50.845) 0.082 East 1.889 (0.521 to 6.846) 0.333 Central 1 . Frequency of involvement in donation after brain death 6 or more times per year 141.204 (9.901 to 2013.767) <0.001 2–5 times per year 43.66 (5.125 to 371.925) 0.001 Once per year 17.751 (1.901 to 165.778) 0.012 Once every few years 7.144 (0.818 to 62.405) 0.075 Never 1 . Discussion Center with a DBD program Yes 16 (29.6%) 38 (70.4%) 0.016 No 13 (59.1%) 9 (40.9%) Transplant center* Yes 10 (33.3%) 20 (66.7%) 0.484 No 19 (41.3%) 27 (58.7%) OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared ith hi h d hi h i Fi h t t t Table 6 Participants’ characteristics and comfort level with transplant competencies Variable OR (95%CI) VL/L/Ave H/VH P Health Sector Ministry of Health 1.115 (0.302 to 4.107) 0.87 Ministry of National Guards - Health Affairs 0.937 (0.24 to 3.665) 0.926 Ministry of Defense and Aviation - Armed Forces Hospitals 0.523 (0.127 to 2.148) 0.369 King Faisal Specialist Hospital & Research Centre 2.09 (0.294 to 14.861) 0.461 Ministry of Higher Education 1 . Region West 1.853 (0.529 to 6.488) 0.335 South 0.921 (0.151 to 5.598) 0.928 East 1.526 (0.449 to 5.189) 0.498 Central 1 . OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test ence interval, VL Very low, L Low, Ave Average, H High, VH Very high se tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and d very high using Fisher exact test Discussion Frequency of involvement in donation after brain death * 6 or more times per year 4 (57.1%) 3 (42.9%) 0.804 2–5 times per year 19 (54.3%) 16 (45.7%) Once per year 10 (66.7%) 5 (33.3%) Once every few years 8 (57.1%) 6 (42.9%) Never 4 (80%) 1 (20%) Center with a DBD program * Yes 31 (57.4%) 23 (42.6%) 0.616 No 14 (63.6%) 8 (36.4%) Transplant center * Yes 12 (40%) 18 (60%) 0.006 No 33 (71.7%) 13 (28.3%) OR Odds ratio, CI Confidence interval, VL Very low, L Low, Ave Average, H High, VH Very high *Asterisks represent those tests that did not fit the proportional odds assumption; therefore, very low, low, and average results were combined and compared with high and very high using Fisher exact test Table 6 Participants’ characteristics and comfort level with transplant competencies Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Page 9 of 11 Fig. 1 Potential barriers to organ and tissue donation Fig. 1 Potential barriers to organ and tissue donation Fig. 1 Potential barriers to organ and tissue donation Fig. 1 Potential barriers to organ and tissue donation donation, precisely DBD. The key finding was that pediatric intensivists exhibited low comfort levels with organ donation competencies, and their comfort was asso- ciated with the frequency of exposure to organ donation patients. Our findings may have significant implications for organ donation reform programs. First, the pediatric critical care community is aware of the need in expanding the role of intensive care physicians in the organ donation process. Second, the findings of low comfort level in sev- eral competencies with infrequent involvement of pediatric intensivists in DBD, combined with low organ donation rates in the country, suggest a role for donation physician specialists and mandatory comprehensive dona- tion education programs for intensivists and include all competencies with a focus on family conversation tech- niques and national laws, ethical, religious views around brain death. These strategies of comprehensive donation education program and dedicated donation physician spe- cialists could increase rates of organ donation and meet the need for transplantation. principles of donation. Specifically designed organ dona- tion training [42, 47]. In the UK, all staff involved in organ donation receive mandatorily specifically designed organ donation training [44]. Discussion Our study presents several strengths, including the ap- plication of statistical and methodological approaches to organ donation competencies and related factors for the first time in Saudi Arabia. The relatively higher response rate (76%) compared to similar studies [23], a represen- tation of responses across all health sectors and regions of Saudi Arabia, bolsters our findings. Findings from this study are relevant and potentially generalizable to other countries with low organ donation rates that are seeking to reform their organ donation programs. It should be noted while medical education in the organ donation process and family conversation to critical care physi- cians and implementing donation physician specialists are key interventions at critical care level, they need to be complemented by interventions at hospitals, commu- nities, and national levels for a successful organ donation program. This study has a limitations. As with any sur- vey: there might be a gap between the studied percep- tions and reality. Nevertheless, such limitation is expected to produce higher comfort with competencies than in reality, supporting our finding of low comfort in organ donation competencies. Abbreviations DBD: Donation after brain death; DCD: Donation after circulatory death; ICU: Intensive care unit; PMP: PMP Per million of Population; SCFHS: Saudi Commission for Health Specialties; SCOT: Saudi Center for Organ Transplantation Abbreviations DBD: Donation after brain death; DCD: Donation after circulatory death; ICU: Intensive care unit; PMP: PMP Per million of Population; SCFHS: Saudi Commission for Health Specialties; SCOT: Saudi Center for Organ Transplantation Abbreviations DBD: Donation after brain death; DCD: Donation after circulatory death; ICU: Intensive care unit; PMP: PMP Per million of Population; SCFHS: Saudi Commission for Health Specialties; SCOT: Saudi Center for Organ Transplantation Conclusion g We thank Jennifer Hancock, Ken Lotherington, Sam Shemie, Amber Appleby, and Richard Hall for their permission to use the study survey. Thanks, too, to all the participants involved in this study for their cooperation and support. In conclusion, this study was conducted to assess the comfort levels in the core competencies of organ Page 10 of 11 Page 10 of 11 Page 10 of 11 Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Kazzaz and Da’ar BMC Medical Education (2020) 20:358 Funding Th d 15. Donation and Transplantation Institute. Transplant procurement management - donation and transplantation institute. International registry in organ donation and transplantation. 2018. 15. Donation and Transplantation Institute. Transplant procurement management - donation and transplantation institute. International registry in organ donation and transplantation. 2018. The study was funded by the King Abdullah International Medical Research Center (KAIRMC) [SP19/107/R]. The source of funding had no involvement in the study design; collection, analysis, and interpretation of data; and writing of the report. 16. Shemie SD, MacDonald S. Canadian Blood Services-Canadian critical care society expert consultation group. Improving the process of deceased organ and tissue donation: a role for donation physicians as specialeists. CMAJ. 2014;186:95–6. 16. Shemie SD, MacDonald S. Canadian Blood Services-Canadian critical care society expert consultation group. Improving the process of deceased organ and tissue donation: a role for donation physicians as specialeists. CMAJ. 2014;186:95–6. p g The authors declare no conflicts of interest. The authors declare no conflicts of interest. 23. Hancock J, Shemie SD, Lotherington K, Appleby A, Hall R. Development of a Canadian deceased donation education program for health professionals: a needs assessment survey. Can J Anaesth. 2017;64:1037–47. Received: 20 April 2020 Accepted: 28 September 2020 26. Curtis JR, Engelberg RA, Wenrich MD, Shannon SE, Treece PD, Rubenfeld GD. Missed opportunities during family conferences about end-of-life care in the intensive care unit. Am J Respir Crit Care Med. 2005;171:844–9. 27. Williams MA, Lipsett PA, Rushton CH, Grochowski EC, Berkowitz ID, Mann SL, et al. The physician's role in discussing organ donation with families. Crit Care Med. 2003;31:1568–73. Authors’ contributions 11. Schaeffner ES, Windisch W, Freidel K, Breitenfeldt K, Winkelmayer WC. Knowledge and attitude regarding organ donation among medical students and physicians. Transplantation. 2004;77:1714–8. YK conceptualized and participated study design, coordinated data collection, carried out the initial analyses, drafted the initial manuscript, and read and approved the manuscript as submitted and agree to be accountable for all aspects of the work. OD participated in study design, guided the methodology coordinated and supervised data collection and analyses, reviewed and edited manuscript, read and approved the manuscript as submitted and agree to be accountable for all aspects of the work. Both authors read and approved the manuscript as submitted and agree to be accountable for all aspects of the work. 12. Palaniswamy V, Sadhasivam S, Selvakumaran C, Jayabal P, Ananth SR. Organ donation after brain death in India: a trained intensivist is the key to success. Indian J Crit Care Med. 2016;20:593–6. 13. Rocker GM. Organ and tissue donation in the intensive care unit. CMAJ. 2002;167:1248–9. 13. Rocker GM. Organ and tissue donation in the intensive care unit. CMAJ. 2002;167:1248–9. 14. Kutsogiannis DJ, Asthana S, Townsend DR, Singh G, Karvellas CJ. The incidence of potential missed organ donors in intensive care units and emergency rooms: a retrospective cohort. Intensive Care Med. 2013;39: 1452–9. 14. Kutsogiannis DJ, Asthana S, Townsend DR, Singh G, Karvellas CJ. The incidence of potential missed organ donors in intensive care units and emergency rooms: a retrospective cohort. Intensive Care Med. 2013;39: 1452–9. Availability of data and materials h d d d/ l d d The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 17. Committee on Hospital Care, Section on Surgery, Section on Critical Care. Policy statement--pediatric organ donation and transplantation. Pediatrics. 2010;125:822–8. 17. Committee on Hospital Care, Section on Surgery, Section on Critical Care. Policy statement--pediatric organ donation and transplantation. Pediatrics. 2010;125:822–8. 18. Schein AE, Carvalho PR, Rocha TS, Guedes RR, Moschetti L, La Salvia JC, et al. Evaluation of intensivists' knowledge on brain death. Rev Bras Ter Intensiva. 2008;20:144–8. Consent for publication 21. Saudi Center for Organ Transplantation. Annual report 2016 deceased donation after brain death in the Kingdom of Saudi Arabia. 2016. 21. Saudi Center for Organ Transplantation. Annual report 2016 deceased donation after brain death in the Kingdom of Saudi Arabia. 2016. Ethics approval and consent to participate 18. Schein AE, Carvalho PR, Rocha TS, Guedes RR, Moschetti L, La Salvia JC, et al. Evaluation of intensivists' knowledge on brain death. Rev Bras Ter Intensiva. 2008;20:144–8. The study was approved by the King Abdullah International Medical Research Center (KAIRMC) Institutional Review Board [SP19/107/R]. Informed written consent was obtained from each participant prior to participation in the study. 19. Al-Attar B, Shaheen F, Salam MA, Al-Sayyari A, Babiker A, Zakaria H, et al. Implications of ICU stay after brain death: the Saudi experience. Exp Clin Transplant. 2006;4:498–502. 19. Al-Attar B, Shaheen F, Salam MA, Al-Sayyari A, Babiker A, Zakaria H, et al. Implications of ICU stay after brain death: the Saudi experience. Exp Clin Transplant. 2006;4:498–502. 20. Shaheen FA. Organ transplantation in Saudi Arabia. Transplantation. 2016; 100:1387–9. 20. Shaheen FA. Organ transplantation in Saudi Arabia. Transplantation. 2016; 100:1387–9. 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https://openalex.org/W2732575153	https://zenodo.org/records/2664753/files/11151.pdf	English	null	Microstructure and High Temperature Deformation Behavior of Cast 310S Alloy	Zenodo (CERN European Organization for Nuclear Research)	2,014	cc-by	328	World Academy of Science, Engineering and Technology International Journal of Industrial and Manufacturing Engineering Vol:8, No:6, 2014 World Academy of Science, Engineering and Technology International Journal of Industrial and Manufacturing Engineering Vol:8, No:6, 2014 World Academy of Science, Engineering and Technology International Journal of Industrial and Manufacturing Engineering Vol:8, No:6, 2014 International Scholarly and Scientific Research & Innovation 8(6) 2014 Microstructure and High Temperature Deformation Behavior of Cast 310S Alloy Authors : Jung-Ho Moon, Myung-Gon Yoon, Tae Kwon Ha ut o s : Ju g o oo , yu g Go oo , ae o a Abstract : High temperature deformation behavior of cast 310S stainless steel has been investigated in this study by performing tensile and compression tests at temperatures from 900 to 1200°C. Rectangular ingots of which the dimensions were 350×350×100 in millimeter were cast using vacuum induction melting. Phase equilibrium was calculated using the FactSage®, thermodynamic software and database. Thermal expansion coefficient was also measured on the ingot in the temperature range from room temperature to 1200°C. Tensile strength of cast 310S stainless steel was 9 MPa at 1200°C, which is a little higher than that of a wrought 310S. With temperature decreased, tensile strength increased rapidly and reached up to 72 MPa at 900°C. Elongation also increased with temperature decreased. Microstructure observation revealed that σ phase was precipitated along the grain boundary and within the matrix over 1200°C, which is detrimental to high temperature elongation. and Manufacturing Engineering Vol:8, No:6, 2014 waset.org/abstracts/11151 g Keywords : stainless steel, STS 310S, high temperature deformation, microstructure, mechanical properties Conference Title : ICMET 2014 : International Conference on Manufacturing Engineering and Technology Keywords : stainless steel, STS 310S, high temperature deformation, microstructure, mechanical properties Conference Title ICMET 2014 International Conference on Manufacturing Engineering and Technology Conference Location : Istanbul, Turkey Conference Location : Istanbul, Turkey Conference Dates : June 19-20, 2014 Conference Dates : June 19-20, 2014 International Scholarly and Scientific Research & Innovation 8(6) 2014 ISNI:0000000091950263 1
https://openalex.org/W4281653658	https://bmcpregnancychildbirth.biomedcentral.com/counter/pdf/10.1186/s12884-022-04778-w	English	null	Exploring solutions to improve antenatal care in resource-limited settings: an expert consultation	BMC pregnancy and childbirth	2,022	cc-by	8,795	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 https://doi.org/10.1186/s12884-022-04778-w Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 https://doi.org/10.1186/s12884-022-04778-w Abstract Background: Shortage or low-quality antenatal care is a complex and “wicked” problem relying heavily on con- textual, socio-cultural, environmental and intersectional aspects. We report the outcome of an expert consultation discussing solutions to improve antenatal care quality, access and delivery in low- and middle-income countries, and providing recommendations for implementation. Methods: The social ecological model was used as an analytical lens to map and interpret discussion points and proposed solutions. In addition, a conceptual framework for maternal and neonatal health innovation based on the building blocks of the World Health Organization health system and the Tanahashi Health Systems Performance Model provided a logical overview of discussed solutions. Results: Many barriers and norms continue to hinder antenatal care access. From values, beliefs, traditions, customs and norms, to poor resource allocation, there is a need of reshaping health systems in order to provide high qual- ity, respectful maternal and childcare. The burden of poor maternal health, morbidity and mortality is concentrated among populations who are vulnerable due to gender and other types of discrimination, have financial constraints and are affected by humanitarian crises. Conclusions: In order to address maternal health issues, good quality and evidence-based services should be guaranteed. Investments in strengthening health systems, including data and surveillance systems and skilled health workforce, should be considered an essential step towards improving maternal health services. Keywords: Antenatal care (ANC), Expert consultation, Social ecological model (SEM), Maternal and neonatal health (MNH) ANC) is important for the health of the mother and the development of the unborn baby. Inadequate care during this time breaks a critical link in the continuum of care, and affects both women and babies [1, 2]. Exploring solutions to improve antenatal care in resource‑limited settings: an expert consultation Carlotta Gamberini1, Federica Angeli2 and Elena Ambrosino1* Correspondence: e.ambrosino@maastrichtuniversity.nl Correspondence: e.ambrosino@maastrichtuniversity.nl 1 Institute for Public Health Genomics, Department of Genetics and Cell Biology, School for Oncology and Developmental Biology (GROW), Faculty of Health, Medicine and Life Sciences, University of Maastricht, Maastricht, The Netherlands 1 Institute for Public Health Genomics, Department of Genetics and Cell Biology, School for Oncology and Developmental Biology (GROW), Faculty of Health, Medicine and Life Sciences, University of Maastricht, Maastricht, The Netherlands Full list of author information is available at the end of the article Full list of author information is available at the end of the article *Correspondence: e.ambrosino@maastrichtuniversity.nl 1 Institute for Public Health Genomics, Department of Genetics and Cell Biology, School for Oncology and Developmental Biology (GROW), Faculty of Health, Medicine and Life Sciences, University of Maastricht, Maastricht, The Netherlands Background ANC) is important for the health of the mother and the development of the unborn baby. Inadequate care during this time breaks a critical link in the continuum of care, and affects both women and babies [1, 2]. Pregnancy is a crucial period and the changes happening in this phase have the potential to impact maternal and newborn health immediately, as well as later in life. As such, high quality care during pregnancy (antenatal care, f Lack of availability of ANC is a complex, “wicked” problem, which is highly dependent on contextual, socio- cultural, socio-ecological, intersectional aspects, with no straightforward definition or solution [3]. The provision and utilization of ANC services is subject to many differ- ent elements, such as lack of quality care; socio-demo- graphic and socio-cultural characteristics of patients, as Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 2 of 11 Page 2 of 11 (United Kingdom, Netherlands, India, Bangladesh, South Africa, Kenya, Ethiopia and Colombia).h education, occupation, ethnicity, social relationships and patients’ income level; logistical aspects, such as waiting time and location of facilities; and lastly social perception of general health, illness and diseases [4]. The goals were to bring together an interdisciplinary group of experts to discuss solutions to improve ANC quality, access and delivery in LMICs and generally in resource-constrained settings, and to give recommen- dations for implementation. The first objective was to exchange information and experiences related to ANC across settings, with the use of expert’s presentations. Following the presentations, participants were asked to further discuss in smaller groups the second objec- tive, which was to recognize challenges and experiences of ANC access and delivery in the settings where the experts have worked and practiced in, taking into con- sideration the different factors that influence health, such those in the social ecological model (SEM) [7]. The third and last objective of the event was to reflect on solutions to improve ANC access and delivery, in a specific setting, or as a general model. Despite the ambition to reduce global maternal mor- tality ratio (MMR) to less than 70 per 100,000 live births set by the United Nation Sustainable Development Goal (SDG) [2], the world will fall short of this target by more than 1 million lives with the current pace of progress [5]. The high number of maternal deaths in some areas of the world reflects inequalities in access to quality health ser- vices and highlights the gap between high-income coun- tries and low-middle income countries (LMICs), where the burden of MMR is the highest. There is a continued urgent need for maternal health to remain high on the global health and development agenda [6].fi To address these issues, experts from different fields working in maternal, newborn and reproductive health gathered for an expert consultation entitled “New approaches to improve antenatal care in resource limited settings”. The event was held virtually on June 14 2021 and was attended by 24 participants from eight countries In order to achieve the second goal, the SEM, shown in Fig. 1, was used as the analytic lens to further discuss the subject, and to organize the study findings. The SEM Fig. Experiences from ANC in different settingshf Experiences from ANC in different settings The experiences of ANC in different settings around the globe (Sub Saharan African countries, Eastern Europe, Bangladesh and Colombia) were shared and discussed by five experts in short presentations. Participantsh The 24 participants in attendance at the workshop included experts on maternal, neonatal and child health, sexually transmitted diseases, gynaecology, public health, health systems and services, economics and health tech- nology assessment (Table 1). 1 Revised Social Ecological Model, depicting the items discussed in the workshop ig. 1 Revised Social Ecological Model, depicting the items discussed in the workshop Page 3 of 11 Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 has five levels: the individual level, which focuses on the person’s attitude and beliefs towards care; the organi- zational level, which focuses on the role of healthcare systems; the community level, which focuses on the vari- ous organizations in the area; and lastly the policy level, which focuses on programs and policies [7]. Finally, a conceptual framework for MNH innovation based on the World Health Organization’s (WHO) health system building blocks and the Tanahashi model of measuring health systems performance (Fig. 2) was chosen to pro- vide a logical overview of the innovations discussed, for the third and last objective [8]. server. Each presentation was transcribed using word processing software (Otter.ai software), and anonymized. No recording was made during the discussions in smaller groups, notes from these discussions were taken by selected participants. Each transcript and set of notes were subsequently uploaded into Atlas.ti (Version 9.1.3.0, Berlin, Germany). The transcripts and notes served as the basis for the the- matic content analysis. The thematic analysis focused on patterns and themes that highlight the subjective view- points of the participants. The transcripts were coded using an ‘abductive approach’. Following the initial cod- ing process, codes were grouped into categories to form a working analytical framework, which was used to help code further transcripts. Finally, the SEM and the modi- fied conceptual framework for MNH innovation were utilized to further discuss the subject, and to organize the study findings. Results Experiences from ANC in different settingshf Data collection and analysis This observation supports the need for a more tailored ANC to better fit the reality of resource-limited settings, re-evaluat- ing the traditional western model, which has mostly been adopted so far. In most Western countries, ANC has traditionally consisted of a series of one-on-one appointments with a midwife, an obstetrician, or a gen- eral practitioner (GP) in a hospital setting. In order to provide ANC based on the individual needs of women, health care providers need to have sufficient resources, and this, in turn, is dependent on organizational norms and values of the setting. Thus, this model is inadequate in LMICs and in general in resource limited settings. All around, experts acknowledged that in order to make the required improvements, healthcare systems should be strengthened, since facilities are often overcrowded, overloaded, they lack skilled healthcare workers, equip- ment or essential drugs. Further, a need to improve the referral system was pointed out, as well as to strengthen the healthcare service delivery at the point-of-care in order to reach rural areas, and the most vulnerable women. focuses on the clinical assessments and services [9], is becoming inadequate in most LMICs. This observation supports the need for a more tailored ANC to better fit the reality of resource-limited settings, re-evaluat- ing the traditional western model, which has mostly been adopted so far. In most Western countries, ANC has traditionally consisted of a series of one-on-one appointments with a midwife, an obstetrician, or a gen- eral practitioner (GP) in a hospital setting. In order to provide ANC based on the individual needs of women, health care providers need to have sufficient resources, and this, in turn, is dependent on organizational norms and values of the setting. Thus, this model is inadequate in LMICs and in general in resource limited settings. All around, experts acknowledged that in order to make the required improvements, healthcare systems should be strengthened, since facilities are often overcrowded, overloaded, they lack skilled healthcare workers, equip- ment or essential drugs. Further, a need to improve the referral system was pointed out, as well as to strengthen the healthcare service delivery at the point-of-care in order to reach rural areas, and the most vulnerable women. fl Another category of barriers that many experts recog- nized refers to cultural barriers, which were differing the most across settings. Data collection and analysis Data was extracted from the experts’ presentations, dis- cussing ANC delivery in Europe, South-east Asia, South America and Sub-Saharan Africa. Moreover, additional data was gathered during the second part of the event, when the participants were asked to further discuss the second objective of the workshop in smaller groups.h i Recurring points conveyed by several speakers were the many barriers that communities encounter in the context of ANC. Although country-level differences were observed, women and their families face similar chal- lenges across settings. The most recognized ones are barriers in access to care, such as those experienced by The audio component of each presentation was recorded using Zoom recording service, with each recording uploaded and stored on a secure university Fig. 2 Conceptual framework for Maternal and Newborn health (MNH) innovation adapted from the Tanashi [8] Fig. 2 Conceptual framework for Maternal and Newborn health (MNH) innovation adapted from the Tanashi [8] Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 4 of 11 Table 1 Participants’ affiliations and focus of work Sector Country Field of work Academic institution Bangladesh Maternal, sexual and reproductive health and rights Hospital institution Curacao Obstetrics and gynaecology Academic institution Ethiopia Reproductive, and health services management Academic institution India Gynaecology Academic institution Kenya Public health Academic institution and non-governmental organization Netherlands/Kenya Public health Academic institution Netherlands Health Sciences Academic institution Netherlands Reproductive and maternal health, sexually-transmitted infections Academic institution Netherlands Maternal health and sexually transmitted infections Academic institution Netherlands Health Services Research Academic institution Netherlands Patient care optimization Academic institution Netherlands Sexually transmitted infections Hospital institution Netherlands Gynaecology Private sector Netherlands Design for sustainability Academic institution South Africa Sexual and reproductive health Academic institution South Africa Public health aspects of sexually transmitted infections Academic institution South Africa Respectful maternal care, barriers to quality of care Academic institution United Kingdom Sexual and reproductive health, family planning, HIV/AIDS Academic institution United Kingdom Global health Academic institution United Kingdom Economic evaluation and health technology assessment Academic institution United Kingdom Public relations and management Academic institution United Kingdom Global health Academic institution United Kingdom Public health Academic institution United Kingdom Healthcare management Table 1 Participants’ affiliations and focus of work women living in rural areas experiencing long distances to the healthcare facilities, lack of means of transporta- tion, effects of floods and poor roads. focuses on the clinical assessments and services [9], is becoming inadequate in most LMICs. Data collection and analysis Examples of these are lack of aware- ness or different perceptions of ill health, lack of knowl- edge about pregnancy complications and ANC, religious practices and beliefs shaping women’s healthcare seeking behaviour, and spousal support and involvement. Financial barriers were also highlighted as important obstacles that women and their families have to consider. Out-of-pocket payments (direct payments for medical expenses paid by individual users), informal payments and, broadly, poverty and inequality have a substantial effect on the health of mothers and children. f Violence has also a central role in the access to ANC. Besides being a key driver to pregnancy complica- tions, violence (including gender based-violence) leaves mothers with traumatic and stressful experiences often remaining unaddressed. What transpired from the speakers’ experiences was that a traditional ANC based model, which generally Page 5 of 11 Page 5 of 11 Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 ANC access and delivery: challenges and experiences at different levels Individual level Organization of health services & resource alloca‑ tion Poor allocation and shortage of healthcare resources, such as essential drugs and essential equip- ment, are cited by the experts as one of the challenges to ANC utilization. Additionally, mismanagement of funds and resources by the local governments and authorities have been recognized as another factor contributing to poor ANC services. Health perception Across regions, women were reported to often have a limited understanding on the purpose of early ANC and therefore the right time to seek care during pregnancy. This lack of understanding might be influenced by a perception that ANC is primar- ily provided to detect or treat diseases and was suggested to associate with women’s educational level literacy rate. The experts highlighted how health systems in their set- tings show gaps between the recommended practices and the care that patients actually receive. These gaps and related inconsistencies make it difficult to collect accu- rate data at all levels, thus diminishing the opportunity for programmatic interventions to benefit the population. Moreover, as the COVID-19 pandemic transformed the way people access health care, with care quickly mov- ing to telehealth and fear of contracting the virus, many women have gone without health care services. Among the discussed settings, this has been particularly the case in India and Bangladesh, where the pandemic has highlighted and aggravated long-standing inequities in healthcare availability and access. Health workers’ attitudes and shortage of staff In many countries the shortage of skilled healthcare workers, and consequently a tired, overworked and fatigued workforce, is contributing to poor ANC. Moreover, coupled with lack of maternal care training, what some experts have experienced on the field is the disrespectful care towards expectant mothers and abuse that some women have to endure while seeking care. Sociocultural beliefs Individual women’s ANC seek- ing behaviour was also reported to be restricted by the significant role of family members in health care decisions. Usually, mothers-in-law and husbands are the main decision-makers in determining the need for women’s ANC. Other important factors influencing the utilization of maternal health services are religious and socio- cultural norms as well as gender stereotypes, which affect the decision-making process of seeking care dur- ing pregnancy and postnatal care. The need to attend to domestic chores and care for children at home pre- vent some women from seeking maternal healthcare services. Community level Stigma and empowerment, gender‑based disparities The emergence of stigma and gender-based disparities at the individual level are closely related with the community. Many women experience some form of mistreatment, as abuse, neglect and discrimination when seeking care. Operational barriers Physical barriers, such as living in rural and/or remote areas distant to the health facili- ties, lack of means of transportation, and obstacles, such as floods and poor roads, are few of the barriers that the experts have identified. As discussed before, women’s ANC seeking behaviour is restricted by the significant role of their family members in health care decisions. Communities and community leaders play a significant role in influencing and affecting women access to maternal health services, especially in rural communities. Gender norms, values and expecta- tions about how women and men should be and behave are usually shaped by the community. Although these norms are specific to particular cultures and societies, there is strict gender role distribution when it comes to the issue of taking care of deliveries and childbirth. Financial barriers are also important obstacles that women have to consider and might make expectant mothers financially dependent on their families. Indeed, often women are economically reliant on their husbands and are not engaged in any vocation, making it hard to have independency from their families. Health-related stigma is a complicated phenomenon rooted in social inequity and power imbalance. Discrimi- nation, oppression and marginalization, as enacted and reinforced by the community, frequently have negative From out-of-pocket payments and informal payments, poverty and inequality all have a substantial effect on the health of mothers and children. Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 6 of 11 innovation based on the WHO’s health system and the building blocks and the Tanahashi model of measuring health systems performance (Fig. 2) was used to present the discussion points along the continuum of care to expectant mothers. social, psychological, behavioural and medical conse- quences for people needing care. Stigma and discrimi- nation at community level and the consequent fear of judgment, affect the willingness of women to access ANC services. Urban vs rural differences Many of the experts have highlighted the urban vs rural differences in the deter- minants of maternal health service utilization. Health service deliveryh The experts have stated that innovative approaches in ANC should aim at improving the quality of health ser- vice delivery along the continuum of care. Delivering and ensuring access, quality and safety of care across different locations and over time is deemed fundamen- tal. Experts agreed that more attention should be drawn at implementing a care model that requires an in depth understanding of the user’s perspective, which should be inclusive and aiming at reducing the challenging barri- ers to care. Additionally, it was acknowledged that men- tal health care needs to be integrated into all elements of health, particularly in ANC, where evidence suggests that increasing resources for mental health care is linked to better health outcomes for both mothers and their babies. Thus, the emphasis was put on the development of counselling packages and community education on maternal and neonatal health, which should better inte- grate the health needs of the population and available resources, as healthcare providers, medicines and money. Community level They have suggested that the inadequate use of ANC services is more of a rural phenomenon, which is linked to indi- vidual level challenges discussed above, as woman’s edu- cation level and autonomy. Inadequate ANC was mostly prevalent among poor and low empowered women who reside in rural areas. Policy level Midwife‑led and preventive care Many experts have acknowledged and recognized the role that healthcare providers, such as midwifery maternity care, can have in addressing psychosocial risk factors, which are often not given enough recognition. In fact, inadequate psy- chosocial assessment and or support can have adverse effects on both the mother and the foetus. Maternity care involving midwifes as the primary care provider leads to positive health outcomes for both mothers and their babies. In addition to improving maternal and neona- tal health, better integration of midwifes into the health systems across regions can facilitate the reduction of pri- mary care shortages across such areas. Medical products and health technologies Equitable access to medical products and health technol- ogies of assured quality, safety and efficacy is fundamen- tal for a functioning health system. Strategies to make novel medical products and health technologies available in LIMICs are not implemented enough. Barriers to the efficient implementation of new technologies have been described by the experts as a governmental and stake- holder problem, which should establish international norms and standards to promote the quality of medi- cal products. More support should be offered, through guidelines and strategies that can maximize patient and staff safety and that can promote equitable access to it. Many experts have acknowledged that the reliable col- lection of data in LMICs is challenging due to multiple factors, such as human resource, capital and technologi- cal factors. More investments should be made to improve data collection and consequently to increase the utiliza- tion digital health and digital health tools, which have the potential to meet the challenge of shortage in human resources, and to reach poor, rural and undeserved areas. Therefore, the experts have highlighted that the integra- tion and incorporation of health technologies in the pri- mary care system, has the potential to ensure universal access to maternal-health related information e.g., by delivering financial incentives through mobile money The concept of preventive care in the form of early risk identification was also discussed, highlighting the impor- tance of collaboration and communication between healthcare professionals and patients in the provision of comprehensive and holistic care. Regulatory challenges, decentralization The effective- ness of ANC services depends on the availability of a quality antenatal model that is implemented by the local government. Experts highlighted how poor regulatory mechanisms, or insufficient capacity to enforce regula- tions, contribute to the difficulty in assuring quality of care in public and private ANC clinics. Leadership and governance Innovative leadership and governance initiatives related to the formation of partnerships and the establishment and implementation of national ANC policies have been discussed. Across sectors of government, and with actors outside government, including civil society, innovative approaches to generate support for policy and influence key determinants of health, should be considered. Part- nerships for ANC include public-private cooperation between government and regional or international asso- ciations, to enhance capacity and quality of ANC deliv- ery. In order to do so, leadership should facilitate the collection of quality-assured, timely routine data, man- aged by innovative new interventions that would improve and strengthen the accuracy of routine real-time health data, contributing evidence for ANC policy-making. Moreover, a multidisciplinary approach to health financing has been also discussed. In fact, the experts have expressed a positive view on the promotion of inter- national dialogue to increase ANC financing across set- tings, from domestic and external sources, and to ensure that the new sources contribute to the development of sustainable national financial institutions. Health workforce As discussed above, the healthcare workforce is often tired and overworked and staff shortages are becoming a reality in many rural settings across LMICs. Coun- tries and stakeholders should harness more innovative workforce approaches, involving novel and/or additional training programs to improve the supply side of maternal care. Countries should finance the scaling-up of educa- tion programs for healthcare staff in a realistic and sus- tainable manner across settings. ANC access and delivery: solutions to improve it Based on the initial presentations and experts’ experi- ences, participants were asked to think of an innova- tive approach to improve ANC access and delivery at different levels. The conceptual framework for MNH Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 7 of 11 forum for participants to develop skills and confidence, share experiences and resources. technologies and enabling remote access of maternal health services. This could allow more individuals, living in rural, remote and marginalized communities, to access services which remain under-utilized, such as ANC. The empowerment of women and CHWs, and the role of the group consultation model, was highlighted across experts as a fundamental and innovative step aimed at reducing the logistical and economic burden of ANC attendance. In fact, group ANC models, which aim to put women at the centre of service provision in order to improve women’s access, engagement and sat- isfaction with care, can increase convenience for women and providers and can make care delivery more efficient. For example, as long wait-times for care have been men- tioned to hinder MNH services, scheduling group ses- sions in advance could help to reduce this challenge. This solution, in addition to allowing all women to receive the recommended care, it allows women to access counsel- ling opportunities, which benefits both women and the healthcare providers. Such collaboration between provid- ers and women enables both efficient and comprehensive care delivery, which may improve care provision, experi- ence, and utilisation while also providing opportunities to ensure continuity of care [10]. Health financing dd fi Addressing financial constraints and the financial bar- riers that limit the access to quality care has been high- lighted by the experts across settings as an important concern across health system. The creation of innovative financial programs that aim at attenuating financial bar- riers to ANC and at improving the coverage of such care should be central in government’s policy plans. The con- cept of “value for money” has also been touched upon, becoming a central topic in the development of a com- prehensive ANC policy and its delivery. The first level in generating value is to secure the value of health policies, especially to measure the value for money in the choice of policies to fund. In this context, the value of money refers to using economic methods (usually cost-effective- ness analysis) to measure the health promotion achieved at a particular level of spending. Community ownership and participation Healthcare provid- ers’ statements on reproductive health issues need to be adapted to different social contexts, including those with low levels of education and income. Furthermore, there is a need to continue to focus on community education and awareness campaigns on the importance of early participation in ANC. Educational interventions target- ing both men and women have been reported to improve the health-oriented behaviour of pregnant women and improve birth preparedness and complication readiness. In addition to home visits, education could be offered at various political and social gatherings in the community. In Tanzania, community health workers are reported to play an important role in promoting men’s participation in maternal and child health issues [21]. A key question for practitioners and policy makers is how to improve women’s perception of the importance of pregnancy care. Any intervention should be culturally relevant. Train- ing programs in cultural literacy and sensitivity should be developed to improve healthcare utilization among women. Stakeholders may want to include cultural skills and sensitivity training in its health education curricu- lum when training new health professionals [22]. Finally a recurrent theme in the expert consultation Similarly, men control the economic resources of the household, which in turn influences women’s choice and ability to use maternal and child health services. Men and the community still view pregnancy as a typically feminine domain and do not feel involved. Pregnancy is still a stressful event that disrupts the link between families and communities. Women are often burdened by physical, psychological, and financial hardships, cou- pled with inadequate care of ANC, skilled birth attend- ance coverage and transport facilities. These results are consistent with previous studies [13]. Consistent with the literature, many experts have emphasised that women’s beliefs and attitudes play a role in deciding whether to initiate or continue ANC. There are numerous shared cultural experiences when the patient seeks care; their cultural background, the cul- ture of their provider, and the medical culture. Patient have different beliefs, attitudes, values and behaviours to healthcare providers. The culture of the medical com- munity is often at odds with the patient’s culture; i.e., education, means of transport, occupation and socio- demographic factors, and when they fail to recognize these differences, they may deliver low-quality care. Such differences can also influence women’s decision making, acceptance of care and can make it difficult for patients to follow advice from the medical community [14]. Community ownership and participation Across regions, experts emphasized that many barriers and norms, largely associated with community cultural beliefs, and influenced by the traditional household set- up, limit expectant mothers’ autonomy to seek care. In fact, in many instances, women are expected to pri- oritise the health of their family over their own, and to continue to work both outside and inside the home [11]. Furthermore, socially constructed gender norms continue to hinder men’s participation in pregnancy and childbirth. Men control decision-making at home, which influences the timing of ANC attendance [12]. Innovative approaches are increasingly aimed at strength- ening community health mechanisms that improve links with primary health care. Across settings, it was recog- nized that community-based interventions, facilitated by community health workers (CHW), and women’s groups, can be a really useful tool in strengthening ANC. Wom- en’s groups aim to empower and support their members; engaging in conversation with peers helps normalize the experience of pregnancy and share knowledge in the community. The group format also promotes self-efficacy and social support for pregnant women by creating a Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 8 of 11 Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Programs to promote health education among expect- ant mothers with low levels of education are necessary to raise awareness among rural women about the ben- efits of optimal ANC. In order to do this, it is important to strengthen the existing role of CHWs, who are able to provide appropriate health education and create con- nections between vulnerable populations and health- care providers [20]. Thus, it is imperative that significant efforts be made to improve the quality of ANC by pro- viding pregnant women with appropriate counselling, including supportive listening, advice, and relevant infor- mation. Involving mothers as active participants in the decision-making about their care would also help make changes in health care, from one based on provider- dominated dialogue to one that involves clients in the decision-making process. This requires a transition in the role of health workers from one of authority to one that is based on collaboration and partnership between patients and providers. Pregnancy information should also be provided in a form that is easy to understand and accessible to users of ANC services. Solutions Reshaping the health system requires political leader- ship and policy change, hospitals that can provide high quality, respectful maternal and childcare, health systems that can break down barriers to access, and empowered populations that can demand high quality care [25, 26]. Improving nurses’ and midwifes’ knowledge of compre- hensive care and its contribution to the quality of care is an important issue that needs to be addressed in many countries where existing nursing practices are unsat- isfactory and the doctor-led care is commonly applied in clinical environments. The central role of nurses and midwives in delivering respectful, caring, friendly and helpful ANC can be seen in many studies throughout the literature. For example, many studies have showed that the presence of providers who were caring and sympa- thetic, and familiar with patient’s cultural practices and communities, were essential factors in encouraging ANC demand and usage [27, 28]. Through the identification of the needs of patients who are neglected by sole use of the doctor-led care recovery can accelerate, hospital stays can shorten and the costs reduced [29]. From the need of new technologies, to address the healthcare staff shortages occurring in resource-limited areas, addressing financial barriers that many coun- tries encounter is fundamental. Thus, it is important to ensure value for money through an integrated people- centred health service approach. This approach should consciously incorporate the perspectives of individuals, families and communities and should see them as partici- pants and beneficiaries of a trustworthy health systems that address their needs and preferences in more holis- tic way [33]. Different examples of people-centred and integrated health services can be found in literature. In Mali, primary care networks have been developed, which are made up of community-owned, community-operated primary care centres with the support of government- run district health teams. In rural South Africa, nurse-led chronic disease management programmes focusing on people with high blood pressure, diabetes, asthma have supported patient education, self-management support and improved surveillance leading to improved control of disease. In South Sudan, Uganda and Zambia there have been cases of integrated community case for the manage- ment for malaria, pneumonia and diarrhoea to reduce child mortality, involving community health workers who assess and treat children with serious illnesses [34]. Community ownership and participation Thus, especially in pregnancy care, a doctor should have a basic understanding of patients’ needs and communicate with them effectively in a way that makes them feel comfort- able. Doctor’s interventions must consider broader eco- nomic, geographical and social factors that might affect an individual’s access to services. Finally, it is essential to have cohesiveness between culturally-appropriate ser- vices and other health care providers that women and their families encounter along the continuum of care through pregnancy [15]. Additionally, another significant aspect highlighted by the experts in respect to the challenges of ANC delivery, was the reported limited understanding that women have on the purpose of early ANC. This lack of understand- ing was suggested to associate with women’s educational level and literacy rate. In fact, it has been shown that the increase in women’s educational level is a major motiva- tor for increasing the likelihood of her ANC attendance [16, 17]. This suggests that educated women are more likely to have adequate knowledge of prenatal care ser- vices and understand the importance of early booking for ANC as well as attending the recommended eight visits [18]. Thus, they tend to value ANC and will use preg- nancy care services more, compared to less educated women. Longer time in school can also develop women’s ability to reach out to health workers to ask questions and discuss possible health issues [19]. Finally, a recurrent theme in the expert consultation was namely that geographical inaccessibility contributes to the late onset of ANC attendance. This finding has also been reported in other studies. In fact, Nsibu and colleagues showed statistically significant associations between the place of residence and attendance to the first prenatal visit in the first trimester [23]. Addition- ally, a great discrimination in the allocation of resources and in the availability of rural and urban health facili- ties was described. Poor reediness to provide antenatal/ Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 9 of 11 Page 9 of 11 natal health services and supplies is hindering the ANC landscape. In fact, differences have been documented in availability of equipment and supplies needed for ante- natal and natal services between urban facilities and rural ones [24]. intention. In fact, because people living in developing countries don’t frequently access medical care, they pre- fer face-to-face meeting with doctors, instead of remote mode of communication. Community ownership and participation Additionally, they have noted that, due to already inadequate and poor resource, people associate a perceived sense of risk with the adoption of telemedicine [31]. It is important to ensure greater use of ANC services, by establishing health care facilities in catchment areas in rural communities, employing more qualified health workers to provide medical care to women in their com- munities, and ensuring adequate transportation infra- structures and services. Stakeholders need to ensure that maternity care services are closer to home, which could be achieved, especially in rural and hard-to-reach areas, through mobile clinics that would help many women with financial difficulties access prenatal care. Nonetheless, digital health technologies overall hold promises for addressing major public health backlogs and for strengthening health systems in LMICs. New technologies have the potential to harness clinical and public health, and more research is needed around emerging ones, including artificial intelligence, big data, cloud, cybersecurity, telemedicine and wearable devices to demonstrate their potential use in remote settings. For example, a recent case reported by Runckle et al. [32], have showed that the use of wearable sensor technology in prenatal care, was well received by both patients and providers, which responded favourably to the implemen- tation of such technology, especially in rural underserved populations. Consent for publication Oral and written informed consent was obtained from participants in relation to both workshop participation and to prospective use of data for publication purposes. i This expert consultation emphasized ANC delivery – especially in resource-constrained setting – as a wicked problem [37]. The experts’ views highlighted how the ANC challenge takes a different form, depending on the vantage point and domain of observation: in some contexts, it is an issue of shortage of healthcare infra- structures, in others gender bias, domestic violence and power dynamics are predominant antecedents, in yet other domains women education and the charac- teristics of the built environment are perceived as the Availability of data and materials The authors declare that the data supporting the findings of this study are available within the article and its supplementary information files. Solutions As discussed earlier, barriers to access maternal health services using telehealth relate to common challenges, as limited access to broadband in rural areas, cost of the equipment, scheduling time with providers [30]. Due to the scarcity of health resources, particularly in developing countries where those are often very limited, only appro- priate (effective, safe and feasible) technologies should be implemented and used. There are different opportunities to use telemedicine to expand access to maternal health care for women living in rural areas; however, its accept- ance remains limited. Based on the current literature, technological anxiety and perceived risk act as significant barriers to telemedicine usage. Kamal and colleagues [31] have demonstrated that technological anxiety had a sig- nificant negative relationship with telemedicine usage Ensuring equal access to quality health services that meet the broad needs of individuals and communities requires a fundamental change in the way health services are planned, funded and delivered. Thus, it is essential to engage and empower individuals, families and communi- ties so that they have the opportunity, skills and resources to develop into articulate and empowered users of health Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 10 of 11 services. Hence, policy interventions should actively pro- mote health literacy, shared decision-making and patient self-management contribute to the health services that people value most. For instance, a study investigating the association between access to health care and women’s empowerment in Myanmar, have found that, especially in rural areas, women’s empowerment was an important factor of one’s ability to access care [35]. predominant foci of attention. More holistically, and in line with the socio-ecological view, complex inter- actions among all these elements are foundational to understand and properly address ANC delivery in dif- ferent settings. Solutions also need to be multifaceted, and mindful that one intervention might lead to unex- pected adverse consequences, according to the wicked problem framework [38, 39]. For example, whilst digital technologies hold enormous potential for improving ANC services in remotes, without adequate financial support and policy intervention there is high risk of increasing income-based inequity in ANC services and outcomes. Abbreviations l ANC: Antenatal care; MMR: Maternal mortality ratio; SDG: Sustainable develop- ment goals; LMIC: Low-middle income country; HIC: High income country; SEM: Social ecological model; MNH: Maternal and newborn health; GP: Gen- eral practitioner; CHW: Community health worker. Conclusion Th The expert consultation offered the opportunity to share experiences around challenges of maternal and newborn care from different settings, and brainstorm on possible approaches to innovative solution. Overall, the event strengthened the idea that the burden of poor maternal health, morbidity and mortality is concentrated among vulnerable populations. The findings suggest several courses of action for improving ANC services and deliv- ery in LMICs and rural areas. Good quality, equitable, evidence-based maternal health services that respond to local needs and are capable of meeting emerging chal- lenges should be prioritized. Investments in strength- ening health systems, including data and surveillance systems, facility capability, and a skilled health workforce should be considered a fundamental step towards better maternal health services.h Acknowledgments We would like to express our gratitude to all the experts who took part in the consultation for their valuable and constructive. Funding This paper contributes to the idea that ANC services and delivery in LMICs and in resource-limited settings continue being disrupted by different challenges and bar- riers. The COVID-19 pandemic had further highlighted these obstacles [36]. Innovative measures are required to address these obstacles and ensure women are not denied access to available, accessible, acceptable, and high-qual- ity maternal healthcare services in normal situations and in spite of emergencies, such as COVID-19. The workshop was funded by The York-Maastricht strategic partnership. Ethics approval and consent to participate The workshop was approved and supported by The York-Maastricht strategic partnership. All workshop attendees were informed prior to the workshop outset that presentations would be recorded, that notes would be taken of the discussions and that results would be synthesized in a report. The methodology was carried out in accordance with relevant guidelines and regulations. Efforts to train and motivate healthcare providers to adopt online, remote approaches such as use of tele- medicine are critical. Similarly, initiatives to expand the involvement of frontline maternal healthcare providers and to deliver information on the availability of services strategies are crucial to mobilise and secure community confidence in the safety of maternal care services.h Authors’ contributions FE & EA: study conception and design: CG: data collection; FE, EA, CG: analysis and interpretation of results; CG: draft manuscript preparation; All authors reviewed the results and approved the final version of the manuscript. Received: 8 March 2022 Accepted: 20 May 2022 References 22. World Health Organization D of MPS. Working with individuals, families and communities to improve maternal and newborn health. 2010. 1. UNICEF. Healthy Mothers, Healthy Babies: Taking stock of maternal health. New York; 2019. Available from: https://data.unicef.org/resources/healthy- mothers-healthy-babies/ 23. Nsibu CN, Manianga C, Kapanga S, Mona E, Pululu P, Aloni MN. 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Author details 1 Institute for Public Health Genomics, Department of Genetics and Cell Biology, School for Oncology and Developmental Biology (GROW), Faculty of Health, Medicine and Life Sciences, University of Maastricht, Maastricht, The Netherlands. 2 University of York Management School, University of York, York, UK. Received: 8 March 2022 Accepted: 20 May 2022 Gamberini et al. BMC Pregnancy and Childbirth (2022) 22:449 Page 11 of 11 Page 11 of 11 References Higher educational attainment associated with optimal antenatal care visits among childbear- ing women in Zambia. Front Public Heal. 2016;4(June):1–7. 37. Waddell S. Societal change systems: a framework to address wicked prob- lems. J Appl Behav Sci. 2016;52(4):422–49. 37. Waddell S. Societal change systems: a framework to address wicked prob- lems. J Appl Behav Sci. 2016;52(4):422–49. 16. Muyunda B, Makasa M, Jacobs C, Musonda P, Michelo C. 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https://openalex.org/W2151354690	https://hal.science/hal-01102615/file/13595_2014_Article_373.pdf	English	null	Alley coppice—a new system with ancient roots	Annals of forest science	2,014	cc-by	14,904	Alley coppice-a new system with ancient roots Christopher D. Morhart, Gerry C. Douglas, Christian Dupraz, Anil R. Graves, Michael Nahm, Pierluigi Paris, Udo H Sauter, Jonathan Sheppard, Heinrich Spiecker To cite this version: Christopher D. Morhart, Gerry C. Douglas, Christian Dupraz, Anil R. Graves, Michael Nahm, et al.. Alley coppice-a new system with ancient roots. Annals of Forest Science, 2014, 2 (71), pp.527 - 542. 10.1007/s13595-014-0373-5. hal-01102615 Distributed under a Creative Commons Attribution 4.0 International License Alley coppice—a new system with ancient roots Christopher D. Morhart & Gerry C. Douglas & Christian Dupraz & Anil R. Graves & Michael Nahm & Pierluigi Paris & Udo H. Sauter & Jonathan Sheppard & Heinrich Spiecker Christopher D. Morhart & Gerry C. Douglas & Christian Dupraz & Anil R. Graves & Michael Nahm & Pierluigi Paris & Udo H. Sauter & Jonathan Sheppard & Heinrich Spiecker Received: 28 October 2013 /Accepted: 25 March 2014 /Published online: 6 May 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com & Methods Based on a comprehensive literature review, we compare the advantages and disadvantages of the alley cop- pice system and contrast the features with well-known existing or past systems of biomass and wood production. Abstract & Context Current production from natural forests will not satisfy future world demand for timber and fuel wood, and new land management options are required. & Results We describe and discuss the basic aspects of alley coppice, its design and dynamics, the processes of competi- tion and facilitation, issues of ecology, and areas that are open for future research. & Aims We explore an innovative production system that combines the production of short rotation coppice in wide alleys with the production of high-value trees on narrow strips of land; it is an alternative form of alley cropping which we propose to call ‘alley coppice’. The aim is to describe this alley coppice system and to illustrate its potential for produc- ing two diverse products, namely high-value timber and ener- gy wood on the same land unit. & Conclusion Based on existing knowledge, a solid founda- tion for the implementation of alley coppice on suitable land is presented, and the high potential of this system could be shown. Handling Editor: Douglass Jacobs HAL Id: hal-01102615 https://hal.science/hal-01102615v1 Submitted on 13 Jan 2015 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 Annals of Forest Science (2014) 71:527–542 DOI 10.1007/s13595-014-0373-5 REVIEW PAPER Handling Editor: Douglass Jacobs Contribution of the co-authors Christopher D. Morhart: designing the experiment, writing the paper and supervising the work. Anil R. Graves: designing the experiment and writing the paper. Gerry C. Douglas: designing the experiment and writing the paper. Christian Dupraz: designing the experiment and writing the paper. Michael Nahm: designing the experiment, writing the paper and coordinating the research project. Pierluigi Paris: designing the experiment and writing the paper. Udo H. Sauter: designing the experiment and coordinating the research project. Contribution of the co-authors Christopher D. Morhart: designing the experiment, writing the paper and supervising the work. p g p p p g Anil R. Graves: designing the experiment and writing the paper. Gerry C. Douglas: designing the experiment and writing the paper. Christian Dupraz: designing the experiment and writing the paper. Michael Nahm: designing the experiment, writing the paper and coordinating the research project. g p j Pierluigi Paris: designing the experiment and writing the paper. Udo H. Sauter: designing the experiment and coordinating the research project. 1 Introduction Mixed plantations offer a variety of multiple benefits, both at productive and environmental levels. There can be a signif- icant yield increment at stand level, with overall productivity being dramatically increased in mixed designs in comparison with monocultures; this is highlighted in the case of eucalypts in mixtures with nitrogen-fixing tree species such as A. mearnsii and A. falcataria (DeBell et al. 1997; Forrester et al. 2005). However, contrasting negative results might also be obtained, such as in the case of eucalyptus (Eucalyptus robusta Sm.) with Casuarina equisetifolia L. or with Leucaena leucocephala (Lam.) de Wit (Parrotta 1999). It is predicted that by the middle of the twenty-first century, the greatest proportion of human wood consumption will come from planted forests, most of them intensively managed (Sedjo 2001). It has been shown that the natural forests of the world do not have the capacity to sustainably meet the current demand for timber and for fuel wood (Fenning and Gershenzon 2002). Additionally, in recent decades, the inter- est in renewable energy has grown, and thousands of hectares of short-rotation coppice (SRC) have been planted worldwide to satisfy biomass requirements (Fiala and Bacenetti 2012; Schweier and Becker 2012). At the same time, the demand for high-quality timber, such as common ash (Fraxinus excelsior L.) and sycamore (Acer pseudoplatanus L.), has outstripped supply and needs to be increased (Becker and Klädtke 2009). An innovative approach to boost capacity is to combine the production of SRC and valuable timber trees on the same land unit so as to capture all available light more efficiently. Much has been written about the management and environmental effects of SRC and agroforestry as separate land management systems. However, the combined approach of alley coppice has so far received much less attention both in theory and in practice (Unseld 2007; 2009; Clinch et al. 2009; Morhart et al. 2010; Cardinael et al. 2012). Such a combined production system has similarities with several systems which are well known in conventional forestry (mixed plantations and coppice-with-standards), in biomass forestry (SRC) and in agroforestry (alley cropping). We discuss the main features of these known systems separately, and we evaluate the char- acteristics which may apply in a system of alley coppice. Another important productive advantage of mixed planta- tions is improved rates of individual tree growth and stem quality. These advantages are extremely important when pro- ducing high-quality timber. A. R. Graves Centre for Environmental Risks and Futures, Cranfield University, Bedfordshire, Cranfield MK43 0AL, UK M. Nahm: U. H. Sauter Forest Research Institute Baden-Württemberg (FVA), Wonnhaldestr. 4, 79100 Freiburg, Germany P. Paris Consiglio Nazionale delle Ricerche; Istituto di Biologia Agroambientale e Forestale, Via G. Marconi, 2, 05010 Porano (TR), Italy p j Jonathan Sheppard: writing the paper. Heinrich Spiecker: designing the experiment and writing the paper. A. R. Graves Centre for Environmental Risks and Futures, Cranfield University, Bedfordshire, Cranfield MK43 0AL, UK C. D. Morhart (): J. Sheppard: H. Spiecker Chair of Forest Growth, Albert-Ludwigs-University Freiburg, 79085 Freiburg, Germany e-mail: christopher.morhart@iww.uni-freiburg.de C. D. Morhart (): J. Sheppard: H. Spiecker Chair of Forest Growth, Albert-Ludwigs-University Freiburg, 79085 Freiburg, Germany e-mail: christopher.morhart@iww.uni-freiburg.de M. Nahm: U. H. Sauter Forest Research Institute Baden-Württemberg (FVA), Wonnhaldestr. 4, 79100 Freiburg, Germany G. C. Douglas Teagasc—Agriculture and Food Development Authority, Kinsealy Research Centre, Malahide Rd., Dublin 17, Ireland G. C. Douglas Teagasc—Agriculture and Food Development Authority, Kinsealy Research Centre, Malahide Rd., Dublin 17, Ireland C. Dupraz Institut National de la Recherche Agronomique (INRA), 2, Place Viala, 34060 Montpellier, France 528 C.D. Morhart et al. Keywords High-value timber production . Short-rotation coppice . Alley cropping . Agroforestry . Coppice with standards . Biodiversity . Biomass . SRC countries such as black alder (Alnus glutinosa (L.) Gaertn.) and hybrid poplar ‘Roxbury’ (Populus nigra L. × Populus trichocarpa Torr. and Gray) in Canada (Côté and Camiré 1987), and wild cherry (Prunus avium L.) and other broad- leaves in Chile (Loewe et al. 2013). 1 Introduction Many broadleaves have poor api- cal dominance, and mixed plantations can significantly im- prove stem straightness due to canopy stratification, which results in modulated light competition, suppressing epicormic branches and enhancing branching habit (Mohni et al. 2009; Loewe et al. 2013). These positive interactions on stem form are crucial, especially for timber species for which genetically improved material is not yet available, due to their long breeding cycles or to difficulties in vegetative propagation. The risk of pest damage (fungi, bacteria and insects) can have significant productive and environmental consequences which affect the financial success of forest plantations. Mixing different species in plantation stands reduces pest risk by enhancing genetic diversity at stand level. However, this aspect of mixed stands is particularly difficult to study, be- cause pest damage can happen episodically and be affected by the vegetation structure at landscape level. Loewe et al. (2013) have reported contrasting results for the same mixed planta- tion design in different locations of the same region. Two mechanisms are involved in reducing pest risk in mixed stands: the dilution of the host concentration for a pest organ- ism and habitat diversification which allows greater popula- tions of natural enemies of pests to be supported (Watt 1992). Therefore, the habitat diversification provided by mixed spe- cies plantations creates important environmental services for biodiversity conservation, and is especially important for the restoration of degraded lands. Mixed plantations, with simul- taneous planting of pioneer to climax tree species, have prov- en to be a sustainable approach to restoring natural vegetation. This has been shown both on spoiled mining soils in Brazil (Parrotta and Knowels 1999) and Germany (Lögters and Dworschak 2004), and on abandoned agricultural lands in Sri Lanka (Ashton et al. 2001) where mixed plantations have been superior to alternative approaches using sequential 1.2 Coppice with standards Coppice with standards has been alternatively named as stored coppice, composite forest or compound coppice. Coppice with standards consists of two major structural elements. The majority of trees form an even-aged understory and follow a traditional coppice culture management, in combina- tion with selected trees constituting the larger uneven-aged trees as ‘standards’ representing the over-storey. In Germany, the first documented appearance of the coppice with standards system can be dated to 600AD, in France to the Middle Ages, and records from the twelfth century exist in England (Troup 1928). Similarly in eastern European countries, the system was well known and important for landowners. Coppice with standards has enjoyed a long tradition and reached a peak around the year 1900 covering 3 % of the total land area in the present day Czech Republic and Slovakia (Machar 2009). In some parts of England, coppice with standards was considered to be the most important form of woodland management from an economic point of view up until the middle of the nine- teenth century (Rotherham and Jones 2000), and in Germany, coppice with standards was the most important broadleaf forest in the seventeenth and eighteenth centuries (Gross and Konold 2010). A coppice system provides small wood for fuel and other uses within short rotations periods (10–30 years), depending on the site and tree species (Hamm 1900). For the regeneration of coppice with standards, the methods utilised were vegetative reproduction and regenera- tion from seed, with the planting of trees being least common. The harvest of the coppice was carried out by clear-cutting all trees with the exception of selected trees left as future stan- dards. Standards were managed to produce large-diameter timber over long rotations but also to provide seeds for natural regeneration or even to help to protect the coppice understory against frost (Troup 1928). An additional function, important in the past, was the non-timber value, i.e. the production of fodder for animals, especially pigs and sometimes game (Burschel and Huss 1997). The number of standards in ancient forests was heavily dependent on the desired production goal; a general rule of 30–100 standards per hectare between all Research has shown that mixed plantations, if well de- signed and planned, can provide multiple productive, financial and environmental benefits (Evans 1984; Hemery 2001). 1.1 Mixed plantations Forest plantations respond more quickly and efficiently to management inputs in comparison to traditionally managed forest stands (West 2006). However, forest plantations can be characterised by significant biological and economic risks, especially when establishing monocultures, with uniform ge- netic composition at stand and/or landscape level. To mitigate these risks, the mixing of different tree species in plantation stands is a beneficial alternative to a monoculture planting design. There are numerous successful examples of mixed plantations in the tropics, for example, eucalyptus (Eucalyptus globulus Labill.) and acacia (Acacia mearnsii (Vahl) Benth.) in Australia, (Forrester et al. 2005), Brazil and Congo (Bouillet et al. 2013); Eucalyptus saligna Sm. and Albizia falcataria (L.) Fosberg in Hawaii (DeBell et al. 1997); and in temperate 529 Alley coppice—a new system with ancient roots introduction of species of different characteristics or stages of succession. agricultural and marginal lands using a wide range of native timber tree species more adapted to very specific local site conditions (Hall et al. 2011). For this innovative approach, mixed plantations would seem to be most suitable because they can combine efficient use of site resources with the benefit of multiple products in varying rotations (Redondo- Brenes and Montagnini 2006). Future research should be extended to designing operational scale plantation trials for testing innovative mixtures with an emphasis on simplicity of management regarding mechanisation and cultural operations, and a clear focus on marketability of the final and intermediate products (Magagnotti et al. 2010). In addition to biodiversity, carbon sequestration is another key environmental service of mixed species plantations. Trees are efficient sequesters of carbon, with carbon storage capac- ity increasing according to stand age and ecosystem total biomass (Magnani et al. 2007). The composition of tree spe- cies is also another important factor in carbon sequestration. Research evidence showed that mixed species plantations have the potential to store more carbon than monocultures in fewer years. Examples of this include 11-year-old E. globulus/ A. mearnsii mixtures in Australia (Forrester et al. 2006) and 12- to 13-year-old mixtures of nine native indigenous tree species in Costa Rica (Redondo-Brenes and Montagnini 2006). The greater carbon storage capacity of mixed planta- tions in comparison to monoculture plantations is dependent on greater carbon sequestration in the vegetation and soil, for example, with Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) and alder (Alnus cremastogyne Burk.) (Wang et al. 1.1 Mixed plantations 2009), in the leaf litter just above ground level (eucalyptus with acacia, Forrester et al. 2006), and just below ground level (Castanopsis hystrix A. Dc. with Pinus massoniana Lamb. in China (He et al. 2013)). According to Redondo-Brenes and Montagnini (2006), long-term carbon storage capacity seems to be dependent on the successful interaction between fast- growing tree species and slower growing species, such as high-quality timber trees in the mixtures. This poses questions regarding the trade-off between carbon sequestration and ex- cessive water consumption for forest plantations (Vanclay 2009), with faster growing trees requiring more water for growth (Spring et al. 2005). Forrester et al. (2010) demon- strated higher water consumption in eucalyptus and acacia mixtures, but also an improved water use efficiency of the eucalyptus trees in the same mixture. Lauteri et al. (2006) showed evidence of functional roots stratification in water use in mixed walnut (Juglans regia L.) alder (Alnus cordata (Loisel.) Duby) plantations in temperate Italy. More research on overall water consumption in mixed stands is advocated by Vanclay (2009), specifically regarding the effects of heteroge- neous canopy structures in mixed stands that might decrease the total canopy stand transpiration. 1.2 Coppice with standards So far, these have been underestimated, especially by foresters who may be more interested in productivity and in simplifying management operations in plantations. This approach also tends to focus on a few species that are well suited to mono- culture plantation schemes and to genetic improvement (Kelty 2006). Current concerns regarding climate change and biodi- versity conservation have led to suggestions that the harvest- ing pressure on forest stands could be decreased by implementing plantation forestry and agroforestry on 530 C.D. Morhart et al. classes has previously been proposed (Evans 1984). Never- theless, it was recognised that the quantity of standard trees must be dictated by the coppice layer’s light requirement (Hartig 1820). One advantage of the system was the rapid growth of the standards and their above-average growth in volume and value (Nyland 2002). Standards could be selected from coppice stock, but they were selected preferably from trees of seedling origin (Troup 1928). Compared with trees grown in close-canopied high forests, the standards had shorter boles and a higher proportion of branch wood (Troup 1928). The architecture of trees is dependent on the length of the rotation and silvicultural practices, and it can be modified by the application of artificial pruning employed at the correct developmental phase. In this ancient silvicultural system, the presence of standards created a more diverse array of species and age classes, and provided improved habitat structures over those provided by pure coppice systems. Furthermore, the outward appearance of the coppice area which was regularly cut was softened by the presence of standard trees both pre- and immediately post-harvest. The rotation age of coppice was dependent on the tree species that were selected as stan- dards and the shade tolerance of the coppice crop. The stan- dard rotation was always a multiple of the coppice rotation (Hamm 1900; Troup 1928). The general characteristics of a coppice with standards system is that all species used should be able to resprout from a cut stool while displaying fast growth. Species that demanded high-light conditions are less suitable for the coppice layer, while the ideal standards should display a degree of self pruning and have naturally thin and high crowns, such features provided better growing conditions for the coppice (Troup 1928). 1.2 Coppice with standards Standards could be selected from coppice stock, but they were selected preferably from trees of seedling origin (Troup 1928). Compared with trees grown in close-canopied high forests, the standards had shorter boles and a higher proportion of branch wood (Troup 1928). The architecture of trees is dependent on the length of the rotation and silvicultural practices, and it can be modified by the application of artificial pruning employed at the correct developmental phase. In this ancient silvicultural system, the presence of standards created a more diverse array of species and age classes, and provided improved habitat structures over those provided by pure coppice systems. Furthermore, the outward appearance of the coppice area which was regularly cut was softened by the presence of standard trees both pre- and immediately post-harvest. The rotation age of coppice was dependent on the tree species that were selected as stan- dards and the shade tolerance of the coppice crop. The stan- dard rotation was always a multiple of the coppice rotation (Hamm 1900; Troup 1928). The general characteristics of a coppice with standards system is that all species used should be able to resprout from a cut stool while displaying fast growth. Species that demanded high-light conditions are less suitable for the coppice layer, while the ideal standards should display a degree of self pruning and have naturally thin and high crowns, such features provided better growing conditions for the coppice (Troup 1928). The typical species used within this system included: oak (Quercus spp.), ash, sycamore, wild cherry, poplar (Populus spp.), beech (Fagus sylvatica L.), hazel (Corylus avellana L.), alder (A. glutinosa), hornbeam (Carpinus betulus L.), birch (Betula spp.), lime (Tilia spp.), walnut (Juglans spp.) and sometimes conifers (Troup 1928; Burschel and Huss 1997). Often, combinations of the above species were chosen. In the past, oaks were important for boat and building construction, but they also provided acorns as fodder for pigs. For this reason, oak was a common tree utilised for standards in Europe (Troup 1928), and remnants can be frequently seen today. 1.2 Coppice with standards The typical species used within this system included: oak (Quercus spp.), ash, sycamore, wild cherry, poplar (Populus spp.), beech (Fagus sylvatica L.), hazel (Corylus avellana L.), alder (A. glutinosa), hornbeam (Carpinus betulus L.), birch (Betula spp.), lime (Tilia spp.), walnut (Juglans spp.) and sometimes conifers (Troup 1928; Burschel and Huss 1997). Often, combinations of the above species were chosen. In the past, oaks were important for boat and building construction, but they also provided acorns as fodder for pigs. For this reason, oak was a common tree utilised for standards in Europe (Troup 1928), and remnants can be frequently seen today. In modern times, the decreasing demand for fuel wood caused by a growth in coal use has reduced the interest in the decreasing area coverage of coppice with standards. In France, such coppice systems still cover 37 % of the forest area (Vallet et al. 2006). In addition, coppice with standards remains a commonly utilised system in many Mediterranean areas, using a range of oak species (Messier et al. 2013) and, therefore, still constitutes an important silvicultural system. Systems similar to coppice with standards are rarer in the tropics than in Europe. However, renewed interest in coppice with standards has resulted in testing of eucalyptus in Rwanda (Reynders 1984), teak (Tectona grandis L.F.) in Ghana (Adu- Anning and Blay Jnr. 2001) and in India (Singh and Singh 2011), as well as sal (Shorea robusta C.F. Gaertn.) in the Himalayan regions of India (Negi 2000). The coppice with standards system is not necessarily restricted to broadleaf species. In Korea, a system utilising pitch pine (Pinus rigida Mill.) standards with a black locust (Robinia pseudoacacia L.) coppice understory has been reported (Stewart 1980). At present, coppice with standards systems are seldom found as commercial ventures in Europe, but they can be considered to be interesting alternatives to other currently employed silvi- cultural systems (Suchant et al. 1996). Furthermore, due to their species diversity and the canopy structure created through their unique management, they also provide high ecological and protective values (Suchant et al. 1996; Machar 2009; Messier et al. 2013). classes has previously been proposed (Evans 1984). Never- theless, it was recognised that the quantity of standard trees must be dictated by the coppice layer’s light requirement (Hartig 1820). One advantage of the system was the rapid growth of the standards and their above-average growth in volume and value (Nyland 2002). 1.3 Alley cropping Whilst these modifications reduced the value of alley cropping as a soil fertility-enhancing technique, they did allow alley cropping to be operationalised within the constraints of the farmer, so that the system could be used to satisfy a wider range of livelihood needs (Graves et al. 2004). In the temperate zone, where fertilisers are easily available and soil fertility is less of a concern, alley cropping frequently takes the form of combinations of high-value trees cultivated in rows and separated by wide alleys that are cropped with annual or perennial plant species. Such silvoarable systems have recently been the subject of major research efforts in Europe aimed at developing computer simulation tools (Van der Werf et al. 2007; Graves et al. 2010a; Keesman et al. 2011), to identify and quantify their major economic (Graves et al. 2007, 2010b) and environmental benefits (Palma et al. 2007a, 2007b) as well as to determine how farmers might respond to such innovations (Graves et al. 2008). The wide spacing between the rows of trees in silvoarable systems increases rates of growth and can, with an appropriate pruning regime, increase the value of the timber through an enhanced form and increased bole length. In the southern USA, cotton, peanut, maize, soybean, wheat and oats are important crops for alley cropping, and these are normally combined with trees such as pines (Pinus spp.) and pecan (Carya illinoensis K. Koch) (Wanvestraut et al. 2004; Zamora et al. 2007). The increasing demand for timber of high quality in Europe, and the decline in tropical hardwood avail- ability, has stimulated the development of silvoarable systems specifically intended for high-quality timber production However, farmers have adapted alley-cropping practices when they are able to modify them to meet specific require- ments. In the Philippines, Garcia et al. (2002) reported that farmers increased alley widths, used single rather than double hedgerows, reduced pruning and mulch application, and used various tree species in the hedgerows in order to satisfy a range of other needs. Whilst these modifications reduced the value of alley cropping as a soil fertility-enhancing technique, they did allow alley cropping to be operationalised within the constraints of the farmer, so that the system could be used to satisfy a wider range of livelihood needs (Graves et al. 2004). Results concerning the land equivalent ratio (LER) (Mead and Willey 1980) of modern alley cropping systems have raised interest in the system. 1.3 Alley cropping Alley cropping is an agroforestry practice that involves the planting of row crops or pasture in alleys formed by single or multiple rows of trees or shrubs (Garret and Buck 1997; Gillespie et al. 2000). The rows of trees provide various benefits, such as high-quality timber if the trees are allowed to mature (often called silvoarable systems (Dupraz 1994), crop fertilisation through use of leguminous trees (Kang et al. 1981) or biomass for energy (Tsonkova et al. 2012), if the trees are coppiced. Alley cropping is a modern form of agro- forestry that can be designed to be compatible with crop mechanisation. It is typically a two-storey agroforestry sys- tem, where light competition between the trees and crops plays a major role in the productivity of the method (Talbot and Dupraz 2012). Alley cropping was initially developed in Nigeria during the 1970s at the International Institute for Tropical Agriculture (Kang 1997), primarily as a soil fertility-enhancing technique (Graves et al. 2004). Rows of trees or shrubs were pruned prior to crop planting and then pruned again periodically during crop growth to prevent shading. The pruned biomass was then used as a green manure or mulch in the intercrop area (Graves et al. 2004). It was initially envisaged that fast- growing nitrogen-fixing trees or shrubs would create soil conditions similar to those produced during the fallow phase of shifting cultivation systems, because they could cycle In modern times, the decreasing demand for fuel wood caused by a growth in coal use has reduced the interest in coppice systems. Over time, most of the coppice with stan- dards systems has been converted into high forests, often accompanied by the planting of fast-growing conifer species, with a focus on production of large-diameter timber trees. The remainder was left unmanaged and grew into broadleaf- dominated forests. This is especially true in England where large areas of former oak and hazel coppice (Moss et al. 1910) were abandoned following the First and Second World Wars (Hopkins and Kirby 2007). There are, however, exceptions to Alley coppice—a new system with ancient roots 531 (Dufour et al. 2013). In contrast to fruit trees, it is considered that there is no critical stage at which the growth of timber trees in alley cropping systems can be impaired by competi- tion from crops (Dupraz 1994). 1.3 Alley cropping Dupraz and Liagre (2008) re- cently published results of the first-ever alley cropping exper- iment to be monitored from tree establishment through to final tree harvest. A LER of 1.4 was found for this poplar-wheat system. This ratio means that a 100-ha farm using an alley cropping system of walnut trees and cereals could produce as much cereal and walnut timber as a 140-ha farm where the trees and cereals are cropped separately. Therefore, alley cropping with timber trees is considered to be more ecologi- cally efficient relative to monoculture production of the same components. Explanations for this relatively high productivity include more efficient capture of light due to lagged phenol- ogy of trees and crops (Graves et al. 2010b; Talbot and Dupraz 2012) and the plasticity of tree root systems that are displaced by crop root competition to deeper horizons, thus reducing competition for water and nutrients (Mulia and Dupraz 2006). Some guidelines for the design of sustainable alley cropping systems have been produced through the use of computer simulation models (Talbot 2011). For example, it is recom- mended that tree rows should be spaced at twice the height of the mature trees, so that the yield of the alley crops will not decrease significantly during the first half of the tree life cycle, and so that they will remain high enough to provide economic profit until the trees are harvested. In the temperate zone, where fertilisers are easily available and soil fertility is less of a concern, alley cropping frequently takes the form of combinations of high-value trees cultivated in rows and separated by wide alleys that are cropped with annual or perennial plant species. Such silvoarable systems have recently been the subject of major research efforts in Europe aimed at developing computer simulation tools (Van der Werf et al. 2007; Graves et al. 2010a; Keesman et al. 2011), to identify and quantify their major economic (Graves et al. 2007, 2010b) and environmental benefits (Palma et al. 2007a, 2007b) as well as to determine how farmers might respond to such innovations (Graves et al. 2008). The wide spacing between the rows of trees in silvoarable systems increases rates of growth and can, with an appropriate pruning regime, increase the value of the timber through an enhanced form and increased bole length. 1.3 Alley cropping In Italy, silvoarable systems of hybrid poplars and cereal crops were developed in the Po Valley in northern Italy and subsequently adopted throughout northern Europe by the 1980s. The system incorporated maize, soybean and cereals between the tree rows during the first 2 years of a tree rotation lasting between 7 and 10 years, but declined because European Union grant systems for plant- ing trees on arable land did not permit intercropping (Eichorn et al. 2006). In France, the system became widespread in the eighteenth century and is still used on approximately 6,000 ha of alluvial soils throughout the country. In the UK, large hybrid poplar plantations in southern England were developed for the production of matches in the 1950s (Beaton 1987; Dupraz and Newman 1997). The alleys were intercropped with cereals for 8 years, then under-sown with a grass and clover mixture, and grazing until year 20; after which, light interception by the tree canopy prevented sufficient pasture development. The poplars were then harvested when about 25 years old. nutrients, suppress weeds and reduce erosion. Early experi- mental results, obtained in humid climates and fertile soils, were promising, with significant intercrop yield increases (Atta-Krah and Sumberg 1988). But the results in semi-arid regions were less favourable and where environmental re- sources were scarce; long-term benefits to fertility appeared to be offset by short-term competition for water and nutrients (Rao et al. 1998). Although uptake of alley cropping in humid parts of Africa was initially high, many farmers subsequently abandoned it. Douthwaite et al. (2002) reported that in Nigeria, no new adoption was seen to occur after the 1990s; the high labour requirements for establishing and managing the woody com- ponent, and incorporating its biomass in the intercrop soil, were viewed as major constraints. Further studies have report- ed similar findings, with farmers stating that the labour needed for pruning was an especially problematic aspect of alley cropping (David 1995; Craswell et al. 1997). cropping (David 1995; Craswell et al. 1997). However, farmers have adapted alley-cropping practices when they are able to modify them to meet specific require- ments. In the Philippines, Garcia et al. (2002) reported that farmers increased alley widths, used single rather than double hedgerows, reduced pruning and mulch application, and used various tree species in the hedgerows in order to satisfy a range of other needs. 1.3 Alley cropping In the southern USA, cotton, peanut, maize, soybean, wheat and oats are important crops for alley cropping, and these are normally combined with trees such as pines (Pinus spp.) and pecan (Carya illinoensis K. Koch) (Wanvestraut et al. 2004; Zamora et al. 2007). The increasing demand for timber of high quality in Europe, and the decline in tropical hardwood avail- ability, has stimulated the development of silvoarable systems specifically intended for high-quality timber production The effect of trees in alley cropping systems is also of interest environmentally (Palma et al. 2007c). Trees are capa- ble of capturing and recycling fertiliser nutrients from deeper soil horizons than crops, and this helps to improve nutrient use efficiency as well as mitigate groundwater contamination by 532 C.D. Morhart et al. excess nutrients (Rowe et al. 1999; Allen et al. 2004). Inten- sive agricultural production has caused groundwater and sur- face water contamination through nitrogen leaching and is a major environmental problem. Agroforestry systems, and al- ley cropping in particular, have been identified as a means by which the nitrogen contamination of water can be reduced (Jose et al. 2004; Palma et al. 2007c). Other environmental benefits also exist. The tree rows provide habitat for several fauna and flora species, and help protect and enhance biodi- versity in arable landscapes (Palma et al. 2007c). Evidence suggests that the biological control of several crop pests could potentially be achieved using silvoarable systems, but knowl- edge on how this can be reliably managed is lacking, and more research is needed (Smits et al. 2012). system. In alley coppice, the timber trees are aligned while they are randomly distributed in most coppice with standards. Furthermore, alley coppice systems are intensively managed and better suited to agricultural land rather than forest land. The alley coppice system consists of narrow strips of land for production of high-value timber trees in rows at wide spacing combined with wide alleys between the rows of trees for production of SRC (Fig. 1). The main features of an alley coppice system and the ancient coppice with standards are compared and summarised in Table 1. A major difference between the two systems, as noted in Table 1, is the shorter rotation with alley coppice and the optimised design, to com- bine the mechanised production of coppice biomass and high- value timber trees. 1.3 Alley cropping The coppice with standards system was historically practised on less fertile forest sites, while current alley coppice systems may fit to more fertile sites formerly used for agriculture; this facilitates shorter harvesting cycles and production of timber trees of high value for industrial transformation such as veneering, according to past investiga- tion conducted in Italy on common walnut and wild cherry (see Zanuttini et al. 2006). However, more research is needed concerning the relationship between wood quality and growth rates of temperate valuable hardwoods. 1.4 Short-rotation coppice Biomass production in short rotations of 3–5 years involves growing species which can freely coppice, produce high yields of biomass on agricultural land and be mechanically harvested. The aspects of SRC forestry which are applicable to the proposed alley coppice system are described below in “Short rotation coppice production in alley coppice.” The general features of SRC are well known and reviewed (Verwijst and Nordh 1992, Brown et al. 1996, Verwijst and Telenius 1999, McCracken et al. 2001, Aylott et al. 2008, Wickham et al. 2010, Filat et al. 2010, Paris et al. 2011; Fiala and Bacenetti 2012; Morhart et al. 2013b). 2.1 High-value timber production between alley coppice 2.1 High-value timber production between alley coppice The goal of high-value timber production is a straight and branch-free bole of sufficient quality for use as veneer wood. The crop tree species proposed for this purpose should be capable of producing a high-value product and include wild cherry, common ash, sycamore, sweet chestnut (Castanea sativa Mill.), walnut (including Juglans nigra L. and hybrids), 1.5 The presentation of an innovative combined system: alley coppice 1.5 The presentation of an innovative combined system: alley coppice 1.5 The presentation of an innovative combined system: alley coppice The combination of growing SRC together with high-value timber trees is an alley cropping system with SRC as the intercrop, and is therefore close to the coppice with standards Fig. 1 The alley coppice system combining valuable wood production in narrow land strips with a SRC component in wide alleys. The distance between the valuable trees on the tree line is equivalent to the crown width (CW) of a mature tree Fig. 1 The alley coppice system combining valuable wood production in narrow land strips with a SRC component in wide alleys. The distance between the valuable trees on the tree line is equivalent to the crown width (CW) of a mature tree 533 Alley coppice—a new system with ancient roots Table 1 Comparison of management systems: the ancient coppice with standards and the alley coppice system System component Coppice with standards Alley coppice system Coppice Species Oak (Quercus spp.), ash (Fraxinus spp.), maple (Acer spp.), cherry (Prunus spp.), poplar (Populus spp.), beech (Fagus sylvatica), hazel (Corylus avellana.), alder (Alnus spp.), hornbeam (Carpinus betulus), birch (Betula spp.), lime (Tilia spp.), walnut (Juglans spp.) Poplar (Populus spp.), willow (Salix spp.), alder (Alnus spp.), black locust (Robinia pseudoacacia), Eucalypt (Eucalyptus spp.) Rotation length 10–30 years 2–5 years Regeneration Mostly natural (coppice and seedlings) Planted Products Small-sized construction timber, fuel wood Fuel/fibre wood Management Harvesting Management of accompanying ground flora, planting, harvesting Standards Species Same as coppice species Cherry (Prunus spp.), ash (Fraxinus spp.), maple (Acer spp.), walnut (Juglans spp.), Sorbus spp. Rotation length 80–150 years 30–70 years Regeneration Selected natural (vegetative, seed-origin) Planted Products Timber Premium timber for veneers Management Thinning, harvesting Planting, thinning, pruning, harvesting, weed control be chosen as future crop trees. Nevertheless, the initial tree density is comparable with other agroforestry systems and should be as low as possible consistent with a final harvest of 50 to 80 mature trees per hectare (Balandier and Dupraz 1998). It is also desirable to plant the timber trees in advance or at the same time as establishing the coppice crop. This should facilitate an early establishment of the timber trees with minimal early competition. 1.5 The presentation of an innovative combined system: alley coppice The fast-growing SRC may dom- inate the high-value timber trees initially, so we suggest a managed crop-free strip of 3 m on both sides of the high- value timber trees. The production of high-quality timber is the ultimate goal for the timber tree strips, so pruning is of absolute necessity and should be performed until the required branch-free bole length has been reached (Balandier 1997; Balandier and Dupraz 1998). This part of the tree accounts for 90 % of the total tree value (Dupraz and Liagre 2008), and the application of a pruning treatment has been observed to more than double the value of the final timber crop (Pryor 1988). The aim is to produce logs for the veneer industry that are a multiple of the required veneer length of 2.5 m. The target branch-free bole length should be between 5 and 10 m, but this will be dependent on site quality. The distance between the strips stocked with valuable timber trees should be wide enough so as not to reduce the growth of the SRC component. It has been shown that a spacing between the tree rows equal to twice the final height of the timber trees allows crop yields to remain profitable until final tree harvest (Talbot 2011). If the height of the timber trees exceeds the width of the alley, it is very possible that most crops will no longer be profitable. However, it should be noted that these results, obtained for winter annual crops, should not be extrapolated to SRC. It is likely that SRC production will in fact be more adversely alder (Alnus spp.) and Sorbus spp. To be able to produce valuable timber trees in combination with SRC, it is essential that the trees be given appropriate space for crown develop- ment. The required spacing for a given final target diameter of the tree stem can be calculated from the crown projection area, based on the close correlation that exists between stem diam- eter at breast height and crown width. This can serve as the basis for calculating an appropriate spacing for trees within the tree rows and the width of the coppice alleys which are between the rows of crop trees. 1.5 The presentation of an innovative combined system: alley coppice Hein and Spiecker (2009) suggest a simple species-based thinning rule utilising a con- stant value based on the arithmetic mean of trees with a diameter at breast height of 30 and 60 cm, assuming crown cover of 70 % for ash and sycamore, and 50 % for wild cherry. The mean distance between crop trees or standards is depen- dent on the mean radial increment. It should be set at between 20 and 28 times the target diameter at breast height (ash, sycamore and wild cherry). For example, for a target diameter of 50 cm at breast height for wild cherry, this rule dictates a final distance of 12.5 m between trees. Dupraz and Liagre (2008) agree that trees should not be planted too closely on the tree line and recommend distances between 4 and 10 m de- pending on the expected tree canopy size of mature trees. Using an optimal spacing as described above, high annual diameter increments of 1 cm per year can be achieved (Spiecker 2006). Adequate spacing of trees ensures that tree canopies do not close or shade each other out until the point of harvest; this minimises dead branches and the potential for fungal infection (Oosterbaan et al. 2009). To ensure a high success rate of the timber trees, we advocate the planting of trees in groups of two or three at a 2-m spacing within the rows so that poor trees can be removed and only the best trees can 534 C.D. Morhart et al. the same way as for the pure SRC plantations as described above. Harvesting cycles for the SRC component are generally 2–3 years, but can be more. Direct chipping in the field is the prevalent form of harvest. Wood chips, often with a high moisture content (50 % and more) must undergo artificial drying since direct combustion of fresh biomass is not desirable. Alternatively, harvested stems can be left in situ for field drying, with subsequent collection, transportation and chipping of the dry biomass. This harvesting system requires lighter and smaller machines. The number of SRC rotations with profitable yields is generally limited to 4–6 rotations (Geyer 2006; Al Afas et al. 2008), and restoring land from SRC to agricultural land use can be accomplished by stump removal. 2.2 Short-rotation coppice production in alley coppice 2.2 Short-rotation coppice production in alley coppice The proposed alley coppice system is flexible and can be designed to suit the land manager’s needs. If SRC is the primary production goal, the tree rows can be widely spaced to maximise the SRC cropping area. The ideal orientation of the tree rows is north–south to allow maximum sun penetra- tion to the coppice crop and minimal shading by trees. How- ever, specific site characteristics, such as slope, contours and field layout, that could change the optimal row orientation should be accounted for in the planning stages of the alley coppice system. The cropping area must be designed to allow for mechanical harvesters, and the system should incorporate a tree strip with a minimal width of 3 m to accommodate the high-value timber trees. The coppice species of choice de- pends on geographic location and soil parameters. Poplar cultivars are mostly used in south and central Europe (e.g. Italy, Spain and Germany), whereas willow (Salix spp.) is more adapted to northern latitudes (e.g. Sweden, UK, Ireland and northern Poland). Other tree species suitable for SRC include Eucalyptus spp. and R. pseudoacacia, which are cur- rently used less frequently, but seen in countries such as Portugal (Reede et al. 2003) and Hungary (Rédei 1998). Miscanthus may also be an option. Planting density of coppice is generally higher than 5,000 stems per hectare, and manage- ment operations are fully mechanised from establishment to harvest. Management operations include herbicide treatments during the establishment phase, fertiliser application between the SRC cycles, irrigation in southern latitudes and occasional pesticides application when necessary. On soils with low nutrient content, fertilisation can be critical as the frequent harvest of large quantities of biomass deplete the soil of nutrients (Lars 2002; Guidi et al. 2008a; Morhart et al. 2013a). Where necessary, fertiliser should be applied at least once during each rotation, usually in the spring following each harvest of the SRC crop. Fertiliser can take the form of organic sewage sludge, chemical input or material for phytoremediation purposes. Moreover, the availability of soil water plays a deci- sive role in determining SRC yield (e.g. Murach et al. 2008). Hence, irrigation is essential for stabilising yields in areas of southern Europe prone to unreliable precipitation (Guidi et al. 2008b; Pistocchi et al. 2009). 2.2 Short-rotation coppice production in alley coppice Drip irrigation is generally rec- ommended for alley cropping and for SRC intercrop systems in southern European and is considered to be a less expensive and more efficient form of irrigation (Ayars et al. 1999; Wie et al. 2008). The alley areas being used for SRC can be designed in 3 Dynamics of an alley coppice system 3.1 Description of developmental stages over 60 years in an alley coppice plantation using poplar as the short-rotation coppice and with wild cherry as the valuable hardwood 3.1 Description of developmental stages over 60 years in an alley coppice plantation using poplar as the short-rotation coppice and with wild cherry as the valuable hardwood To provide an example scenario, wild cherry was chosen as a common species producing a high-value timber product, in combination with poplar which is frequently employed within SRC plantations in central and southern Europe. In this scenar- io, four important development stages of an alley coppice plantation have been characterised. Figure 2 illustrates wild cherry grown for timber production in strips and poplar clones in wide alleys for SRC biomass production. Each stage is shown using a proportional scale, with wild cherries assumed to be 2 m high at planting, and poplar SRC cuttings assumed to be 20 cm high at planting. The main objective for the high-value wood production is a branch-free bole length of 10 m and a target diameter of 60 cm. The growth rates and development of diameter, height and crown expansion for the valuable timber trees and SRC have been deduced from the literature (Röös 1993; Spiecker 1994; Ali 2007; Hein and Spiecker 2009). 3.2 Growth stages and management operations of alley coppice & Stage 1 (Fig. 2a): & Stage 1 (Fig. 2a): At the end of the first year, the poplar SRC and wild cherry trees have grown without competing for light. The branches of the poplar SRC are still small. Weed control is essential around the trees to assist establishment during the first years. 1.5 The presentation of an innovative combined system: alley coppice An overview of the main advantages and disadvantages of the alley coppice system in comparison with traditional SRC can be found in Table 2. affected by light competition from the trees than most winter cereals, since these have a significant portion of their growth cycle when the trees are leafless. Nevertheless, Clinch et al. (2009) showed that some shading from bigger trees can also be beneficial to the growth of SRC, probably because it lowers evapotranspiration, thus increasing soil moisture content. & Stage 2 (Fig. 2b): & Stage 2 (Fig. 2b): & Stage 2 (Fig. 2b): After the third year, the SRC poplar alley coppice has reached a height of about 6 m and is harvested. Cutting the 535 Alley coppice—a new system with ancient roots Table 2 The main advantages and disadvantages of the alley coppice system in comparison with traditional SRC Advantages Disadvantages Economic advantages Economic disadvantages • Increased productivity (LER >1) • Production of high-quality wood with a high market value • Combination of short and long-term returns • Product diversification • Decreased economic risk • Potentially lower productivity of the SRC areas • Reduced land area for SRC • Long-term engagement in one system • Uncertainty of future market for valuable timber • Relatively expensive planting material • Lack of good planting material with improved stem form • Need for prolonged and more intensive management operations (weed control, tree pruning) • Thinning is often unprofitable and difficult to mechanise Environmental advantages • Lower chemical inputs in comparison to SRC • Provision of additional environmental services (carbon sequestration, water filtration, biodiversity, landscape amelioration) • Increased biodiversity • Improved habitat structure • Reduced erosion and improved nutrient recycling • Labour extensive • Possibility of supporting rare light-demanding tree species • Higher acceptance of the public • Increased productivity (LER >1) • Production of high-quality wood with a high market value • Combination of short and long-term returns • Product diversification • Lower chemical inputs in comparison to SRC • Improved habitat structure • Reduced erosion and improved nutrient recycling • Labour extensive • Possibility of supporting rare light-demanding tree species • Higher acceptance of the public may even be changed to an annual cropping system, akin to more conventional agroforestry practice. SRC at this stage gives the wild cherries growing space. The wild cherry will have reached a height of approxi- mately 3 m, so the poplar will not compete substantially for light if coppiced every 3 years. The first phase of tree pruning is carried out. The sudden exposure of tree stems to full sunlight might also stimulate the growth of epicormic shoots or even cause stem damage such as sunburn. Epicormic shoot production may be mitigated by using targeted pruning systems such as selective prun- ing (i.e. the preferential removal of large diameter and steeply angled branches (Springmann et al. 2011)). Stage 3 (Fig. 2c): If the initial wild cherry trees are planted in groups within the rows, it will be necessary to thin them between years 5 and 15 to leave those trees with the best stem forms. After 20 years and regular branch pruning, a branch-free bole of up to 10 m in height will be produced. After 20 years, the wild cherry trees should be allowed to develop their crowns unrestricted (i.e. without further pruning). Interventions are only necessary if forks or steeply angled branches occur. Taking into account that the commercial duration of the SRC component is approx- imately 15–20 years, depending on the site specific con- ditions and the harvesting cycle, new coppice planting is necessary after this time. This can be used to modify the spacing of SRC material as well as the variety or species of SRC, therefore allowing for greater flexibility of the sys- tem and its possible adaptation to climate change, eco- nomic changes and other future changes. The intercrop & Stage 2 (Fig. 2b): Sun- burn damage may be mitigated by applying white latex paint to stems. In comparison with whorl-wise pruning, selective pruning can significantly reduce the survival rate of epicormics and may help shade the stem (Springmann et al. 2011). g y p Stage 4 (Fig. 2d): & Stage 4 (Fig. 2d): The final objective of high-value timber production has been achieved. The target diameter of 60 cm has been reached, and the valuable timber trees can be harvested. The third generation SRC component will also be harvest- ed and can be removed at this point after reaching its optimal commercial rotation. Depending on the growth rate of individual trees, this goal could be achieved within about 60 years, with harvesting occurring over several years. It should be noted that many modification of the species used and the temporal and spatial aspects of the alley coppice system are possible, thus providing many different system variants. Alternatives also exist to the establishment of the alley coppice system. For example, this could include the introduction of a SRC component into an already- established alley cropping system that includes mature or semi-mature timber trees. Alternatively, it could include the planting of valuable timber trees within a pre-existing SRC plantation, through the removal of an appropriate number of SRC rows. Stage 3 (Fig. 2c): 4.1 Competition for light in alley coppice systems Complementary use of light is a feature of resource use during the first years of an alley coppice system. Timber tree planta- tions make little use all of the available light resource during the initial years, since they are planted at a wide spacing, and the light captured by the herbaceous vegetation between the trees does not bring any significant economic return. Utilising this unused light resource for an economically productive intercrop is one of the advantages of agroforestry systems. As the rotation progresses, the timber trees will start to com- pete for light and dominate the SRC component, depending on the width chosen for the coppice alleys. Whilst this might often be detrimental to SRC growth, in some situations, the shade may have some beneficial effects in drought prone areas. It is, therefore, difficult to make general predictions about the balance between the positive and negative effects of light competition. The outcomes are likely to be site- specific and require new research investigations. 4 Competition and facilitation processes in alley coppice systems The inter-species relationships in plant mixtures vary in time and space, and the ability to evaluate all aspects in the alley coppice system requires modelling solutions. However, the modelling tools currently used in agronomy and forestry are not adapted to simulating multi-species systems, and whilst C.D. Morhart et al. 536 6 m 1 2 3 4 5 6 7 8 9 10 6 m Valuable Wood Short rotation coppice [m] Valuable Wood 1 2 3 4 5 6 7 8 9 10 6 m 6 m [m] Valuable Wood Short rotation coppice Valuable Wood 5 10 15 6 m 6 m [m] Valuable Wood Short rotation coppice Valuable Wood 5 10 15 20 25 30 6 m 6 m [m] Valuable Wood Short rotation coppice Valuable Wood c d b a he alley 1st year ), after the he 60th C. . o a t et a . 6 m 1 2 3 4 5 6 7 8 9 10 6 m Valuable Wood Short rotation coppice [m] Valuable Wood 1 2 3 4 5 6 7 8 9 10 6 m 6 m [m] Valuable Wood Short rotation coppice Valuable Wood 5 10 15 6 m 6 m [m] Valuable Wood Short rotation coppice Valuable Wood c b a ey year er the 0th Short rotation coppice Valuable Wood Short rotation coppice 5 10 15 20 25 30 6 m 6 m [m] Valuable Wood Short rotation coppice Valuable Wood d 537 Alley coppice—a new system with ancient roots some agroforestry models are available (Van Noordwijk and Lusiana 1999; Van der Werf et al. 2007; Graves et al. 2010b), validated computer-based models of alley coppice systems are not widely distributed for temperate conditions. Talbot and Dupraz (2012) have shown how a dynamic model of plant interaction can be used to identify key processes responsible for the productivity of an agroforestry system. The results for a walnut-wheat system underlined the impor- tance of several processes, such as root plasticity and turn- over, and shade for critical phases of crop growth, that had not been identified as significant in earlier research. The same approach with SRC as an alley crop may help identify appro- priate management options to increase the efficiency of the alley coppice mixture. 4.1 Competition for light in alley coppice systems 4.2 Competition for soil resources in alley coppice systems Below-ground competition may be fierce between SRC and timber trees. Plants compete for a broad range of soil resources including water and at least 20 mineral nutrients. Although SRC and timber trees have very similar requirements, they may be able to exploit different layers of the soil with their root systems, as shown for other agroforestry systems (Van Noordwijk et al. 1996). Mixtures of leguminous and non- leguminous tree species could be desirable, since using an N-fixing SRC species such as alder or black locust could stimulate the growth of the timber trees by making more nitrogen available in the tree root zone. However, nutrient removal through the frequent harvest of SRC biomass with relatively high nutrient concentration (Lars 2002; Guidi et al. 2008a; Morhart et al. 2013a) may induce nutrient deficiencies in the timber trees, and furthermore, the nutrient demand of SRC will progressively increase and rise to a maximum level the year before SRC harvest. An increasing amount of research has been devoted to the valuing of ecosystem services using both monetary and non- monetary approaches. However, these assessments are chal- lenging and are hindered by lack of rigorous understanding of the mechanisms that underlie the provision of ecosystem ser- vices and understanding of social preferences for those ser- vices. A key component determining the quality and the quan- tity of ecosystem services is considered to be the biodiversity present within a given ecosystem. Among the key beneficial effects of both SRC and agroforestry on intensively managed agricultural land is an increase of biodiversity (e.g. Fry and Slater 2009; Quinkenstein et al. 2009; Reeg et al. 2009; Schulz et al. 2009; Mosquera-Losada et al. 2011; Baum et al. 2012; Kaonga 2012). Therefore, it is likely that alley coppice systems will also provide increased biodiversity. Furthermore, it is likely that alley coppice plots will also constitute key elements of the ‘green network’ which will connect natural features of the landscape in intensive agricultural land, in order to facilitate the movement of animals (Rigueiro-Rodríguez et al. 2009). For these reasons, it is envisaged that the alley coppice approach will help to improve the quality and quantity of ecosystem 4.4 Biodiversity, ecosystems services and climate change Agriculture has a diverse and complex relationship with soci- ety, pivoting around its use of natural resources and the provision of ecosystem services. Ecosystem services provide a variety of benefits that have value for society and provide a well-being for humans. Examples of ecosystem services in- clude provisioning services (for example, production of food, wood and water), regulating services (for example, climate and air quality regulation) and cultural services (for example, provision of aesthetic value and recreation). These are underpinned by supporting services for the ecosystem, such as pollination, nutrient and water cycling, and soil formation (Millennium Ecosystems Assessment 2005; European Academies Science Advisory Council 2009). Whilst agricul- tural landscapes can produce a range of ecosystems services for the well-being of society, intensively managed monocul- ture landscapes are generally considered to be depleted eco- systems (Benton et al. 2003), specialised in provisioning services, but providing relatively few of the other ecosystem services from which people benefit. 4.3 Integrating competition and facilitation processes in a dynamic model of interactions 4.3 Integrating competition and facilitation processes in a dynamic model of interactions Relatively few computer models have been developed for mixtures of tree species (Bartelink 2000). Dynamic simulation models need to integrate competition for different resources in time and space, so as to predict the impact of highly non-linear responses to compete for specific environmental resources. 538 C.D. Morhart et al. services provided from agricultural land. Some of the ways in which this might be undertaken are described in Table 3. Ad- ditional methods to improve biodiversity within an alley cop- pice system could include the planting of shrubs and wild flowers in the tree strips, the use of a variety of different tree species of high-value timber trees, the use of trees of different tree ages on the same tree strip and the use of different clones and ages of the SRC component. It is generally agreed that climate change will affect the functioning of both natural and cultivated ecosystems (Parmesan and Yohe 2003; Tubiello et al. 2007), and this is best mitigated by a policy of species diversification. Diversity within natural ecosystems is thought to contribute to a greater resilience to changing environmental conditions (Elmqvist et al. 2003; Allan et al. 2011). Cultivated ecosystems are mostly monocultures, and therefore considered to be more vulnerable to a changing environment (Malézieux et al. 2009); it could therefore be deduced that the increase in plant diversity in alley coppice systems will result in improving their adaptive capacity to climate change. However, there is a degree of uncertainty associated with this position, and given the importance of the topic, the following section examines how alley coppice system might respond to climate change in more detail (Lin 2011; Smith et al. 2013). environmental stress, including the greater root plasticity de- veloped by plants when inter-species competition occurs, greater resource use efficiency and modifications to the micro-climate that could help to reduce plant stress. The forest-like micro-climate created by the SRC component of the alley coppice system might be beneficial to the widely spaced timber trees, especially during very dry and hot weath- er conditions that are forecast to increase in the future. Con- versely, the shady environment provided by the mature trees for the SRC component may help the SRC cope with very high evaporative demands. Table 3 Estimates for improve- ment of ecosystem services from alley coppice systems relative to traditional forms of agricultural management 4.3 Integrating competition and facilitation processes in a dynamic model of interactions However, the SRC component might also cause an increased water deficit in the soil, and woody coppice is known to be more water-demanding than annual crops (Petzold et al. 2009). Potential benefits could thus be negated by the effects of competition for water. Predicting the final outcome between these positive and neg- ative effects is a challenge that needs to be addressed using both field experiments and computer modelling. Processes such as plant root plasticity, three dimensional compensation strategies of plants, micro-climate modifications and phenol- ogy as influenced by shade and organ temperature may also modify the functioning of the associated plants in alley cop- pice systems. The abiotic risks induced by climate change and their effects on monoculture crop yields have recently been ob- served (Lin et al. 2008) and may partly explain why crop yields are currently stagnating in Europe (Brisson et al. 2010). Recent research has shown that agroforestry systems may be more resilient than monoculture systems to abiotic distur- bances caused by climate change (Talbot et al. 2009). 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The implementation of alley coppice on suitable land appears to hold some advantages compared to traditional land use con- cepts such as pure SRC plantations or agroforestry. Table 3 Estimates for improve- ment of ecosystem services from alley coppice systems relative to traditional forms of agricultural management Function Services Welfare benefits Provisioning Livestock products Diversification of product lines Stabilisation of rural employment Additional renewable energy wood source Additional timber production Regulating Climate regulation Carbon sequestration in timber trees SRC as a carbon-neutral biomass source Decreased anthropogenic nitrogen input Water filtration Reduced nitrate and nutrient leaching Increased ground water quality Air filtration Pollutant absorption (city boundaries) Cultural Landscape appreciation Increased aesthetic value Sense of increased ecologic value and sustainability Supporting Refugium and nursery/pollination Increased biodiversity (invertebrates, mammals, birds, plants) Soil formation and retention Improved soil quality and fertility, reduced erosion Nutrient cycling Improved nutrient cycling 539 Alley coppice—a new system with ancient roots Atta-Krah AN, Sumberg JE (1988) Studies with Gliricidia sepium for crop/livestock production systems in West Africa. Agrofor Syst 6: 97–118 Furthermore, it could provide additional biomass for both the energy and the material wood industry, contribute to the diversification of farmers’ product lines, and increase biodi- versity, soil quality, and ecosystem services. However, poten- tial disadvantages may be the increased water demand of alley coppice systems, the danger of devaluing sunburn on the stems of the high-value timber trees and the long management cycles that might dissuade farmers from establishing alley coppice systems on their fields. At present, only a few alley coppice systems are in existence, and knowledge on their management, productivity and biological aspects are poorly understood. In particular, the interaction between high-value timber trees and SRC has undergone little research. 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https://openalex.org/W2293098307	https://europepmc.org/articles/pmc4924704?pdf=render	English	null	Expression of androgen receptor in non-muscle-invasive bladder cancer predicts the preventive effect of androgen deprivation therapy on tumor recurrence	Oncotarget	2,016	cc-by	4,208	Oncotarget, Vol. 7, No. 12 Oncotarget, Vol. 7, No. 12 www.impactjournals.com/oncotarget/ Expression of androgen receptor in non-muscle-invasive bladder cancer predicts the preventive effect of androgen deprivation therapy on tumor recurrence Koji Izumi1,2,, Yusuke Ito1,, Hiroshi Miyamoto3, Yasuhide Miyoshi4, Junichi Ota2, Masatoshi Moriyama2, Tetsuo Murai5, Hiroyuki Hayashi6, Yoshiaki Inayama7, Kenichi Ohashi8, Masahiro Yao1 and Hiroji Uemura4 1 Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama, Japan 2 Department of Urology, Yokohama Municipal Citizen’s Hospital, Yokohama, Japan 3 Departments of Pathology and Urology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 4 Department of Urology, Yokohama City University Medical Center, Yokohama, Japan 5 Department of Urology, International Goodwill Hospital, Yokohama, Japan 6 Department of Pathology, Yokohama Municipal Citizen’s Hospital, Yokohama, Japan 7 Department of Pathology, Yokohama City University Medical Center, Yokohama, Japan 8 Department of Pathology, Yokohama City University Graduate School of Medicine, Yokohama, Japan * These authors have contributed equally to this work Koji Izumi1,2,, Yusuke Ito1,, Hiroshi Miyamoto3, Yasuhide Miyoshi4, Junichi Ota2, Masatoshi Moriyama2, Tetsuo Murai5, Hiroyuki Hayashi6, Yoshiaki Inayama7, Kenichi Ohashi8, Masahiro Yao1 and Hiroji Uemura4 1 Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama, Japan 2 Department of Urology, Yokohama Municipal Citizen’s Hospital, Yokohama, Japan 3 Departments of Pathology and Urology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 4 Department of Urology, Yokohama City University Medical Center, Yokohama, Japan 5 Department of Urology, International Goodwill Hospital, Yokohama, Japan 6 Department of Pathology, Yokohama Municipal Citizen’s Hospital, Yokohama, Japan 7 Department of Pathology, Yokohama City University Medical Center, Yokohama, Japan 8 Department of Pathology, Yokohama City University Graduate School of Medicine, Yokohama, Japan * These authors have contributed equally to this work Correspondence to: Koji Izumi, email: koji.izumi522@gmail.com Keywords: bladder cancer, recurrence, androgen receptor, androgen deprivation therapy Department of Pathology, Yokohama City University Graduate School of Medicine, Yokohama, Japa These authors have contributed equally to this work Correspondence to: Koji Izumi, email: koji.izumi522@gmail.com Keywords: bladder cancer, recurrence, androgen receptor, androgen deprivation therapy Received: November 04, 2015 Accepted: January 29, 2016 Published: February 12, 2016 ABSTRACT Our recent retrospective study revealed a significantly reduced risk of bladder cancer (BC) recurrence in men who received androgen deprivation therapy (ADT) for their prostate cancer. However, whether androgen receptor (AR) signals contributed to the preventive effect of ADT remained unclear because ADT could reduce serum estrogens as well. The purpose of this study is to investigate the associations between the expression of AR/estrogen receptors (ERs) and BC recurrence in patients treated with ADT. We immunohistochemically stained 72 BCs and 42 corresponding normal urothelial tissues. AR/ERα/ERβ were positive in 44(61%)/22(31%)/39(54%) tumors and 35(83%)/24(57%)/34(81%) corresponding normal urothelial tissues, respectively. There were no statistically significant correlations between AR/ERα/ ERβ expression and clinicopathological features of BC. With a median follow-up of 31.3 months, 12 (43%) of 28 patients with AR-negative tumor versus 11 (23%) of 44 patients with AR-positive tumor experienced BC recurrence. Thus, patients with AR- positive tumor had a significantly lower risk of BC recurrence (P=0.031), compared with those with AR-negative tumor. Meanwhile, the expression of ERα/ERβ in tumors and that of AR/ERα/ERβ in normal urothelial tissues were not significantly correlated with BC recurrence. A multivariate analysis revealed AR positivity in tumors as an independent prognosticator (hazard ratio: 0.27; 95% confidence interval: 0.11-0.67) for BC recurrence. These results indicate that ADT prevents BC recurrence via the AR pathway, but not via the ERα/ERβ pathways. Patient characteristics AR/ERα/ERβ expression in BCs and corresponding normal tissues, in relation to patient/tumor characteristics, is shown in Tables 1 and 2. There were no statistically significant correlations between each receptor expression in tumors or normal tissues and clinicopatholgical features of BCs. RESULTS bacillus Calmette-Guérin (BCG), and approximately 10% of them eventually progressed to muscle invasion [2]. In addition, developments in treatment modalities for BC did not result in an improvement in disease mortality rates for several decades [3]. Therefore, new treatment is urgently needed to prevent both recurrence and progression of BC. AR/ERα/ERβ expression in tumors and corresponding normal tissues Using preclinical models, we and others have revealed the involvement of androgen receptor (AR) signaling in BC development [4-6]. However, prognostic significance of AR expression in BC recurrence is still controversial [7-11]. Moreover, there is no significant gender-specific difference in the expression levels of AR [7], suggesting that circulating serum androgens may be critical for AR-stimulated BC development.i We immunohistochemically evaluated 72 BCs and 42 corresponding normal tissues for the expression of AR, ERα, and ERβ. Positive signals for all three receptors were detected predominantly in the nuclei of neoplastic (Figure 1) or non-neoplastic (figure not shown) urothelial cells. AR/ERα/ERβ were positive in 44(61%)/22(31%)/39(54%) tumors and 35(83%)/24(57%)/34(81%) corresponding normal-appearing urothelial tissues, respectively. In accordance with our previous findings [7], AR (P = 0.020)/ERα (P = 0.006)/ERβ (P = 0.005) expression was significantly lower in tumors than in non-neoplastic urothelial tissues. Recently, we demonstrated first clinical evidence indicating that androgen deprivation therapy (ADT) prevented BC recurrence [12]. In brief, in patients with double primary cancers of the prostate and bladder, ADT for their prostate cancer, mostly with a LHRH analogue, significantly reduced the risk of BC recurrence [hazard ratio (HR): 0.29]. LHRH analogues suppress secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland resulting in suppression of androgens from the testis. Since estrogens in men are mainly produced by aromatization of testosterone, LHRH analogue deprives estrogens as well [13]. Estrogens-mediated estrogen receptor (ER) signals have also been implicated in BC development [14-16]. Even in men, estrogens whose serum levels are similar to those in postmenopausal women have physiological functions, such as bone health, regulation of body fat, and sexual function [17]. INTRODUCTION 80% of patients are present with non-muscle-invasive BC (NMIBC) at the initial diagnosis, 36-51% of them recur despite of currently available adjuvant instillation therapy, such as intravesical instillation of an anthracycline or Men have 3-4 times higher incidence of bladder cancer (BC) than women [1]. Although approximately www.impactjournals.com/oncotarget Oncotarget 14153 RESULTS Oncotarget Oncotarget Expression of AR, ERα, and ERβ in BCs and tumor recurrence Thus, whether AR signals were responsible for the preventive effect of ADT on BC recurrence was still open question. The purpose of this study is to investigate the associations between AR/ERα/ERβ expression in surgical specimens and BC recurrence in patients treated with ADT. We next performed Kaplan-Meier analyses coupled with log-rank tests to assess possible associations between staining of AR, ERα, and ERβ in BCs and tumor recurrence. With a median follow-up of 31.3 months, 12 (43%) of 28 patients with AR-negative tumor versus 11 (23%) of 44 patients with AR-positive tumor experienced BC recurrence. Median time to recurrence was 8.2 (interquartile range [IQR]: 4.7-20.4) months. Thus, patients with AR-positive tumor had a significantly lower Figure 1: Immunohistochemistry of AR, ERα, and ERβ in urothelial tumors. Original magnification: x400. Immunohistochemistry of AR, ERα, and ERβ in urothelial tumors. Original magnification: x400. Figure 1: Immunohistochemistry of AR, ERα, and ERβ in urothelial tumors. Original magnification: x400. Figure 1: Immunohistochemistry of AR, ERα, and ERβ in urothelial tumors. Original mag www.impactjournals.com/oncotarget Oncotarget 14154 Table 1: Correlations between AR/ERα/ERβ expression in tumors and clinicopathological features of BC relations between AR/ERα/ERβ expression in tumors and clinicopathological features of BC Table 1: Correlations between AR/ERα/ERβ expression in tumors and clinicopathological features of BC Table 1: Correlations between AR/ERα/ERβ expression in tumors and clinicopathological feat Figure 2: Recurrence-free survival according to the expression of AR/ERα/ERβ in tumors. Figure 2: Recurrence-free survival according to the expression of AR/ERα/ERβ in tumors. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 14155 Table 2: Correlations between AR/ERα/ERβ expression in normal urothelial tissues and clinicopathological features of BC Table 3: Univariate and multivariate analyses for BC recurrence in ADT patients Table 3: Univariate and multivariate analyses for BC recurrence in ADT patients Table 3: Univariate and multivariate analyses for BC recurrence in ADT patients Oncotarget 14156 risk of BC recurrence (5-year actuarial recurrence-free survival: 73% v 57%; P = 0.031, Figure 2A), compared with those with AR-negative tumor. On the other hand, ERα (Figure 2B) or ERβ (Figure 2C) positivity in tumors did not significantly correlate with tumor recurrence. suggesting that ERβ expression is not associated with the preventive effect of ADT but may be an independent prognosticator on BC recurrence. Consistent with our previous observations [7], ERα expression did not predict BC recurrence, suggesting no significant correlation between ERα signals and the ADT effect. To see whether AR expression was an independent predictor of recurrence, multivariate analysis was performed with Cox model, including AR positivity and other well-known clinicopathological risk factors for BC recurrence (Table 3). Of these, AR positivity (HR: 0.27; 95% confidence interval (CI): 0.11-0.67) as well as multiple tumor (HR: 3.12; 95%CI: 1.22-7.95) was found to be an independent prognosticator of BC recurrence. Although a recent review article introduced some clinical trials of new targeted therapies for BC, none of these were approved for BC treatment to date [18]. The authors assumed the lack of patient stratification by each target expression led underestimation of the efficacy of these new therapies. In our previous study [12], although we did not stratify the patients by AR expression, ADT surprisingly prevented BC recurrence with HR of 0.29. The current study then suggests that appropriate patient selection further improves the efficacy of AR inactivation in BC treatment at least for preventing recurrence of NMIBC. Expression of AR, ERα, and ERβ in corresponding normal tissues and tumor recurrence Since de novo tumorigenesis is considered to be one of the mechanisms for BC recurrence, we then investigated associations between AR/ERα/ERβ expression in corresponding normal urothelial tissues and tumor recurrence. However, as shown in Figure 3, any of receptor expression in normal tissues did not significantly correlate with tumor recurrence. For the treatment of prostate cancer, there are two major ways to target AR signals; surgical/chemical castration and anti-androgens. Castration is often associated with reduced health-related quality of life (e.g. fatigue, loss of physical capacity, emotional distress and sexual function) and bone mineral density, compared with anti-androgen monotherapy [19]. Therefore, anti-androgen monotherapy is an alternative for non- metastatic locally advanced prostate cancer, although castration with or without anti-androgen is the standard procedure. Although phase I trial targeting AR in muscle- invasive BC was initiated in the US [20], to the best of our knowledge, prospective studies for NMIBC have not yet been performed. As an adjuvant therapy, anti-androgen monotherapy might be better than castration in terms of adverse effects for the future clinical trials. Furthermore, AR knockdown in urotehlial cells by intravesical instillation of antisense oligonucleotides, as shown with those of heat shock protein 27 in BC [21], is a potential Oncotarget PATIENTS AND METHODS AR expression as a predictor of better prognosis seen in the present study appears to be contrary to the tumor promoting effect of AR signals in BC [4-6]. A potential reason is that AR signals and other pathways complementarily stimulate BC development. Controversial results of an association between AR expression in BCs and their recurrence [7-11] support this hypothesis. If that is the case, when patients receive ADT, BC recurrence is induced by other pathways that are more dominant in AR- negative tumor compared with AR-positive tumor. Tissue samples We retrospectively retrieved 72 BCs and 42 corresponding normal urothelial tissues obtained by transurethral resection from patients who received ADT for their prostate cancer from 2001 to 2012 in four hospitals (Yokohama City University Hospital, Yokohama City University Medical Center, Yokohama Municipal Citizen’s Hospital, and International Goodwill Hospital). Fifteen patients received LHRH monotherapy whereas others received combined androgen blockade consisting of LHRH analogue and anti-androgen. Median duration of ADT was 25.7 (IQR: 9.8-55.1) months. None of these patients received systemic chemotherapy or underwent radical cystectomy. Appropriate approval from the institutional review board at each institution was obtained before use of the tissue samples. A recent retrospective study involving patients who received ADT for prostate cancer showed a significantly lower incidence of subsequent BC development [37]. In addition, a large scale prospective study revealed that finasteride reduced the risk of BC [38]. These clinical findings support the involvement of AR/ERα/ERβ signals in bladder tumorigenesis. Since de novo tumorigenesis might be one of the causes of BC recurrence, we also investigated associations between AR/ERα/ERβ expression in corresponding normal urothelial tissues and tumor recurrence. Since biopsy of normal urothelial tissues was not routinely performed, the sample size was much smaller than that of BC tissues. Consistent with our previous observations [7], there were no statistically significant correlations between each receptor expression in corresponding urothelial tissues and clinicopatholgical features of BC. Furthermore, any of receptor expression in corresponding urothelial tissues did not significantly correlate with tumor recurrence. This is possibly because: 1) tumor dissemination is more dominant way of recurrence; 2) adjacent urothelial tissues are no longer normal; or 3) the sample size is too small. Further investigation using larger sample size is needed to confirm the correlations between the expression of steroid hormone receptors in normal urothelium and BC recurrence. DISCUSSION In the current study, we investigated very specific patients with double primary cancers of the bladder and prostate who received ADT for their prostate cancer and showed that AR positivity in BCs correlated with a reduced risk of tumor recurrence. These findings strongly support that AR signals contributed to the preventive effect of ADT on BC recurrence. On the other hand, similar to the results in patients without ADT [7], ERβ positivity in tumors showed a marginally higher risk of recurrence, Figure 3: Recurrence-free survival according to the expression of AR/ERα/ERβ in normal tissues. ure 3: Recurrence-free survival according to the expression of AR/ERα/ERβ in normal tissues. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 14157 treatment option to avoid systemic adverse effects associated with ADT currently used for the treatment of prostate cancer. i a subset of the patients received ADT for their prostate cancer, a prospective cohort study involving men with double cancers may not be practical. Future clinical trials determining the efficacy of ADT in BC recurrence are still needed to confirm our findings. Recently, some large-scale genome-wide profiling studies proposed new classifications of BC [22-25]. Interestingly, these independent studies showed significant overlaps which improved understanding of molecular features of BC [26]. Among these molecules, epidermal growth factor receptor/ERBB2 [27], Wnt/β-catenin [28], p53 [29], UDP-glucuronosyltransferase-1A [30], CD24 [31], GATA3 [32], and Slug [33] are shown to be activated or inhibited by AR signals in BC or non-neoplastic urothelial cells. In addition, recent studies have revealed the association between AR signals and Nur77 [34], ELK1 [35], or IL-8 [36] in BC. It is thus likely that AR signals contribute to modulating multiple independent pathways in BC cells. CONCLUSIONS We here report the expression of AR, but not ERα or ERβ, in BC is associated with tumor recurrence in patients treated with ADT. Together with our previous observations, ADT with anti-androgen monotherapy and/ or LHRH analogue is suggested to prevent BC recurrence via the AR pathway. In addition, AR expression in BC can be a useful marker in selecting patients for ADT. ACKNOWLEDGMENTS 11. Mir C, Shariat SF, van der Kwast TH, Ashfaq R, Lotan Y, Evans A, Skeldon S, Hanna S, Vajpeyi R, Kuk C, Alkhateeb S, Morote J, van Rhijn BWG, et al. Loss of androgen receptor expression is not associated with pathological stage, grade, gender or outcome in bladder cancer: a large multi-institutional study. BJU Int. 2011; 108: 24-30. We thank Tamiyo Taniguchi and Rie Shimizu for her assistance in tissue preparation. The authors have no conflicts of interest to disclose. The authors have no conflicts of interest to disclose. Statistical analyses 7. Miyamoto H, Yao JL, Chaux A, Zheng Y, Hsu I, Izumi K, Chang C, Messing EM, Netto GJ, Yeh S. Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder. BJU Int. 2012; 109: 1716-26. The Fisher exact test was used to evaluate the association between categorized variables. Nonparametric two group comparison was carried out using Mann- Whitney U test to assess differences in continuous variables. Survival rates in patients were calculated by the Kaplan-Meier method, and comparison was made by log-rank test. The Cox proportional hazards model was used to determine statistical significance of predictors in a multivariate setting. P < 0.05 was considered to be statistically significant. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics. 8. Tuygun C, Kankaya D, Imamoglu A, Sertcelik A, Zengin K, Oktay M, Sertcelik N. Sex-specific hormone receptors in urothelial carcinomas of the human urinary bladder: a comparative analysis of clinicopathological features and survival outcomes according to receptor expression. Urol Oncol. 2011; 29: 43-51. 9. Nam JK, Park SW, Lee SD, Chung MK. Prognostic value of sex-hormone receptor expression in non-muscle invasive bladder cancer. Yonsei Med J. 2014; 55: 1214. 10. Mashhadi R, Pourmand G, Kosari F, Mehrsai A, Salem S, Pourmand MR, Alatab S, Khonsari M, Heydari F, Beladi L, Alizadeh F. Role of steroid hormone receptors in formation and progression of bladder carcinoma: a case-control study. Urol J. 2014; 11: 1968-73. Immunohistochemistry Immunohistochemical staining was performed, as described previously [7]. Sections (4 μm thick) were immunohistochemically labeled, using a primary antibody to AR (N20 clone, dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ERα (E115 clone, dilution 1:100; Epitomics, Burlingame, CA, USA), and ERβ (14C8 clone, dilution 1:50; Abcam, Cambridge, MA, USA). All the stains were manually scored by one pathologist (H.M.) blinded to patient identity. The expression of each receptor was considered positive when more than 10% of urothelial tumor cells or non-neoplastic urothelial cells were immunoreactive or at least 1% of urothelial tumor cells or non-neoplastic urothelial cells showed moderate to strong intensity [7]. The main limitations of the current study include its retrospective design with limited study population. However, since the incidence of primary BC in prostate cancer patients in our cohort was only 1.2% [12] and only www.impactjournals.com/oncotarget Oncotarget 14158 REFERENCES 13. 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https://openalex.org/W4300474224	https://inf.grid.by/jour/article/download/17/19	Russian	null	THE EFFECT OF DISTORTIONS AND FRAGMENTATION ON RESULTS OF CONTENT-BASED RETRIEVAL OF COLOR IMAGES	DOAJ (DOAJ: Directory of Open Access Journals)	2,016	cc-by	4,199	Введение Появившиеся в 1990-х гг. методы и программные средства поиска цветных изображений по содержанию стали достаточно эффективным ответом на вызовы наступающей «цифровой эры» в области съемки, накопления, хранения, поиска, обработки и анализа цифровых изобра- жений и видеоданных [1, 2]. Известные трудности в формировании запроса на поиск в неин- дексированных базах изображений без использования словесных описаний, ключевых слов и каких-либо количественных признаков привели к тому, что одной из популярных парадигм в технологии поиска стал так называемый поиск по образцу, когда запрос формулируется по принципу «найди изображения, наиболее похожие на это». Целью данной работы является экспериментальное исследование влияния на качество поиска цветных изображений таких факторов, как изменение размеров изображений и про- странственные искажения, а также использование фрагментов (частей) изображений в качестве изображений-образцов для поиска. Работа имеет практическую направленность и не претенду- ет на полноту охвата всего спектра возможных причин и параметров, оказывающих влияние на результаты поиска цветных изображений в базах данных по содержанию. Однако предполага- ется, что полученные в результате проведенных вычислительных экспериментов зависимости и количественные оценки могут оказаться полезными для инженеров и программистов, разра- батывающих соответствующие прикладные программные комплексы. УДК 004.9 УДК 004.9 УДК 004.9 В.А. Ковалев ВЛИЯНИЕ ИСКАЖЕНИЙ И ФРАГМЕНТАЦИИ ИЗОБРАЖЕНИЙ-ОБРАЗЦОВ НА КАЧЕСТВО ПОИСКА ЦВЕТНЫХ ИЗОБРАЖЕНИЙ ПО СОДЕРЖАНИЮ Рассматривается проблема поиска по образцу цветных изображений в больших базах данных с использованием дескрипторов, построенных на матрицах совместной встречаемости цветов. Из- вестно, что качество функционирования соответствующих систем поиска изображений зависит не только от используемых методов и алгоритмов, но и от широкого круга различных «шумовых» фак- торов. Приводятся результаты экспериментального исследования влияния на качество поиска таких факторов, как изменение размеров изображений и их пространственное искажение, а также ис- пользование фрагментов исходных изображений в качестве образца поиска. ИНФОРМАТИКА ИНФОРМАТИКА 2015 № 2 № 2 апрель-июнь апрель-июнь 1.2. Способ описания содержания изображений Для описания содержания изображений использовались матрицы совместной встречаемости цветов. Основываясь на общей концепции матриц совместной встречаемости, предложенной в ра- ботах [3, 4], и следуя методике конструирования конкретных их вариантов, изложенной в [4, 5], в качестве дескрипторов изображений были выбраны трехмерные матрицы совместной встречае- мости пар цветов WC типа «цвет – цвет – расстояние», которые могут быть определены как WC = \|\| wc(с(i), с(k), di,k ) \|\|, 2 wc(с(i), с(k), di,k ) = card{ i, kR2 \| ik, di,k=round(d(i, k)), di,k  D, wc(с(i), с(k), di,k ) = card{ i, kR2 \| ik, di,k=round(d(i, k)), di,k  D, xk=(xi+x), yk=(yi+y), D  x  D, 0  y  D, y(D+1)+x > 0 }. =(xi+x), yk=(yi+y), D  x  D, 0  y  D, y(D+1)+x > 0 }. xk=(xi+x), yk=(yi+y), D  x  D, 0  y  D, y(D+1)+x > 0 }. В приведенном определении через (i, k) обозначена произвольная пара пикселов, иденти- фицируемая с помощью их индексов (номеров) i и k, которые расположены на расстоянии di,k друг от друга. Параметры c(i) и c(k) соответствуют цветам этих пикселов, а wc представляет со- бой количество (частоту встречаемости) пикселов с указанными цветами на расстоянии di,k. Весь диапазон RGB-цветов квантуется путем равномерного разбиения на заданное количество интервалов, а межпиксельное расстояние, округляемое до ближайшего целого значения, изме- няется в пределах от 1 до D. Соответственно card обозначает количество, а round – операцию округления до ближайшего целого. Неравенства, представленные в последней строке опреде- ления, формализуют порядок перебора соседей k текущего пиксела i по принципу вперед и вниз от текущего. Дальнейшие детали об алгоритме перебора можно найти в работе [5]. В силу того что при традиционных восьми битах на каждый из RGB-каналов цветовое пространство имеет весьма большие размеры, на практике матрицы встречаемости цветов яв- ляются сильно разряженными, с большим количеством нулевых элементов. Это позволяет ис- пользовать достаточно грубые схемы квантизации с относительно небольшим количеством ин- тервалов цветов. В данной работе использовалась схема с тремя битами на каждый из трех цветовых каналов, т. е. количество интервалов (квантов) цветов равнялось 512. Адаптивные схемы редуцирования цветового пространства не применялись, поскольку используемые изо- бражения не имели доминирующих цветов. Таким образом, элементы матрицы wc описывают «элементарные» цветовые сегменты, их границы и, опосредованно, пространственное располо- жение сегментов на изображении. 1.1. Тестовая база изображений Технические характеристики. Тестовая база состояла из 10 000 цветных (RGB) изобра- жений относительно небольших размеров, имеющих прямоугольную форму с горизонтальной или вертикальной ориентацией. Горизонтальные размеры варьировали в пределах от 222 до 512 пикселов, а вертикальные – в пределах от 164 до 382. Средний размер (площадь) изображе- ний был эквивалентен изображению квадратной формы размером 350×350 пикселов. Содержание изображений. Используемая база изображений создавалась сотрудниками Фраунхоферовского института компьютерной графики (Германия) с целью тестирования эф- фективности разрабатываемых методов, алгоритмов и программных средств поиска изображе- ний по содержанию общего характера. Для обеспечения полноценного тестирования содержа- ние изображений не ограничивалось какой-либо предметной областью или тематикой. Кроме того, тестовые изображения не имели никакой специфики относительно предпочтительной цве- товой гаммы, наличия только реальных или только искусственных объектов, использования стандартизованных условий съемки, доминирования определенных текстурных свойств, объек- тов какой-либо определенной формы и т. п. Используя бытовую лексику, содержание тестовой базы может быть охарактеризовано как «изображения всего чего угодно». ВЛИЯНИЕ ИСКАЖЕНИЙ И ФРАГМЕНТАЦИИ ИЗОБРАЖЕНИЙ-ОБРАЗЦОВ 32 1.2. Способ описания содержания изображений На примере базы данных из 20 000 изображений было экспе- риментально показано [6], что такое представление является достаточно гибким и одинаково хорошо подходит для описания широкого класса сцен, начиная от простых комбинаций цвет- ных объектов на однородном фоне и заканчивая высокочастотными цветными текстурами на- турального происхождения. Важно отметить, что при вычислении матриц совместной встречаемости рассматриваются все возможные пары в локальной окрестности каждого текущего пиксела. При этом пары с одни- ми и теми же цветами, находящиеся на одних и тех же расстояниях, считаются идентичными не- зависимо от порядка встречаемости цветов при переборе пикселов изображения. Фактически это означает, что для каждого из расстояний соответствующий слой результирующего трехмерного массива частот встречаемости представляет собой нижнюю треугольную матрицу. Завершающим шагом вычисления матриц является их нормализация, выполняемая путем деления всех элемен- тов на их сумму. Перечисленные особенности алгоритма вычисления матриц приводят к тому, что дескрипторы совместной встречаемости обладают следующими важными свойствами: а) инвариантность по отношению к таким преобразованиям изображений, как поворот на угол, кратный 90, и зеркальное отражение (из-за перебора всех возможных пар пикселов); а) инвариантность по отношению к таким преобразованиям изображений, как поворот на угол, кратный 90, и зеркальное отражение (из-за перебора всех возможных пар пикселов); б) сдвиг объектов изображений по однородному фону (из-за отсутствия координатной привязки); в) относительная независимость от размера т е возможность сравнивать содержание б) сдвиг объектов изображений по однородному фону (из-за отсутствия координатной привязки); в) относительная независимость от размера, т. е. возможность сравнивать содержание изображений разного размера и формы (из-за нормализации матриц); ) д р д р д у ф у ( у рд р в) относительная независимость от размера, т. е. возможность сравнивать соде изображений разного размера и формы (из-за нормализации матриц); г) возможность поиска по фрагментам изображений, что обусловлено такими факторами, как нормализация матриц и использование метрики L1, в которой вычисляется разность одно- именных элементов матриц, т. е. относительных частот встречаемости некоторых цветов, пред- ставленных как на материнском изображении, так и на его фрагментах. г) возможность поиска по фрагментам изображений, что обусловлено такими факторами, как нормализация матриц и использование метрики L1, в которой вычисляется разность одно- именных элементов матриц, т. е. относительных частот встречаемости некоторых цветов, пред- ставленных как на материнском изображении, так и на его фрагментах. 33 В.А. 1.2. Способ описания содержания изображений КОВАЛЕВ На заключительном этапе вычисления дескриптора каждого изображения набор ненуле- вых элементов соответствующей матрицы совместной встречаемости представляется в виде списка (<номер элемента матрицы>, <значение>) и записывается в файл дескрипторов в поряд- ке возрастания номеров элементов, что обеспечивает их быстрый перебор при сравнении. Сле- дует отметить, что на практике обычно игнорируются не только элементы матрицы, которые строго равны нулю, но и все «шумовые» элементы, значение которых меньше некоторого (дос- таточно малого) порога. Данный прием позволяет существенно сократить количество элемен- тов, включаемых в дескриптор, что значительно уменьшает его размер и повышает скорость работы системы поиска изображений при условии обеспечения практически тех же самых ре- зультатов поиска. Так, например, при установке минимального значения порога, эквивалентно- го 0,2 % от площади изображения, среднее значение количества элементов матрицы, включае- мых в дескриптор, равно 66 [6]. 1.3. Сравнение дескрипторов изображений Дескрипторы изображений, т. е. множества ненулевых элементов дескриптора изображе- ния-образца поиска и каждого из дескрипторов базы данных, сравнивались с помощью метрики L1 [7], т. е. степень различия между изображениями R вычислялась как нормализованная сумма абсолютных значений разностей однотипных элементов сравниваемых дескрипторов. При от- сутствии какого-либо элемента в дескрипторе его значение полагалось равным нулю. Соответ- ственно степень близости изображений в пространстве признаков (степень сходства S) вычис- лялась как S = 1 – R. Следуя сложившейся традиции, для количественной оценки результатов поиска выводились и использовались только первые N = 20 изображений, наиболее близких к заданному образцу. ВЛИЯНИЕ ИСКАЖЕНИЙ И ФРАГМЕНТАЦИИ ИЗОБРАЖЕНИЙ-ОБРАЗЦОВ ВЛИЯНИЕ ИСКАЖЕНИЙ И ФРАГМЕНТАЦИИ ИЗОБРАЖЕНИЙ-ОБРАЗЦОВ 34 Следует особо подчеркнуть, что при визуальной оценке результатов поиска похожих изо- бражений в базах данных пользователи подобных сервисов интуитивно ожидают увидеть среди N наиболее близких изображений действительно «очень похожие» (в соответствии с их индивиду- альными и, надо сказать, весьма различными представлениями о «похожести»). Вместе с тем очевидно, что в результате поиска в N наиболее близких будут включены только изображения из числа фактически присутствующих в используемой базе изображений. Соответственно в общем случае (и особенно в случае небольших баз изображений) совсем не обязательно, что они будут действительно «очень похожи» на заданный образец и будут отвечать ожиданиям пользователя. 2.1. Типичные результаты поиска На рис. 1 показаны примеры образцов поиска и N = 3 наиболее близких результатов, найден- ных в тестовой базе из 10 000 изображений. Последняя колонка иллюстрирует инвариантность ре- зультатов поиска по отношению к таким преобразованиям, как зеркальное отражение и поворот. а) б) Рис. 1. Изображения-образцы (а) и N = 3 наиболее похожих результата поиска для каждого из них вместе с соответствующей количественной мерой сходства S (б) Образец 1 Образец 2 Образец 3 Образец 4 S = 0,724 S = 0,773 S = 0,645 S = 1,000 S = 0,550 S = 0,592 S = 0,626 S = 1,000 S = 0,518 S = 0,663 S = 0,608 S = 0,782 а) б) Рис. 1. Изображения-образцы (а) и N = 3 наиболее похожих результата поиска для каждого из них вместе с соответствующей количественной мерой сходства S (б) 2.2. Влияние пространственных искажений на результаты поиска красную стрелку на рис. 3). Результаты эксперимента по- казаны на рис. 4 кривой красного цвета. 2. Поиск по искаженному образцу. В качестве образца поиска использовался дескриптор искаженного изображения, в то время как поиск наиболее похожих осуществлялся среди исход- ных, неискаженных изображений (см. красную стрелку на рис. 3). Результаты эксперимента по- казаны на рис. 4 кривой красного цвета. 3. Поиск в искаженной базе. Данный эксперимент являлся противоположным экспери- менту 2, т. е. в качестве образца поиска использовался дескриптор исходного изображения, а поиск осуществлялся среди искаженных изображений (см. стрелку зеленого цвета на рис. 3 и зеленую кривую на рис. 4, представляющую точность получаемых результатов). 3. Поиск в искаженной базе. Данный эксперимент являлся противоположным экспери- менту 2, т. е. в качестве образца поиска использовался дескриптор исходного изображения, а поиск осуществлялся среди искаженных изображений (см. стрелку зеленого цвета на рис. 3 и зеленую кривую на рис. 4, представляющую точность получаемых результатов). Проведенные эксперименты показали, что поиск по искаженному образцу среди иска- женных же изображений обеспечивает более высокую воспроизводимость результатов (в среднем на 7–9 % лучше) по сравнению с гибридными схемами, использующими исходные и искаженные изображения одновременно. В частности, для 10 000 запросов на поиск по об- разцу среднее количество корректных результатов среди N = 20 наиболее похожих изображе- ний достигает 87,4 %. 2.2. Влияние пространственных искажений на результаты поиска В качестве типичного примера пространственных искажений изображений в соответст- вующей серии экспериментов была использована операция их редуцирования к некоторому «стандартизованному» размеру: до небольших, в нашем случае 128×128 пикселов, миниатюр (preview), которые часто используются в интерфейсных целях в современных компьютеризиро- ванных сервисах (рис. 2). При этом информация об исходной портретной или пейзажной форме исходных изображений во внимание не принималась. Кроме приведенного варианта простран- ственных искажений в литературе описываются и такие важные случаи, как искажения изобра- жений, связанные с различиями в освещении сцены [8], и некоторые другие. Однако, по мне- нию автора, в данной работе рассматривается гораздо более массовый и понятный способ пространственного искажения изображений из числа встречающихся на практике. Рис. 2. Примеры исходных изображений и их искаженных (уменьшенных до 128×128 пикселов) версий Рис. 2. Примеры исходных изображений и их искаженных (уменьшенных до 128×128 пикселов) версий Схема вычислительных экспериментов по оценке влияния пространственных искажений на результаты поиска представлена на рис. 3, а их результаты – на рис. 4. Процесс тестировани включал серию из трех независимых экспериментов, описанных ниже. р р р 1. Раздельный поиск. Каждое изображение исходной базы данных подавалось на вход программы в качестве образца поиска, и идентификаторы получаемых N = 20 наиболее близких результатов поиска запоминались в качестве «правильных». Та же самая процедура независимо выполнялась над искаженными версиями изображений, и результаты поиска по «искаженной» базе данных сравнивались с результатами, полученными при поиске исходных изображений. Количественная оценка воспроизводимости результатов среди ближайших 4, 8, 12, 16 и 20 наи- более похожих ответов показана на рис. 4 с помощью кривой синего цвета. 35 В.А. КОВАЛЕВ Рис. 3. Общая схема экспериментов по оценке влияния пространственных искажений на качество поиска Уменьшение до 128×128 Сравнение Сравнение Уменьшение до 128×128 Рис. 3. Общая схема экспериментов по оценке влияния пространственных искажений на качество поиска Рис. 4. Графики правильных результатов поиска (в процентах) среди N = 4, 8, 12, 16 и 20 изображений, ближайших к заданному изображению-образцу Правильные результаты, % Результат среди ближайших N Правильные результаты, % Результат среди ближайших N Результат среди ближайших N Рис. 4. Графики правильных результатов поиска (в процентах) среди N = 4, 8, 12, 16 и 20 изображений, ближайших к заданному изображению-образцу Рис. 4. Графики правильных результатов поиска (в процентах) среди N = 4, 8, 12, 16 и 20 изображений, ближайших к заданному изображению-образцу 2. Поиск по искаженному образцу. В качестве образца поиска использовался дескриптор искаженного изображения, в то время как поиск наиболее похожих осуществлялся среди исход- ных, неискаженных изображений (см. 2.3. Поиск по фрагментам изображений На практике довольно часто встречаются ситуации, когда используется не полное исход- ное цифровое изображение, а тот или иной его фрагмент. Более того, в сети Интернет нередко ВЛИЯНИЕ ИСКАЖЕНИЙ И ФРАГМЕНТАЦИИ ИЗОБРАЖЕНИЙ-ОБРАЗЦОВ 36 можно наблюдать случаи, когда некоторая удачная иллюстрация или ее часть заимствуются различными авторами без ссылки и без разрешения ее автора (правообладателя). Поэтому воз- можность автоматического поиска изображений по их фрагментам представляется весьма по- лезной вне зависимости от того, идет ли речь об обнаружении некорректных заимствований или об обычном использовании свободных мультимедийных ресурсов. Следует подчеркнуть, что в отличие от известных работ по проблеме поиска изображе- ний по фрагментам (см., например, [9, 10]), которые посвящены повышению качества поиска за счет использования некоторых специфических фрагментов изображений, в данной работе про- блема выбора каких бы то ни было «характерных» областей (участков, фрагментов) не рас- сматривается. Вместо этого фрагменты различного размера выбираются вслепую, случайным образом, без оценки их информативности и важности относительно других. Таким образом, данный подраздел посвящен экспериментальной оценке эффективности решения задачи поиска исходных изображений по образцу на основе дескрипторов совместной встречаемости в условиях, когда в качестве образца поиска выступает некоторый фрагмент ис- ходного изображения. Естественно, что при этом дается только базовая количественная оценка потенциальной возможности решения подобных задач без рассмотрения всей технологии поис- ка дубликатов в архивах изображений или в сети Интернет, а также других родственных задач. у р р р ру р В рассматриваемом случае экспериментальное исследование потребовало довольно зна чительных вычислительных затрат и состояло из следующих основных этапов: В рассматриваемом случае экспериментальное исследование потребовало довольно зна- чительных вычислительных затрат и состояло из следующих основных этапов: 1. Подготовка фрагментов изображений. Размеры фрагментов изображений варьирова- лись в широком диапазоне от 50 до 1,56 % от площади исходного изображения, включая сле- дующие пять значений: 50; 25; 15; 6,25 и 1,56 %. Для каждого размера и каждого изображения базы данных в позиции, выбираемой на изображении случайным образом, вырезались 10 фраг- ментов, т. е. брались всего 50 фрагментов с каждого изображения или в общей сложности 500 000 фрагментов из 10 000 тестовых изображений используемой базы данных. Типичные примеры фрагментов показаны на рис. 5, а. 100 % а) б) в) Рис. 5. 2.3. Поиск по фрагментам изображений Фрагменты изображений различных размеров (а); количество случаев (в процентах), когда искомое изображение было найдено среди N ближайших (б) либо среди всех N = 20 (в) 100 % 50 % 25 % 15 % 6,25 % 1,56 % Искомое изображение среди N ближайших Правильные результаты среди N ближайших, % Правильные результаты, % Площадь изображения-образца, % 50 % 25 % 15 % 6,25 % 1,56 % 25 % 15 % 6,25 % 1,56 % 50 % а) Правильные результаты, % Площадь изображения-образца, % Искомое изображение среди N ближайших среди N ближайших, % Правильные результаты среди N ближайших, % б) в) Рис. 5. Фрагменты изображений различных размеров (а); количество случаев (в процентах), когда искомое изображение было найдено среди N ближайших (б) либо среди всех N = 20 (в) 37 В.А. КОВАЛЕВ 2. Вычисление дескрипторов изображений. Как уже упоминалось выше, все дескрипторы (трехмерные гистограммы частоты совместной встречаемости) нормализовывались, чтобы из- бежать зависимости от размеров изображений. р р р 3. Собственно проведение поиска по фрагментам. Данный этап выполнялся в виде пяти серий отдельных экспериментов, по одной серии для каждого размера фрагментов. Каждый из 100 000 фрагментов подавался в качестве образца поиска и ближайшие N = 20 результатов за- поминались. Таким образом, в каждой из пяти серий задача сравнения дескриптора фрагмента- образца с дескрипторами 10 000 исходных изображений решалась 100 000 раз путем выполне- ния 109 сравнений. 3. Собственно проведение поиска по фрагментам. Данный этап выполнялся в виде пяти серий отдельных экспериментов, по одной серии для каждого размера фрагментов. Каждый из 100 000 фрагментов подавался в качестве образца поиска и ближайшие N = 20 результатов за- поминались. Таким образом, в каждой из пяти серий задача сравнения дескриптора фрагмента- образца с дескрипторами 10 000 исходных изображений решалась 100 000 раз путем выполне- ния 109 сравнений. 4. Анализ результатов. Исходное изображение считалось найденным по заданному фраг- менту, если оно появлялось на каком-либо месте среди N = 20 изображений, наиболее близких к заданному образцу. Графики, представленные на рис. 5, б, иллюстрируют процент случаев, когда искомое исходное изображение находилось на первом месте, а также среди ближайших 5, 10, 15 или 20 изображений, являющихся результатом поиска. Кроме того, для удобства процент кор- ректных результатов поиска среди N = 20 ближайших изображений как функция размера фраг- мента-образца показан отдельно на рис. 5, в. Следует отметить, что в представленных данных вероятность случайного появления искомого изображения среди наиболее близких результатов поиска не учитывалась в силу ее малости. 2.3. Поиск по фрагментам изображений Как и следовало ожидать, доля корректных результатов поиска исходного изображения зависит от размера фрагмента, используемого в качестве образца. Так, например, при размере фрагмента, составляющем 50 % площади материнского изображения, оно находится в 100 % случаев, причем в подавляющем большинстве из них материнское изображение оказывается на первом месте (см. графики синего цвета на рис. 5). С другой стороны, что довольно неожидан- но, использование в качестве образца фрагментов весьма малого размера, составляющих лишь 1,56 % от площади исходного изображения, тем не менее позволяет найти исходное изображе- ние среди N = 20 результатов в 39,5 % случаев. При этом в 12 % случаев оно оказывается на первом месте (см. кривую черного цвета на рис. 5). Список литературы Liao, C.-J. Complementary retrieval for distorted images / C.-J. Liao, S.-Y. Chen // Pattern tion. – 2002. – Vol. 35. – P. 1705–1722. 8. Liao, C.-J. Complementary retrieval for distorted images / C.-J. Liao, S.-Y. Chen // Recognition. – 2002. – Vol. 35. – P. 1705–1722. g 9. A scheme of fragment-based faceted image search / T. Komamizu [et al.] // Database and Expert Sytems Applications. – 2012. – Vol. 7447. – P. 450–457. 9. A scheme of fragment-based faceted image search / T. Komamizu [et al.] // Database and Expert Sytems Applications. – 2012. – Vol. 7447. – P. 450–457. 10. Li, J. IRM: Integrated region matching for image retrieval / J. Li, J.Z. Wang, G. Wiederhold // Proc. of the 2000 ACM Multimedia Conf. – Los Angeles, 2000. – P. 83–86. 10. Li, J. IRM: Integrated region matching for image retrieval / J. Li, J.Z. Wang, G. Wiederhold // Proc. of the 2000 ACM Multimedia Conf. – Los Angeles, 2000. – P. 83–86. Поступила 08.04.2015 Объединенный институт проблем информатики НАН Беларуси, Минск, Сурганова, 6 e-mail: vassili.kovalev@gmail.com Заключение Экспериментальные результаты, полученные на тестовой базе из 10 000 цветных изображе- ний общего характера, позволяют сделать следующие выводы. Пространственное искажение цветных изображений путем приведения их к миниатюрам стандартизованного размера в 128×128 пикселов приводит к тому, что результирующий набор из N = 20 изображений, ближайших к заданному образцу поиска, может содержать порядка 13–20 % ошибок. В частности, наихудший результат с 20 % ошибок (соответственно точность 80 %) полу- чается при «смешанной схеме», т. е. при поиске по неискаженному образцу в искаженной базе изображений. Противоположная смешанная схема, т. е. поиск по искаженному образцу в исход- ной (неискаженной) базе изображений, занимает среднее, второе место с показателем качества 18,9 % ошибок (точность 81,1 %). Наилучшие результаты (12,6 % ошибок и, соответственно, точ- ность 87,4 %) получаются при «однородной» схеме, т. е. при поиске по искаженному изображе- нию-образцу в искаженной базе изображений. При поиске по фрагменту изображения его оригинала результаты в существенной степе- ни зависят от размера используемого фрагмента. При размере фрагмента, составляющем 50 % от площади исходного изображения, материнское изображение находится среди N = 20 наибо- лее похожих всегда (100 %), причем в подавляющем большинстве случаев (99,3 %) оно оказы- вается на первом месте. При уменьшении фрагмента-образца поиска точность поиска падает. Однако даже при использовании фрагментов, составляющих менее 2 % от площади исходного изображения, рассматриваемый метод обеспечивает автоматический поиск исходного изобра- жения почти в 40 % случаев. Несмотря на то что рассматриваемые дескрипторы в принципе могут использоваться для описания структуры и поиска в базах данных полутоновых изображений, качество их поиска бу- дет сравнительно невысоким. Причина этого заключается в том, что для покрытия всего цветово- го пространства рассматриваемые матрицы совместной встречаемости используют достаточно ВЛИЯНИЕ ИСКАЖЕНИЙ И ФРАГМЕНТАЦИИ ИЗОБРАЖЕНИЙ-ОБРАЗЦОВ 38 грубую схему его разбивки на 888 = 512 цветов. Учитывая, что гамме серого цвета в простран- стве RGB соответствуют цвета с равными цветовыми компонентами r = g = b, максимальное ко- личество различимых уровней серого будет равно восьми. Этого может быть недостаточно для целого ряда задач хранения и поиска полутоновых изображений по содержанию. Более подроб- ные сведения о дескрипторах полутоновых изображений можно найти в [4]. Автор выражает глубокую благодарность доктору Штефану Волмеру и профессору Геор- гиусу Сакасу за предоставленную базу изображений и плодотворные дискуссии по проблеме поиска изображений по содержанию. Список литературы 1. Content-based image retrieval at the end of the early years / A.W.M. Smeulders [et al.] // IEEE Transactions on PAMI. – 2000. – Vol. 22(2). – P. 1349–1380. 1. Content-based image retrieval at the end of the early years / A.W.M. Smeulders [et al.] // IEEE Transactions on PAMI. – 2000. – Vol. 22(2). – P. 1349–1380. ( ) 2. A survey of content-based image retrieval with high-level semantics / Y. Liu [et al.] // Pat- tern Recognition. – 2007. – Vol. 40(1). – P. 262–282. 2. A survey of content-based image retrieval with high-level semantics / Y. Liu [et al.] // Pat- tern Recognition. – 2007. – Vol. 40(1). – P. 262–282. g 3. Kovalev, V.A. Feature extraction and visualization methods based on image class compari- son / V.A. Kovalev // Medical Imaging 1994 Intern. Symp., Image Processing. – 1994. – Vol. 2167. – P. 691–701. 3. Kovalev, V.A. Feature extraction and visualization methods based on image class compari- son / V.A. Kovalev // Medical Imaging 1994 Intern. Symp., Image Processing. – 1994. – Vol. 2167. – P. 691–701. 4. Kovalev, V.A. Multidimensional сo-occurrence matrices for object recognition and matching / V.A. Kovalev, M. Petrou // Graphical Models and Image Processing. – 1996. – Vol. 58, № 3. – P. 187–197. 4. Kovalev, V.A. Multidimensional сo-occurrence matrices for object recognition and matching / V.A. Kovalev, M. Petrou // Graphical Models and Image Processing. – 1996. – Vol. 58, № 3. – P. 187–197. 5. Ковалев, В.А. Анализ текстуры трехмерных медицинских изображений / В.А. Ковалев. – Минск : Белорус. наука, 2008. − 264 с. 5. Ковалев, В.А. Анализ текстуры трехмерных медицинских изображений / В.А. Ковалев. – Минск : Белорус. наука, 2008. − 264 с. 6. Kovalev, V.А. Color co-occurrence descriptors for querying-by-example / V.А. Kovalev, S. Volmer // Intern. Conf. on Multimedia Modelling. – Lausanne, 1998. – P. 32–38. 6. Kovalev, V.А. Color co-occurrence descriptors for querying-by-example / V.А. Kovalev, S. Volmer // Intern. Conf. on Multimedia Modelling. – Lausanne, 1998. – P. 32–38. 7. Ковалев, В.А. Влияние мер близости в пространстве признаков на качество поиска ме- дицинских изображений по содержанию / В.А. Ковалев, А.А. Дмитрук // Информатика. – 2011. – № 2(30). – С. 5–11. 7. Ковалев, В.А. Влияние мер близости в пространстве признаков на качество поиска ме- дицинских изображений по содержанию / В.А. Ковалев, А.А. Дмитрук // Информатика. – 2011. – № 2(30). – С. 5–11. V.A. Kovalev THE EFFECT OF DISTORTIONS AND FRAGMENTATION ON RESULTS OF CONTENT-BASED RETRIEVAL OF COLOR IMAGES This paper is dealing with the problem of content-based image retrieval using color co-occurrence matrices. It is well-known that there are a number of factors influencing the results of image retrieval. The specific contribution of this paper lies on quantitative assessment of the effect of spatial image distortions as well as on estimating the accuracy of retrieving original images using its fragments of different size as query examples. The presented study is capitalized on database containing 10 000 color RGB images of very different content. The necessary experimental results are provided.
https://openalex.org/W3175610977	https://www.e3s-conferences.org/10.1051/e3sconf/202127403003/pdf	English	null	Hydraulic transport in small livestock farms	E3S web of conferences	2,021	cc-by	3,703	* Corresponding author: rahmat1959@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Hydraulic transport in small livestock farms Оktyabr Rakhimov1*, Laylo Аshurova1, and Fotima Artikbekova2 1Karshi engineering-economics institute, Karshi, Uzbekistan 2Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, Tashkent, Uzbekistan Abstract. The article highlights the advantages and benefits of pipeline transport of liquid feed mixtures on small pig farms, the device and the principle of operation and technical characteristics of the improved rotor pump developed by the authors of the article. Methods of experimental research to determine the concentration of feed mixtures, the influence of the shape of the separator on the performance characteristics, the required power and the flow of an improved pump are presented. The results of the experiments showed that one of the bottlenecks limiting the operation of rotary pumps on feed mixtures of increased concentration is the low suction capacity of the pump, that is, the design of the suction pipe of the pump, where an increase in resistance with an increase in the concentration of feed mixtures causes a breakdown of the operating mode and cavitation. To resolve this issue, we used a screw feeder installed in front of the pump suction nozzles. It was found that if the screw feeder provides excess pressure within (about, 0.25 ... 0.3) 105 Pa per suction cavity, the pump operates stably and reliably. Keywords: centrifugal fecal pumps, concentration, volumetric methods, water flow. E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 https://doi.org/10.1051/e3sconf/202127403003 1 Introduction Modern pig farming is the world's leading livestock industry; occupying a leading position in the 'meat' balance. In the structure of world meat production, pork ranks first (38.739.7 %). In Asia, the share of pork in the production of all types of meat is 54.6 %, in Europe 51.0 %, in Russia 32.4 % [1]. The organization of productive, cost-effective pig breeding on small farms can be successfully solved if it is carried out at a high technical level and with a rational animal feeding. Different types of feed consistencies have their own characteristics. Dry food does not require additional preparation, it is convenient for distribution, but it increases the dustiness of the air in the pigsty and causes respiratory diseases in pigs. In addition, when the animals are fed only with dry compound feeds, their digestibility decreases by 4 .. 6 % compared to the use of wet feed mixtures [2-5]. Dry food is the most efficient, but wet feeding is more effective. That is why, in order to preserve technical means and reduce labor costs for maintaining hygiene, you should use dry food, moistened immediately before feeding the animals [6-8]. E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 https://doi.org/10.1051/e3sconf/202127403003 Feeding with liquid feed has a number of advantages: the time for eating the feed is reduced, the loss of feed is reduced, it becomes possible to use finely ground feed, the uniformity of feed and distribution to the feeders is maintained. With a decrease in eatability in sows, the feeding method allows, which is very important, to regulate the nutritional value of the feed in the same volume of bark mixture [9-11]. Feeding with liquid feed also makes it possible to use one of the most economical and reliable methods of transporting feed - hydromechanical, that is, a method when feed is fed into the feeders through pipes in certain doses. Pipeline transport is highly productive, simple in structure, it can be mounted in buildings of any type, it does not require special aft passages, and it increases work culture [12, 13]. The use of pipeline transport for the transportation and distribution of feed mixtures on pig farms requires the use of special feed-transporting pumps with high reliability. Currently, the industry does not produce special pumps for supplying rational liquid feed mixtures, and the use of pumps from other industries does not give effective results. 2 Methods The design of the pump used for pumping liquid feed mixtures was determined by its rheological properties. Depending mainly on the concentration of the dry component. Concentrations of feed mixtures were determined by taking feed mixtures of a known volume from a mixer, followed by removal of moisture, drying and weighing the dry residue. To determine the effect of the shape of the spacer on the performance, the pump is equipped with three replaceable flat, convex and wedge shaped spacers. An IS-203.4 sump was used to determine the required power. The pump flow was determined by volumetric methods using a measuring tank. 1 Introduction Currently, on farms and complexes, the previously produced centrifugal fecal pumps of the 21/2 NF, 4 NF, 6NF types, the NPG-2, NPG-3, 4PS-9 sand pumps, 8FS-17 mixing pumps, and the S-296 piston concrete pumps are widely used, gear pumps D-159-V, screw pumps VNM-18-2, VNM-15-100 and other pumps. But, as noted above, all these pumps are not intended for feeding feed mixtures and they require structural changes, and also, the rotary pump of the Kamur company (Sweden) has become of widespread use while pumping various high-viscosity slurries [14, 15]. Therefore, many farms use various converted and homemade pumps. Therefore, the development of a pump used for feeding feed mixtures with low moisture content is very important. Based on this, the purpose of the study is to develop a new energy-saving design of a rotary pump that allows transporting feed mixtures of increased concentration and low humidity. 3 Results and discussion Based on the analysis of the designs and performance of the pumps used for the supply of viscous slurries, as well as the requirements imposed on them and taking into account the preliminary expirations, the design and technological scheme of a two-rotor rotary pump was adopted as an object of research [16-19], and a rotary pump with a screw feeder was developed for supplying liquid feed mixtures by pipe distributor. 2 2 E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 https://doi.org/10.1051/e3sconf/202127403003 Fig. 1. The proposed scheme of a rotary pump with a screw feeder: 1 – case; 2 – blade; 3 – shaft; 4 – rotor; 5 – additional chamber; 6 – stream splitter; 7 – screw feeder; 8 – loading mouth. Fig. 1. The proposed scheme of a rotary pump with a screw feeder: 1 – case; 2 – blade; 3 – shaft; 4 – rotor; 5 – additional chamber; 6 – stream splitter; 7 – screw feeder; 8 – loading mouth. The pump consists of an assembled casing 1, in which two collapsible rotors 4 rotate in opposite directions. The rotors are rigidly connected to the shaft 3, which is mounted on ball bearings in lateral struts connected to the casing by bolts. One of the shafts (rotors) is connected to the motor through a gearbox and is driven. The second shaft (rotor) is driven with a coordinated rotation from the drive shaft using gear wheels, the gear ratio of which is equal to one. The gears are housed in an oil bath chamber. On each of the rotors 4, five blades 2 are fixed, located at equal distances around the circumference. When the rotors turn, they enter special recesses, thereby ensuring separation of the high pressure zone from the low pressure zone. Elimination of leakage into the surrounding seat and prevention of the ingress of feed mixtures into the oil bath and bearings is ensured by gland seals. The working part of the pump consists of two modular rotors, which rotate with the same angular velocity, but in opposite directions. The consistency of the movement of the rotors is ensured by means of a gear train with the number i = 1, placed outside the working chamber. The displacers are special blades 2 mounted on the rotor. 3 Results and discussion Displacers, they are also cut-off devices, one by one enter the receiving chamber, cut off certain volumes of mass when interacting with the body and move them into the pressure chamber. After the cut-off volume is moved to the pressure chamber, the displacers return again to the receiving chamber to repeat the cycle, ensuring closure and minimal mass transfer from the pressure chamber to the suction chamber. In addition, the pump is equipped with a flow divider 5, which includes additional resistance to rotation of the pump rotor due to the effect of the supplied mass. It is installed on the pump inlet and serves to change the direction of flow in the direction of the rotor rotation. At the same time, the supplied mass does not create braking resistance forces and gives up its kinetic energy, acquired from the screw feeder, to the rotor, thereby reducing the required pump power. In addition, the spacer 5 is made with an adjustable angle between the edges. It is advisable to install a flow divider in the additional pump chamber 6. The results of the experiments showed that one of the bottlenecks limiting the operation of rotary pumps on feed mixtures of increased concentration is the low suction capacity of the pump, that is, the design of the suction pipe of the pump, where an increase in resistance with an increase in the concentration of feed mixtures causes a breakdown of the 3 E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 https://doi.org/10.1051/e3sconf/202127403003 operating mode and cavitation. When cavitation occurs, the pumps are unstable, there is a sharp drop in head and flow. To solve this issue, we used a screw feeder installed in front of the pump suction nozzles. operating mode and cavitation. When cavitation occurs, the pumps are unstable, there is a sharp drop in head and flow. To solve this issue, we used a screw feeder installed in front of the pump suction nozzles. The main reason for the deterioration of the pump performance when feeding a feed mixture with low moisture content is a decrease in the speed of the feed mixture in the suction pipe of the pump. 3 Results and discussion When testing the pump, the maximum volumetric efficiency is (µ = 0.79) when pumping feed families with a moisture content of 68 % with a pressure in the discharge line of 0.45 MPa was obtained at an average speed of the feed mixture in the suction pipe of the pump equal to vср = 0.42 m/s (Figure 2.) The change in the flow rate Qн, the required power N and the efficiency of the pump µ depending on the pressure in the discharge line at a constant rotor speed is graphically shown in Figs 3, 4. Fig. 2. Dependence of the flow rate (Qн) of the pump on the speed of feed mixtures (v) in the suction pipe of the pump. Fig. 2. Dependence of the flow rate (Qн) of the pump on the speed of feed mixtures (v) in the suction pipe of the pump Fig. 2. Dependence of the flow rate (Qн) of the pump on the speed of feed mixtures (v) in the suction pipe of the pump. Fig. 3. Features of a rotary pump with a screw feeder while feeding mixtures with grass paste with a moisture content of 72.8 %. Fig 3 Features of a rotary pump with a screw feeder while feeding mixtures with grass paste with a Fig. 3. Features of a rotary pump with a screw feeder while feeding mixtures with grass paste with a moisture content of 72.8 %. 4 https://doi.org/10.1051/e3sconf/202127403003 E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 Fig. 4. Characteristics of a rotary pump when feeding concentrate feed mixtures with a moisture content of 68 %. Fig. 4. Characteristics of a rotary pump when feeding concentrate feed mixtures with a moisture content of 68 % Fig. 4. Characteristics of a rotary pump when feeding concentrate feed mixtures with a moisture content of 68 %. The study shows that the speed of the feed mixture in the suction cavity of the pump when feeding the feed mixture with a moisture content of 68 % is 0.12 m / s, and the pump flow rate is 21.5 м3/h, that is, 26 % lower than the feed mixture with a moisture content of 80 %. To improve the operation of the pump when feeding feed mixtures with low humidity, additional backwater is required in front of the suction nozzles of the pump, which can be created using a screw feeder. 3 Results and discussion It has been established that the value of the feed mixture velocity in the suction line depends on the productivity of the screw feeder and the cross- sectional area of the pump inlet pipe. The productivity of the screw feeder was regulated by changing the rotational speed of its screw using replaceable sprockets. It was found that if the screw feeder provides excess pressure within (about, 25 ... 0.3) 105 Pa per suction cavity, the pump works stably and reliably. The application of this improved pump in farm production requires careful learning. This requires the use of the most innovative [20, 21] and modern pedagogical [22] technologies of teaching, online training of farmers and telecommunication training projects [23]. In such teaching, lectures are not in a simple traditional form but are problematic and modern lectures [24-26] should be conducted using interactive methods, ensuring the activity of the audience. At the same time, of course, it is important to address the audience with problematic questions during the lesson, to direct them to creative and scientific thinking by creating problematic situations in the lecture. In addition, the creation of online training courses, including the use of technologies such as project style [27], scientific and creative thinking, enriching the online course module platform with electronic resources [28], including video and audio lectures, gives great results. In short, there is a need to pay more attention to education in the use of improved pumps on farms. It is no coincidence that the quality of education is the quality of life [29-34]. 4 Conclusions 1. Our preliminary studies of this pump showed that a rotary pump of this design works stably at a feed mixture moisture content of 70 % or more. However, the introduction of grass paste or juicy components into the feed mixture sharply increases its viscosity, concentration and at the same time reduces the fluidity of the feed mixture by 10 times or more. In this case, the performance of the pump deteriorates, that is, the flow and head of 5 5 E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 https://doi.org/10.1051/e3sconf/202127403003 the pump decrease. To ensure reliable transportation of feed mixtures of low moisture content with green paste, it is necessary to use forced feeding. For this, a screw feeder 7 was installed at the pump inlet. the pump decrease. To ensure reliable transportation of feed mixtures of low moisture content with green paste, it is necessary to use forced feeding. For this, a screw feeder 7 was installed at the pump inlet. p p 2. On the basis of theoretical and experimental studies, the need to supply the pump with a flow divider was substantiated, which excludes additional resistance to the rotation of the pump rotor due to the effect of the supplied mass. The optimal angle α between the working edges of the separator has been established. For this pump, the angle α is 90. 1000 with a rotor diameter of 260 mm. The use of this design of the separator allows reducing the required drive power by 20 % compared to pumps without a separator. 2. On the basis of theoretical and experimental studies, the need to supply the pump with a flow divider was substantiated, which excludes additional resistance to the rotation of the pump rotor due to the effect of the supplied mass. The optimal angle α between the working edges of the separator has been established. For this pump, the angle α is 90. 1000 with a rotor diameter of 260 mm. The use of this design of the separator allows reducing the required drive power by 20 % compared to pumps without a separator. 3. 4 Conclusions Based on the results of the research, the following optimal parameters and operating modes of the pump were determined: flow rate 30 m3/h, discharge pressure 0.4 MPa, pump rotor speed 250 min-1, required power 6.3 kW, specific energy consumption of the technological process not more than 0.21 KWh/m3, labor time spent on the technological process 0.034 h. 4. The use of a new rotary screw pump with screw feeders on small pig farms allows to reduce the metal consumption by 2 times, the energy consumption of the technological process by at least 3.5 times and increase the productivity by 2 times in comparison with the existing centrifugal pumps. 5. Before implementing this improved feed pump and other new techniques on farms, it is advisable to train farmers in online training courses on the basis of modern innovative pedagogical technologies. 5. Before implementing this improved feed pump and other new techniques on farms, it is advisable to train farmers in online training courses on the basis of modern innovative pedagogical technologies. References 1. E. A. Kryshtop, Improving the productive qualities of pigs by mobilizing the internal reserves of the body (Volgograd, Russia, 465, 2011) 2. K. I. Knyazev, Intensive meat feeding of pigs (Moscow, Russia, 222, 1979) 3. A. I. Devyatkin, Rational use of feed in industrial production (Moscow, Russia, 343, 2016) 4. L. G. Boyarsky, Production and use of feed in industrial production, (Moscow, Russia, 542, 2018) 5. Scientific foundations of complete feeding of farm animals, Sat scientific works (Moscow, Russia, 145, 2017) 6. Kh. Kh. Gubeidullin, Improvement of technologies and means of mechanization of distribution of liquid and semi-liquid feed (Ulyanovsk, Russia, 351, 2005) 7. M. R. Baxter, Design of new feeder for pigs, Farm Building Progress, 19-22 (1 8. Е. Roth, Hachlesefiir Sauenhalter und Schweinmaster Agrar, Ubers, 40(9), 66-69 (1989) 9. V. I. Alexandrov, Methods for reducing energy consumption in the hydraulic transport of high concentration mixtures (SPGGI (TU), St. Petersburg, Russia, 117, 2000) 10. V. P. Dokukin, Improving the efficiency (TU), St. Petersburg, Russia, 105, 2005) 10. V. P. Dokukin, Improving the efficiency of the pipeline hydro transport system (SPGGI (TU), St. Petersburg, Russia, 105, 2005) 11. Y. D. Tarasov, V. P. Dokukin, A. K. Nikolaev, Pressure hydro transport installations in the mining industry, Pressure Hydraulic Plants in the Mining Industry, (SPGGI (TU), St. Petersburg, Russia, 104, 2008) 12. A. A. Utkin, Pig feeding systems (GNU VNIIMZh Russian Agricultural Academy, Podolsk, Russia, 226, 2011) 13. F. Abduosiev, Prospects for the development of animal husbandry taking into account scientific and technological progress, (Internet-version of the publications of Charkhi Gardun LLC, 2012) 6 E3S Web of Conferences 274, 03003 (2021) STCCE – 2021 https://doi.org/10.1051/e3sconf/202127403003 14. V. I. Klimov, Hydrotransport of fibrous materials in pulp and paper production (Moscow, Russia, 1971) ( ) 15. V. I. Klimov, Rotary lobe pumps for high-viscosity dairy products (Moscow, Russia, 1970) 16. O. D. Rakhimov, B. M. Khudoyorov, F. M. Mamatov, I. Ergashev, Pump for the supply of liquid feed mixtures, 1089, Patent No 5149, Uzbekistan Republic (1998) 17. O. D. Rakhimov, B. M. Khudoyorov, F. M. Mamatov, U. B. Eshkobilov, Feed pump, 1090. Patent No 5150, Uzbekistan Republic (1998) 18. O. D. Rakhimov, Zh. Kh. Togaev, A. Kh Khuzhakulov, Improved feed pump for farms, Akademy, 6(45), 25-27 (2019) 19. O. D. Rakhimov, Zh. Kh Togayev, A. Kh. Khuzhakulov, Usovershenstvovannyy kormonasos dlya fermerskikh khozyaystv, Academy, 6(45), 25-27 (2019) 20. O. D. Rakhimov, Y. K. 34. R. Kh. Mukhamеtrakhimоv, A. A. Рanсhеnkо, Fеaturеs оf thе quality соntrоl systеm fоr thе соnstruсtiоn оf оutdооr watеr suррly and sеwеragе nеtwоrks, Izvestiya KGASU, 4(42), 360-367 (2017) References Manzarov, A. E. Karshiyev, Description of pedagogical technologies and technology of problem-based learning, Problemy sovremennoy nauki i obrazovaniya, 2(147), 59-62 (2020) DOI: 10.24411/2304-2338-2020-10201 21. O. D. Rakhimov, L. Аshurova, Types of modern lectures in higher education, technology of their design and organization, Problemy sovremennoy nauki i obrazovaniya, 12(157), 41-46 (2020) 22. O. D. Raximov, O. M. Turgunov, Modern educational technologies (Science and Technology, Tashkent, Uzbekistan Republic, 205, 2013) 23. O. D.Raximov, S. S. Eshev, The importance of the telecommunication project on education system, in proceedings of International scientific conference Topical issues of the development of science in the world, Moscow, Russia, 2019 24. O. D.,Raximov, Y. K. Manzarov, A. E. Qarshiyev, Sh. A. Sulaymanova, Description of pedagogical technology and problematic teaching technology, Problemy sovremennoy nauki i obrazovaniya, 2(147), 59-62 (2020) DOI: 10.24411/2304-2338-2020-10201 25. O. D. Rakhimov, Necessity of live modern lectures in higher education and its types, Science problems, 10(58), 60-64 (2020) DOI: 10.24411/2413-2101-2020-11002 26. O. D. Rakhimov, B. F. Nazarov, M. S. Sapaev, Modern lectures in higher educati (TUIT Karshi branch, Karshi, Uzbekistan Republic, 2012) 27. O. D. Raximov, Innovative pedagogical technologies: project style as a technology that improves the quality of education (TUIT Karshi branch, Karshi, Uzbekistan Republic, 80, 2013) 28. O. D. 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https://openalex.org/W3031631046	https://www.matec-conferences.org/articles/matecconf/pdf/2020/10/matecconf_icsc-isatech20_02001.pdf	English	null	Metaheuristics optimized machine learning modelling for estimation of exergetic emissions of a propulsion system	MATEC web of conferences	2,020	cc-by	3,299	* Corresponding author: onder.turan@gmail.com © 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/). Metaheuristics optimized machine learning modelling for estimation of exergetic emissions of a propulsion system Tolga Baklacioglu 1,, Onder Turan 2, and Hakan Aydin3 1Eskisehir Technical University, Faculty of Aeronautics and Astronautics, TR-26470 Eskisehir, Turkey, tbaklacioglu@eskisehir.edu.tr 2Eskisehir Technical University, Faculty of Aeronautics and Astronautics, TR-26470 Eskisehir, Turkey, onderturan@eskisehir.edu.tr 3TUSAS Engine Industries, Eskisehir, Turkey e-mail: tei.hakan@gmail.com Tolga Baklacioglu 1,, Onder Turan 2, and Hakan Aydin3 1Eskisehir Technical University, Faculty of Aeronautics and Astronautics, TR-26470 Eskisehir, Turkey, tbaklacioglu@eskisehir.edu.tr 2Eskisehir Technical University, Faculty of Aeronautics and Astronautics, TR-26470 Eskisehir, Turkey, onderturan@eskisehir.edu.tr 3TUSAS Engine Industries, Eskisehir, Turkey e-mail: tei.hakan@gmail.com Abstract. You This study offers a metaheuristic design for primary parameters and architectures of two models of artificial neural network (ANN) in predicting a business jet aircraft’s exergo-emission parameters, such us exergy destruction ratio (rex,dest) and waste exergy ratio (rwex), at different flight stages. In consideration of this, the development of hybrid genetic algorithm (GA)-ANN models has been achieved by considering real databases of rex, dest and rwex at various power levels. Implementing a metaheuristics-based optimization on the generated multilayer perceptron (MLP) ANN models has produced the most favorable initial network weights, step-size, biases as well as training algorithm’s back-propagation (BP) momentum rate in addition to optimal quantity of neurons in the hidden layer(s) with regard to the topology design. In accordance with an error assessment approach, there exists a close fit linking the reference real data and rwex (linear correlation ratio, R, value of 0.999851) as well as rex,dest (R value of 0.999985) predicted values. MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031402001 1 Introduction With the growth of air travel around the world, environmental impacts and energy consumption of the aviation industry has increasing rapidly. Fuel consumption and aircraft emission are about 3% and 2% of the total fuel usage and total CO2 emissions around the world, respectively. Furthermore, according to the IATA, total greenhouse gas emissions associated with air transportation will be 400–600% higher in 2050 than in four decades [1- 4]. Fuel price and energy efficiency of their engine systems effect the total energy efficiency of air transportation systems. For long-term sustainability, many measures are resorted to control wasted emission containing the optimizing fleet and air route, adapting wing-tip fin by aircraft designers and airliners. As energy usage in next generation aircraft fleet gains MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031402001 importance due to operating costs, aircraft must be optimized for energy efficiency, design technologies, environmental considerations, operating and economic costs [5-12]. Environmental problems, i.e. global warming, noise and atmospheric pollution, are related to energy efficiency. Besides, for the sustainable development and environmental sustainability, energy consumption plays a crucial role. So, energy and exergy concepts can be utilized incorporated with environmental considerations, sustainability and thermodynamics. Nowadays there is a great interest in studying the linkages between energy, exergy, environment and sustainability. Exergetic and energetic metrics are useful dimensions for evaluating aircraft environmental and technological performances [13-17]. For improving performance, environmental and economical characteristics of aircraft, powerful engines and less fuel consumption are inevitable. When energy efficiency of the engine increases and wasted energy decreases, fuel usage decreases at same power level. Obviously, latest high technology and selection of aircraft engine type improve the aircraft fuel efficiency. Latest trends in improving specific fuel consumption and overall efficiency show that aircraft entering aviation sector are around 80% more fuel-efficient using turboprop and high bypass ratio turbofan engines [18-22]. The overall efficiency and specific fuel consumption of aircraft engines are calculated to be using thermodynamic analysis. Unfortunately, first law analysis of thermodynamics does not show correct location information for energy destruction and losses. However, the second law must be applied to the aircraft propulsion system for determining locations and magnitudes of inefficiencies in the engine. The number of articles studied on exergy analysis and exergetic optimization of aircraft power systems has increased in the last decades [13- 21, 23,24]. 1 Introduction Especially, modeling of energetic and exergy efficiencies of turboprop engine components using neuro-evolution algorithms and hybrid genetic algorithm-artificial neural networks is studied [20, 25]. The objective of this study is to accomplish two models of artificial neural network (ANN) in predicting a business jet aircraft’s exergo-emission parameters, such us exergy destruction ratio (rex,dest) and waste exergy ratio (rwex), at different flight stages and five input parameters (fuel and air mass flows, power and torque levels, gas generator shaft speed). This study constitutes the first analysis about determining exergo-emission for aircraft power system at various flight phases. Moreover, there is no study on a detailed measure exergetic emission of turboprop power system using metaheuristics optimized machine learning modeling. Lack of this makes the paper original for aircraft energy systems and during typical flight phases. 2 System description Turboprop engines have a great advantage for propulsive/fuel efficiency and low emissions. Turboprop engines used for regional airlines and military transport transmit the equivalent shaft power required to drive the propeller from engine turbine shaft. In this study, selected cargo aircraft (one of the most notable turboprop aircraft version is CASA/IPTN CN235) is very similar to CN235 aircraft as shown in Figure 1. Schematic of the engine and component description are shown in Figures 2 and 3, respectively. 2 2 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 MATEC Web of Conferences 314, 02001 (2020) https://doi.org/10.1051/matecconf/202031402001 Figure 1. Turboprop engines on the cargo aircraft (modified from [20]) Figure 1. Turboprop engines on the cargo aircraft (modified from [20]) Figure 2. Exergetic control volume on the aircraft Figure 3. Main components of the turboprop power system Figure 1. Turboprop engines on the cargo aircraft (modified from [20]) Figure 1. Turboprop engines on the cargo aircraft (modified from [20]) Figure 2. Exergetic control volume on the aircraft Figure 2. Exergetic control volume on the aircraft Figure 3. Main components of the turboprop power system Figure 3. Main components of the turboprop power system 3 https://doi.org/10.1051/matecconf/202031402001 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 ICSC-ISATECH 2019 3 Overview of an ANN (Artificial Neural Network) Figure 4 Architecture of the exergy destruction ratio and waste exergy ratio machine learning models 3 Overview of an ANN (Artificial Neural Network) Figure 4 Architecture of the exergy destruction ratio and waste exergy ratio machine learning models Figure 4 Architecture of the exergy destruction ratio and waste exergy ratio machine learning models Being a type of ANN, a multilayer perceptron (MLP) (also referred to as feed-forward ANN) is composed of an input layer, an output layer and one or more hidden layers as illustrated in Figure 4. 4 GA (GENETIC ALGORITHM) Optimized BP (Back-Propagation) Feed-Forward ANNs As proposed by a group of scientists led by Rumelhart and McCelland in 1986, the BP-ANN is a type of multilayer feed-forward network established on an error back-propagation algorithm for training in a supervised manner. There exist two stages in the learning process of a BP-ANN; namely, forward and backward propagation. Information is propagated from the input layer and then modified by the weighted connections of the hidden layer into the output layer, which enables the attainment of the input response of the ANN in the output layer. This output value given by the network is compared with the desired output. In the case of an existing error, it returns along the path and adjustment of weights and thresholds is executed in a layer by layer attitude. Being a population driven search algorithm inspired by the biological natural selection, GA mimics the phenomenon of reproduction, crossover and mutation in the process of natural genetics. Adopting a hybrid GA-BP algorithm in this paper, the connection weights, thresholds, and the number of neurons in the hidden layer have been optimized by the GA, which provides the amalgam of GA’s global search virtue and BP’s local search ability in order to prevent the potential inherent problems of BP-ANNs. Moreover, GA optimization has enhanced the learning capability and accuracy of the BP-ANN. The case study cargo aircraft turboprop engine has been tested a number of times for different power settings. The actual values of specifications related to the engine were determined from the parametric evaluation and trials and they were then used to derive the values of waste exergy ratio and actual exergy destruction ratio for this particular engine. The validation and training of the metaheuristically prepared ANN estimation models that were put forward in this study was performed with the help of actual datasets related to waste 4 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031402001 exergy ratio and exergy destruction ratio which matched to parameters of an engine still in the experimental phase. exergy ratio and exergy destruction ratio which matched to parameters of an engine still in the experimental phase. Power (SHP), torque (N.m), speed of the gas generator (rpm), fuel mass flow (kg/s) are classified as explanatory variables, on the other hand the waste exergy ratio and exergy destruction ratio in context of 2 separate ANN models derived in this paper were classified as output variables. p In Figure 5, the lowest possible MSE’s (Mean Square Error) for different rates of momentum in networks with one hidden layer can be seen whereas the same for two hidden layers is depicted in Figure 6. 5(a) 5(a) 5(a) 5 5(b) Figure 5 Training and cross-validation results of the BP algorithm with varied momentum rates for one-hidden-layer networks: a) exergy destruction ratio, b) waste exergy ratio (T: Training, CV: Cross- validation; HL: Hidden layer) 6(a) ATEC Web of Conferences 314, 02001 (2020) https://doi.org/10.1051/matecconf/202031402 SC-ISATECH 2019 MATEC Web of Conferences 314, 02001 (2020) https://doi.org/10.1051/matecconf/202031402001 ICSC-ISATECH 2019 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031402001 5(b) Figure 5 Training and cross-validation results of the BP algorithm with varied momentum rates for one-hidden-layer networks: a) exergy destruction ratio, b) waste exergy ratio (T: Training, CV: Cross- validation; HL: Hidden layer) C ISATECH 2019 5(b) Figure 5 Training and cross-validation results of the BP algorithm with varied momentum rates for one-hidden-layer networks: a) exergy destruction ratio, b) waste exergy ratio (T: Training, CV: Cross- validation; HL: Hidden layer) 6( ) 6(a) 6 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031402001 6(b) Figure 6. Training and cross-validation results of the BP algorithm with varied momentum rates for two-hidden-layer networks: a) exergy destruction ratio, b) waste exergy ratio (T: Training, CV: Cross-validation; HL: Hidden layer) 6(b) Figure 6. Training and cross-validation results of the BP algorithm with varied momentum rates for two-hidden-layer networks: a) exergy destruction ratio, b) waste exergy ratio (T: Training, CV: Cross-validation; HL: Hidden layer) A comparison was drawn between the values of waste exergy ratio and exergy destruction ratio which were predicted by the ideal networks and the actual values which were derived when the turboprop engine was run on the testing datasets of the two ANN models as an experiment. MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 It can be clearly seen from Figures 7(a) and 7(b) that the actual and estimated values of the two ANN models show a close fit. 5 Conclusions An additional new feature of the trial design is that in the design and derivation steps actual values derived during trials and those obtained parametric evaluation of the aircraft’s engine are used. Since, the effectiveness and validity of an ANN model are greatly affected by the network architecture’s initial state and adapted learning algorithm hence in this never before tried method of study use of an ANN designed in a metaheuristic format further increases its precision and capability of generalization. The important results derived from modeling are described ahead: i. For the optimum exergy destruction ratio of BP model, R value of 0.999985 is attained; whereas, in case of GA-BP model, it comes out to be 0.999987. On the other hand, the value of maximum linear correlation coefficient for waste exergy ratio came out to be 0.999851 for ANN models and 0.999869 for GA-ANN models. The fact that the R values are high and the predefined error varieties have low values point towards the fact that the accuracy of the derived ANN models is quite high in context of calculation of parameters related to exergy destruction and waste exergy. i. For the optimum exergy destruction ratio of BP model, R value of 0.999985 is attained; whereas, in case of GA-BP model, it comes out to be 0.999987. On the other hand, the value of maximum linear correlation coefficient for waste exergy ratio came out to be 0.999851 for ANN models and 0.999869 for GA-ANN models. The fact that the R values are high and the predefined error varieties have low values point towards the fact that the accuracy of the derived ANN models is quite high in context of calculation of parameters related to exergy destruction and waste exergy. ii. Both the efficiency and precision have showed an improvement due to application of GA-BP which is a metaheuristic design on prepared ANNs, in addition the time required for convergence was also cut short in the two models. It can be presumed that ANN is similar to a black-box like system with correctly prepared topology and initial values; this will assist in delivering the best possible output and increase the reliability of the model in context of its practical usage and precision. iii. 7 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 https://doi.org/10.1051/matecconf/202031402001 7(a) 7(b) ure 7 Testing dataset validation by the use of optimal ANN model architectures for a) exergy ruction ratio, b) waste exergy ratio p g ECH 2019 7(a) 7(b) re 7 Testing dataset validation by the use of optimal ANN model architectures for a) exer uction ratio, b) waste exergy ratio 7(b) Figure 7 Testing dataset validation by the use of optimal ANN model architectures for a) exergy destruction ratio, b) waste exergy ratio ( ) Figure 7 Testing dataset validation by the use of optimal ANN model architectures for a) exergy destruction ratio, b) waste exergy ratio 8 8 https://doi.org/10.1051/matecconf/202031402001 MATEC Web of Conferences 314, 02001 (2020) ICSC-ISATECH 2019 Ackowledment Authors would like to thanks Eskisehir Tehnical University in Turkey for financial and technical support. This study was supported by Eskisehir Tehnical University Scientific Research Projects Commission under the grant no: 19ADP025. 5 Conclusions It was seen that the accuracy of networks with a single hidden layer was greater than that of networks with two hidden layers for exergy destruction ratio forecast model; whereas, the two-hidden-layer architectures outperformed in the case of waste exergy ratio estimation. iii. It was seen that the accuracy of networks with a single hidden layer was greater than that of networks with two hidden layers for exergy destruction ratio forecast model; whereas, the two-hidden-layer architectures outperformed in the case of waste exergy ratio estimation iv. A new benchmark of level of performance and excellence in context of high precision and results has been set up by use of metaheuristic design in estimation models that aim to determine parameters related to waste exergy and exergy destruction ratios. iv. 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https://openalex.org/W4238684550	https://www.mdpi.com/2075-1680/7/1/2/pdf?version=1515683472	English	null	Acknowledgement to Reviewers of Axioms in 2017	Axioms	2,018	cc-by	390	E i o ia Acknowledgement to Reviewers of Axioms in 2017 Axioms Editorial Office MDPI AG, St. Alban-Anlage 66, 4052 Basel, Switzerland Published: 11 January 2018 Published: 11 January 2018 Peer review is an essential part in the publication process, ensuring that Axioms maintains high quality standards for its published papers. In 2017, a total of 35 papers were published in the journal. Thanks to the cooperation of our reviewers, the median time to first decision was 19 days and the median time to publication was 42 days. The editors would like to express their sincere gratitude to the following reviewers for their time and dedication in 2017: Axioms 2018, 7, 2; doi:10.3390/axioms701000 Akin, Ethan Akram, Muhammad Alcantud, José Carlos R. Ban, Adrian I. Banakh, Taras Onufrievich Bentkowska, Urszula Bianchi, Matteo Breuer, Christoph Brzezinski, Tomasz Carfora, Mauro Carli, Marco Cattani, Carlo Chaiken, Seth Chalishajar, Dimplekumar Chellamuthu, Vinodh Chen, Xianghong Cheng, Qiang Cheng, Yongqiang Choi, Junesang Cohen, Michael Concezzi, Moreno Coquand, Olivier Cordero Fuertes, Juan Antonio Dall’Arno, Michele Demey, Lorenz Di Martino, Ferdinando Dominguez Dafauce, Luis Carlos Dunbar, Robert Ephremidze, Lasha Ernst, Thomas Essafi, Karim Etemad, Katayoon Evans, Michael Fink, Holger Foissy, Loïc Fortuna, Luigi www.mdpi.com/journal/axioms 2 of 2 Axioms 2018, 7, 2 Axioms 2018, 7, 2 A io , , Mls, Karel Moazzemi, Reza Morente-Molinera, J.A. Nowosielski, Adam Palus, Henryk Patoju, Sri Krishna Sudheer Peischl, Bernhard Pekala, Baraba Piasecki, Krzysztof Povstenko, Yuriy Pratt-Hartmann, Ian Przybyła-Kasperek, Małgorzata Quarati, Piero Rascher, Daniel Riera, Juan Vicente Rogosin, Sergei Romon, Pascal Rump, Wolfgang Ruzzenenti, Franco Saikia, Manjil Sambucini, Anna Rita Sandoval, Leonidas Seppi, Andrea Silva, Rui Sivasundaram, Seenith Staiano, Antonino Szymusiak, Anna Tuszynski, Jack Ulanowicz, Robert Uljane, Ingrida Varshney, Lav R. Villanueva, Susana Cubillo Wang, Jianqiang Wang, Jianzhong Weber, Melanie Weit, Yitzhak Wlodarczyk, Zbigniew Yin, Rujie Ying, Mingsheng Zhang, Yang Zhukovsky, Konstantin Ziemba, Paweł © 2018 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2049974202	https://orbit.dtu.dk/files/106430022/Lee_et_al_2015.pdf	English	null	Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway	Scientific reports	2,015	cc-by	15,226	Citation (APA): Lee, J. S., Beuchert Kallehauge, T., Pedersen, L. E., & Kildegaard, H. F. (2015). Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Scientific Reports, 5, Article 8572. https://doi.org/10.1038/srep08572 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology- directed DNA repair pathway ng; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup Document Version Publisher's PDF, also known as Version of record General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research OPEN SUBJECT AREAS: MOLECULAR BIOLOGY BIOTECHNOLOGY GENETIC ENGINEERING Received 20 October 2014 Accepted 26 January 2015 Published 25 February 2015 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2970 Hørsholm, Denmark. Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production. Correspondence and requests for materials should be addressed to H.F.K. (hef@ biosustain.dtu.dk) C C hinese hamster ovary (CHO) cells have been used as the predominant workhorses for production of recombinant therapeutic proteins with complex glycoforms1,2. Traditionally, development of recombin- ant CHO (rCHO) cell lines relies on random integration of a gene of interest (GOI) into the genome, followed by selection of cells carrying the transgene1,2. However, lack of control of gene insertion can give rise to unwanted phenotypic heterogeneity due to the varying accessibility of integration sites for gene expression – termed the position effect variation2,3. In addition, gene amplification methods are traditionally used to increase expression. As a result, these cell lines are often unstable and show reduced production over time4. Due to this variation in expression and genomic composition, subsequent screening of multiple clones is necessary to select proper clones suitable for high and stable expression of recombinant proteins1,2. In this article, we show that targeting of transgenes into specific desirable sites in the CHO genome in a controlled manner reduces the variation in expression, generating a uniform population with stable transgene expression. In order to circumvent uncontrollable gene insertion during rCHO cell line construction, different approaches have been applied for site-directed integration of transgenes using site-specific recombinases, which includes Cre/ loxP system, Flp/FRT system, and phiC31/R4 integrases5–7. Correspondence and requests for materials should be addressed to H.F.K. (hef@ biosustain.dtu.dk) Link back to DTU Orbit Link back to DTU Orbit Citation (APA): Lee, J. S., Beuchert Kallehauge, T., Pedersen, L. E., & Kildegaard, H. F. (2015). Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Scientific Reports, 5, Article 8572. https://doi.org/10.1038/srep08572 this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim. OPEN However, these systems are limited by the need of a prior establishment of platform cell lines with the insertion of recombination site into random or a limited number of specific genomic regions, which is a prerequisite for retargeting to generate cell lines expressing the GOI. With draft genomes of several CHO cell lines recently being made available8,9, it is now possible to efficiently engineer the genomic sequence of CHO cells with engineered nucleases. Customized nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) RNA guided nucleases are technologies readily available to induce targeted insertion/deletion (indel) mutations or precise sequence changes in a broad range of organisms and cell types (reviewed in Ref. 10). Upon site-specific DNA double-strand breaks (DSBs) induced by engineered nucleases, the target locus will typically be repaired by one of two major DNA damage repair pathway: non- homologous end-joining (NHEJ) or homology-directed repair (HDR). Compared with the error-prone NHEJ, which can result in efficient indel mutation generation subsequently leading to knockout of target locus, HDR can be used to modify endogenous loci precisely in the presence of homologous pieces of endogenous/exogenous DNA. Although HDR is typically observed at a lower or more variable frequency than NHEJ, HDR can be leveraged to generate targeted integrations with engineered nucleases in mammalian cells11–15. Notably, CHO cells have previously been described as a cell type recalcitrant to homology-based integration of large DNA constructs, thus targeted integration in CHO cells has been confined to NHEJ based approaches16,17. SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 1 www.nature.com/scientificreports Compared with protein-based genome editing tools with custo- mizable DNA binding specificities, such as ZFNs and TALENS, the newer CRISPR/Cas9 platform is based on simple base-pairing between an engineered RNA and the targeted genomic site, which enables rapid design, ease of use, and low costs18. The CRISPR/Cas9 system, which was initially identified in the bacterial immune system, is composed of two RNA elements, CRISPR-RNA (crRNA) and trans- activating crRNA (tracrRNA), together with the Cas9 nuclease14,19. The active cleavage-complex is generated by base-pairing between the genomic sequence and crRNA, followed by hybridization of the tracrRNA to the crRNA. Subsequently, the Cas9 nuclease associates with the DNA:RNA complex to form the active cleavage complex, which in turn will generate site specific DSBs at the genomic locus19. OPEN A total of 45 clones were recovered, followed by further characterization. 42 out of 45 clones had complete disruptions of the COSMC locus, as indicated by qPCR-based relative copy number analysis compared with genomic DNA from CHO-S wild type cells (Fig. 1e, left panel). Assuming that gene copies must be integer values, the value of relative copy number is expected to be 0, 0.5, or 1 for targeted clonal cells. Three clones without these values were therefore not considered as clonal cells. Relative mCherry copy number result amongst selected clones revealed that most clones harbored the same number of mCherry transgene, except two clones with 2- or 3-fold higher number (Fig. 1e, right panel). Successful targeting of transgenes at the COSMC locus validated our integration method, and prompted us to apply to different genomic loci showing distinct properties. Application of targeted integration platform to the functional hemi- Here we demonstrate efficient targeted gene integration into site- specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring short-homology arms, GOI, and a fluorescent marker gene as an indicator of random integ- ration. Simultaneous introduction of active sgRNAs, Cas9 nucleases, and donor plasmid enabled insertion of a 3.7-kb gene expression cassette at three different loci in CHO cells, following a simple drug-selection. A low level of off-target mutation in most potential off-target sites and homogeneous expression level of GOI was observed in targeted integrants, which supports the robustness and efficiency of the current strategy. OPEN In recent studies, the original type II CRISPR system from S. pyogenes was modified to induce targeted genome editing in eukaryotic cells by fusing the two RNA complexes termed single guide RNA (sgRNA)11,14,19. We previously developed a CRISPR/Cas9 system optimized for CHO cells, and demonstrated high efficiency of indels generated in CHO cells up to 47.3%20. Assuming that the high rate of DSB introduction will induce DNA damage repair pathways, and the concurrent introduction of a donor plasmid will be used as the repair template, the targeted integration at specific genomic sites can be feasible with high fidelity. was chosen for initial validation of the targeted integration strategy. The region targeted by sgRNA2, which gave rise to high indel efficiency in CHO-K1 cells in our previous study20, was selected as the integration site. Based on this site, 59 and 39 homology arms were designed to generate the targeting donor plasmid (Fig. 1a). CHO-S cells were transfected with the vectors encoding CHO codon opti- mized Cas9, COSMC sgRNA2, and donor plasmid. Genomic DNA was extracted either three days after transfection (transient expression) or two weeks after G418 selection (stable expression). No integration events were detected in the transiently transfected pools by the 59/39 junction PCR (Fig. 1b, Lane 1–5). In the stable selected pools, both 59 and 39 junction PCR verified targeted integration of donor DNA into the COSMC locus (Fig. 1b, Lane 7 and 10). In agreement with a previous observation16, linearization of the donor DNA outside of the homology arms resulted in fainter PCR positive bands from 59/ 39 junction PCR compared with those from circular donor DNA (Fig. 1b, Lane 8 and 11). Stepwise decreasing the length of 59 and 39 homology arms flanking the same COSMC integration site from 750 bp to 100 bp indicated that homology arms with a length down to 250 bp were capable of mediating homology-based targeted integration, although the PCR band intensity was gradually decreased (Supplementary Fig. 1). In accordance with the assumption that cells expressing both ZsGreen1-DR and mCherry were the result of random integration, stable cells expressing both mCherry and ZsGreen1-DR, which were isolated by FACS, showed incomplete and faint junction PCR positive bands indicating a mixture of integration events. (Fig. 1b, Lane 13 and 15). To verify precise integration into the genome, the 59/39 genome-donor boundaries were sequenced (Fig. 1c). OPEN Indeed, the sequences of the 59/39 junction PCR amplicons confirmed precise insertion of the target expression cassette into the COSMC locus. We then screened 588 G418-resistant clones and isolated 138 clones, which displayed homogeneous expression of mCherry. 23 clones were excluded due to very slow growth resulting in 115 clones, which were further characterized. 83 out of 115 (72.2%) were 59/39 junction PCR positive (Table 1). Out-out PCR with primers specific for genomic sites revealed that 70 out of 83 junction PCR positive clones (84.3%, i.e. 60.9% in total) were correctly targeted with the intact target integration unit, generating expected size of amplicon (wild type amplicon: 1.6 kb 1 target integration unit: 3.7 kb < 5.3 kb; Fig. 1d, Lane 4, Table 1). As a result, a targeting efficiency of 27.8% was obtained based on the percentage of out-out PCR positive clones in mCherry only expressing clones and a proportion of the mCherry only expressing cells in the stable cell pool (Table 1). These 70 out-out PCR positive clones were expanded and adapted to grow in suspension. A total of 45 clones were recovered, followed by further characterization. 42 out of 45 clones had complete disruptions of the COSMC locus, as indicated by qPCR-based relative copy number analysis compared with genomic DNA from CHO-S wild type cells (Fig. 1e, left panel). Assuming that gene copies must be integer values, the value of relative copy number is expected to be 0, 0.5, or 1 for targeted clonal cells. Three clones without these values were therefore not considered as clonal cells. Relative mCherry copy number result amongst selected clones revealed that most clones harbored the same number of mCherry transgene, except two clones with 2- or 3-fold higher number (Fig. 1e, right panel). Successful targeting of transgenes at the COSMC locus validated our integration method, and prompted t l t diff t i l i h i di ti t ti was chosen for initial validation of the targeted integration strategy. The region targeted by sgRNA2, which gave rise to high indel efficiency in CHO-K1 cells in our previous study20, was selected as the integration site. Based on this site, 59 and 39 homology arms were designed to generate the targeting donor plasmid (Fig. 1a). CHO-S cells were transfected with the vectors encoding CHO codon opti- mized Cas9, COSMC sgRNA2, and donor plasmid. OPEN Genomic DNA was extracted either three days after transfection (transient expression) or two weeks after G418 selection (stable expression). No integration events were detected in the transiently transfected pools by the 59/39 junction PCR (Fig. 1b, Lane 1–5). In the stable selected pools, both 59 and 39 junction PCR verified targeted integration of donor DNA into the COSMC locus (Fig. 1b, Lane 7 and 10). In agreement with a previous observation16, linearization of the donor DNA outside of the homology arms resulted in fainter PCR positive bands from 59/ 39 junction PCR compared with those from circular donor DNA (Fig. 1b, Lane 8 and 11). Stepwise decreasing the length of 59 and 39 homology arms flanking the same COSMC integration site from 750 bp to 100 bp indicated that homology arms with a length down to 250 bp were capable of mediating homology-based targeted integration, although the PCR band intensity was gradually decreased (Supplementary Fig. 1). In accordance with the assumption that cells expressing both ZsGreen1-DR and mCherry were the result of random integration, stable cells expressing both mCherry and ZsGreen1-DR, which were isolated by FACS, showed incomplete and faint junction PCR positive bands indicating a mixture of integration events. (Fig. 1b, Lane 13 and 15). To verify precise integration into the genome, the 59/39 genome-donor boundaries were sequenced (Fig. 1c). Indeed, the sequences of the 59/39 junction PCR amplicons confirmed precise insertion of the target expression cassette into the COSMC locus. We then screened 588 G418-resistant clones and isolated 138 clones, which displayed homogeneous expression of mCherry. 23 clones were excluded due to very slow growth resulting in 115 clones, which were further characterized. 83 out of 115 (72.2%) were 59/39 junction PCR positive (Table 1). Out-out PCR with primers specific for genomic sites revealed that 70 out of 83 junction PCR positive clones (84.3%, i.e. 60.9% in total) were correctly targeted with the intact target integration unit, generating expected size of amplicon (wild type amplicon: 1.6 kb 1 target integration unit: 3.7 kb < 5.3 kb; Fig. 1d, Lane 4, Table 1). As a result, a targeting efficiency of 27.8% was obtained based on the percentage of out-out PCR positive clones in mCherry only expressing clones and a proportion of the mCherry only expressing cells in the stable cell pool (Table 1). These 70 out-out PCR positive clones were expanded and adapted to grow in suspension. Results Thus, this locus SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 2 www.nature.com/scientificreports a Target locus: sgRNA Target (23 bp) // // // 5’ arm SV40 Neo(R) SV40 pA BGH pA EF1α mCherry 3’ arm CMV BGH pA ZsGreen1 // Donor: 3.7 kb HDR 5’ arm SV40 Neo(R) SV40 pA BGH pA EF1α mCherry 3’ arm // // Out In In Out 5’ juncon 3’ juncon 5’ arm 3’ arm a Donor: b c Reference Conservaon 5’ juncon 3’ juncon Target locus: sgRNA Target (23 bp) // // // 5’ arm SV40 Neo(R) SV40 pA BGH pA EF1α mCherry 3’ arm CMV BGH pA ZsGreen1 // Donor: HDR 5’ arm SV40 Neo(R) SV40 pA BGH pA EF1α mCherry 3’ arm // // Out In In Out 5’ juncon 3’ juncon Transient Stable - + - + + - - + + + + + - + - - + + + M M - + M + + + + + - + - + M + + + 5’ juncon 3’ juncon * Cas9 Donor COSMC sgRNA2 + + + - + - M + + + 3’ juncon + - - M 5’ juncon Stable [mCherry+ / ZG1+] 1000bp 1500bp * 1 2 3 4 5 6 7 8 9 10 11 1000bp 1500bp 12 13 14 15 * d + + + + - + + + - + + + M Cas9 Donor COSMC sgRNA2 1500bp 6000bp 5000bp Pool Clone - + - + Juncon PCR 1 2 3 4 e Relave COSMC copy number 0.0 0.2 0.4 0.6 0.8 1.0 1234568 11 12 13 14 16 18 19 20 23 26 28 33 37 38 39 42 44 45 47 50 51 52 53 56 57 58 61 62 63 64 65 69 77 81 84 86 106 108 CHO-S Relave mCherry copy number 0 1 2 3 4 5 1234568 11 12 13 14 16 18 19 20 23 26 28 33 37 38 39 42 44 45 47 50 51 52 53 56 57 58 61 62 63 64 65 69 77 81 84 86 106 108 5’ arm 3’ arm re 1 \| Targeted integration into COSMC locus using CRISPR/Cas9. (a) Schematic illustration of the targeting strategy for the s est. Results Donor plasmid consists of three parts: short homology arms flanking sgRNA target site cleaved by Cas9 (red triangle), mCher tance gene expression cassettes inside homology arms, and ZsGreen1-DR expression cassette outside homology arms. Upon DSB SPR/Cas9, HDR-mediated repair can be used to insert a total size of 3.7 kb of expression cassettes through recombination of the or plasmids. Primer position for 59/39 junction PCR is indicated. (b) Agarose gel of 59/39 junction PCR on transiently transfected c s. An asterisk indicates the use of linearized donor plasmid. M, 1 kb DNA ladder (c) Sanger sequencing of the 59/39 junction PC plicons from the stable cell pool were purified and directly sequenced after PCR amplification. The chromatogram sequence of ju licon was compared with the reference sequence at the genome-donor boundaries. (d) Agarose gel of out-out PCR results of stable c Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (1.6 kb) or targeted integra tive copy number of COSMC and mCherry regions in clonal cells. Each plot shows the relative copy number of each region in co ence sample. Genomic DNA of wild type CHO-S and Clone #1 and was used as the reference for COSMC and mCherry region, respe The error bars represent the standard deviations (n $ 3). Results HDR b Transient Stable - + - + + - - + + + + + - + - - + + + M M - + M + + + + + - + - + M + + + 5’ juncon 3’ juncon * Cas9 Donor COSMC sgRNA2 1000bp 1500bp * 1 2 3 4 5 6 7 8 9 10 11 * + + + - + - M + + + 3’ juncon + - - M 5’ juncon Stable [mCherry+ / ZG1+] 1000bp 1500bp 12 13 14 15 c Reference Conservaon 5’ juncon 3’ juncon d + + + + - + + + - + + + M Cas9 Donor COSMC sgRNA2 1500bp 6000bp 5000bp Pool Clone - + - + Juncon PCR 1 2 3 4 c d e Relave COSMC copy number 0.0 0.2 0.4 0.6 0.8 1.0 1234568 11 12 13 14 16 18 19 20 23 26 28 33 37 38 39 42 44 45 47 50 51 52 53 56 57 58 61 62 63 64 65 69 77 81 84 86 106 108 CHO-S e Relave mCherry copy number 0 1 2 3 4 5 1234568 11 12 13 14 16 18 19 20 23 26 28 33 37 38 39 42 44 45 47 50 51 52 53 56 57 58 61 62 63 64 65 69 77 81 84 86 106 108 Figure 1 \| Targeted integration into COSMC locus using CRISPR/Cas9. (a) Schematic illustration of the targeting strategy for the specific locus of interest. Donor plasmid consists of three parts: short homology arms flanking sgRNA target site cleaved by Cas9 (red triangle), mCherry and neomycin resistance gene expression cassettes inside homology arms, and ZsGreen1-DR expression cassette outside homology arms. Upon DSBs induced by CRISPR/Cas9, HDR-mediated repair can be used to insert a total size of 3.7 kb of expression cassettes through recombination of the target locus with donor plasmids. Primer position for 59/39 junction PCR is indicated. (b) Agarose gel of 59/39 junction PCR on transiently transfected cells and stable cell pools. An asterisk indicates the use of linearized donor plasmid. M, 1 kb DNA ladder (c) Sanger sequencing of the 59/39 junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. Results Development of a HDR mediated targeted integration platform for CHO. To facilitate improved construction of rCHO cell lines, we aimed at developing a CRISPR and HDR mediated targeted integra- tion system to obtain controlled and precise integration of transgenes. The system is based on donor plasmids harboring short homology arms, which flank the Cas9 cleavable sgRNA target site, used as the integration sites (Fig. 1a). The donor plasmids were constructed as follows: Regardless of loci and sgRNAs, the homology arms were designed to be exactly next to the whole 23 bp of sgRNA genomic target sequences (Supplementary Table 1). The length of each 59 and 39 homology arm was set at 750 bp unless otherwise mentioned. An mCherry expression cassette and a neomycin resistance gene expres- sion cassette were placed within the homology arms. A ZsGreen1-DR expression cassette was placed outside the homology arms to detect random integration events. Upon correct targeting of the donor plasmids to the desired locus via HDR, cells will lose ZsGreen1-DR expression and only express mCherry together with neomycin resis- tance. If cells express both mCherry and ZsGreen1-DR after neomycin selection, random integration of the donor plasmid has occurred. This assumption was applied in order to enrich for targeted integrants by excluding clones expressing ZsGreen1-DR. To evaluate correct inte- gration by HDR, 59/39 junction PCR amplicons were designed for each target site (Fig. 1a). We targeted a total of four sites in three different genomic loci of CHO cells; one site in C1GALT1C1 (COSMC) encod- ing the C1GALT1-specific chaperone 1, two sites in Mgat1 encoding the mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyl- transferase, and one site in LdhA encoding the lactate dehydrogenase A. Application of targeted integration platform to the functional hemi- zygous locus, Mgat1. The Mgat1 locus was selected as a second inte- gration site to test the applicability of the targeted integration system at a different genomic locus. Some CHO cell lines contain one func- tional Mgat1 allele that renders this locus more prone to be disrupted by chemical mutagenesis or genome editing tools, leading to func- tional knockout of Mgat121,22. Since Mgat1 adds N-acetylglucosamine Precise targeted integration of transgene at the COSMC locus in CHO cells. The COSMC locus has previously been effectively modified by CRISPR/Cas9 system in CHO cells20. Results Out-out PCR specific for two Mgat1 integration sites veri- fied the correct targeting of target integration unit accompanying the expected size of amplicons (wild type amplicon: 1.9 kb and 2.0 kb 1 target integration unit: 3.7 kb < 5.6 kb and 5.7 kb; Percentage of out- out PCR positive clones from 59/39 junction PCR positive clones 5 90% and 100%, i.e. 29.0% and 27.1% in total; Fig. 2f, Lane 4 and 8, Table 1). As a result, targeting efficiencies of 10.2% and 16.4% for the sgRNA1 and sgRNA5 target sites respectively, were obtained (Table 1). As observed in COSMC locus, Mgat1 locus was also com- pletely disrupted in most targeted clonal cells (20 out of 25 clones), which showed a relative Mgat1 copy number of close to 0 compared with the Mgat1 in CHO-S wild type cells (Fig. 2g, left panel). The mCherry gene dosage was found to be consistent amongst clones except three clones, as shown by relative quantification of mCherry gene (Fig. 2g, right panel). Precise targeted integration at the Mgat1 locus determined that this platform was not restricted to the COSMC locus and/or COSMC-targeting sgRNA. g g The amount of Mgat1-disrupted cells in two stable pools targeted for either sgRNA1 or sgRNA5 site was estimated by analyzing fluorescein labeled RCA-I (F-RCA-I) stained cells (Fig. 2e). In wild type CHO-S cells, the majority of cells (. 94%) were stained with green fluorescent F-RCA-I (Supplementary Fig. 2a). Likewise, the stable pools, only transfected with donor plasmid, showed a complete shift from green fluorescence negative (Q3 1 Q4) to green fluorescence positive popu- lation (Q1 1 Q2) upon F-RCA-I staining, indicating that the majority of cells have functional MGAT1 as expected. On the other hand, the stable pools transfected with Cas9, sgRNA, and donor plasmid revealed a fraction of 4.6–12.5% of green fluorescence negative cells regardless of mCherry expression from donor plasmid, generating red fluorescence. It demonstrated the presence of cells with knockout of the Mgat1 gene (Fig. 2e). Next, 59/39 junction PCR analysis was applied to evaluate the percentage of target specific knock-ins following isolation of G418- resistant and mCherry positive/ZsGreen1-DR negative clones. In con- trast to the high efficiency observed in COSMC locus, we did not obtain junction PCR positive clones, indicating very low efficiency of targeted integration in the Mgat1 locus (Table 1). Results to the Man5GlcNAc2 (Man5) N-glycan structure, Mgat1-disrupted CHO cell lines are capable of producing recombinant proteins with terminal mannose residues and show resistance to Ricinus communis agglutinin-I (RCA-I), a cytotoxic lectin that binds Man5GlcNAc222. In order to select efficient sgRNA target sites for the Mgat1 locus, the indel efficiency of five sgRNAs was analyzed by deep sequenc- ing (Fig. 2a). At post-transfection with Cas9 and sgRNA expression vectors, the five tested sgRNAs generated detectable range of indels from 10.0% to 20.7% in the Mgat1 locus (Fig. 2b). Two sgRNAs, sgRNA1 and sgRNA5, were selected for targeted integration into the Mgat1 locus. CHO-S cells were transfected with Cas9, sgRNA, and donor plasmid to generate two cell pools, one for each sgRNA. Junction PCR analysis of the stable selected pools were performed (Fig. 2c, Lane 9–16) and compared with junction PCR analysis on transiently transfected cells (Fig. 2c, Lane 1–8). Similar to the COSMC locus, 59/39 junction PCR was only positive for the stable pools (Fig. 2c, Lane 10 and 12 for sgRNA1 site; Lane 14 and 16 for sgRNA5 site). Furthermore, stable cells expressing both mCherry and Zsgreen1-DR, denoted by 11, showed less clean and intense bands than cells expressing only mCherry, denoted by 1-, which indicated incomplete integration (Fig. 2c, Lane 17–24). The sequencing results of 59/39 junction PCR amplicons confirmed precise gene insertion occurring at the Mgat1 locus (Fig. 2d). with Cas9, sgRNA, and donor plasmid (Supplementary Fig. 2c). F- RCA-I staining of RCA-I enriched cell pools resulted in the major fraction of green fluorescence negative cells preserved in the popu- lation of Q3 1 Q4, indicating efficient enrichment for the Mgat1- disrupted population (Fig. 2e). Deep sequencing of RCA-I enriched pools confirmed this observation with indel frequencies of 83.5% and 99.0% for the sgRNA1 and sgRNA5 target sites, respectively (Supplementary Fig. 2d). Thus, lectin selection can be applied to increase the frequency of Mgat1-disrupted clones. RCA-I resistant clones were then isolated from two RCA-I resistant stable cell pools in order to analyze Mgat1 targeted integrants. 59/39 junction PCR analysis of clonal cells revealed the presence of 32.3% and 27.1% PCR positive clones for sgRNA1 and sgRNA5 target sites, respectively (Table 1). Results The chromatogram sequence of junction PCR amplicon was compared with the reference sequence at the genome-donor boundaries. (d) Agarose gel of out-out PCR results of stable cell pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (1.6 kb) or targeted integration (5.3 kb). (e) Relative copy number of COSMC and mCherry regions in clonal cells. Each plot shows the relative copy number of each region in comparison to the reference sample. Genomic DNA of wild type CHO-S and Clone #1 and was used as the reference for COSMC and mCherry region, respectively (shown in red). The error bars represent the standard deviations (n $ 3). SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 3 www.nature.com/scientificreports Table 1 \| Summary of targeted integration Target locus Percentage of mCherry1/ ZsGreen1-DR- populationa (%) Total clone number (mCherry1/ZsGreen1-DR-) 59/39 Junction PCR positive Out/Out PCR positivec Targeting efficiencyd (%) COSMC 45.7 6 2.6 115 83 70 27.8 Mgat1b (Before RCA-I enrichment) 62.2 6 2.7/41.1 6 1.0 124/37 0/0 N/A N/A Mgat1b (After RCA-I enrichment) 35.3 6 2.1/60.4 6 6.6 31/59 10/16 9/16 10.2/16.4 LdhA 45.2 6 2.9 61 10 N/A 7.4 aThe numbers indicate the percentage of mCherry1/ZsGreen1-DR- cell population in each stable cell pool measured by imaging cell cytometer. Average values from three independent experiments 6 SD are shown. bTwo numbers separated by diagonal lines indicate the results from two sgRNA target sites (sgRNA1/sgRNA5). cPositive indicates expected size of PCR products occurring by targeted integration. dTargeting efficiency was obtained by multiplying the percentage of mCherry1/ZsGreen1-DR- cell population by the percentage of out/out PCR positive clones amongst total mCherry1/ZsGreen1-DR- clones investigated. In case of LdhA locus, out/out PCR results were not available. Therefore, 59/39 junction PCR results were used instead. aThe numbers indicate the percentage of mCherry1/ZsGreen1-DR- cell population in each stable cell pool measured by imaging cell cytometer. Average values from three independent experiments 6 SD are shown. bTwo numbers separated by diagonal lines indicate the results from two sgRNA target sites (sgRNA1/sgRNA5). cPositive indicates expected size of PCR products occurring by targeted integration. dTargeting efficiency was obtained by multiplying the percentage of mCherry1/ZsGreen1-DR- cell population by the percentage of out/out PCR positive clones amongst total mCherry1/ZsGreen1-DR- clones investigated. In case of LdhA locus, out/out PCR results were not available. Therefore, 59/39 junction PCR results were used instead. Results (Top) sgRNA1 target site (Bottom) sgRNA Mgat1 locus: sgRNA5 // // sgRNA1 sgRNA2 sgRNA3 sgRNA4 a b c d - - - - - - + + - - + + 5’ juncon Cas9 Donor 5 Mgat1 sgRNA1 Transient - - - - M M Mgat1 sgRNA5 - - + + M - + - + - - + + - - - - Stable - - + - + - - + + 3’ juncon - - - M 5’ juncon - - - - + + + - - - + + - 3’ juncon + - - M 5’ juncon + - + - - + - + Donor 1 - - - - + + + + - - - - Stable [Sorted] ++ ++ +- +- ++ ++ +- +- - + + - + - + + - + - + + - + - + + - + + + - + + - M + + + + - + + - + + - - - - 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ e MiSeq Mgat1 sgRNA3 sgRNA2 sgRNA1 sgRNA4 0% 20% 40% 60% 80% 100% wt sequence Indel Replicate #1 Replicate #2 sgRNA3 sgRNA2 sgRNA5 sgRNA4 sgRNA5 sgRNA1 - F-RCA + F-RCA Red ﬂuorescence intensity Green ﬂuorescence intensity Cas9 Donor1 Mgat1 sgRNA1 Mgat1 sgRNA5 - - - - + + - - + + - + - - + - Donor5 + + - - RCA selecon - - - - - + + - + + + + - + - + 5’ Juncon 3’ Juncon Donor1 + Cas9 + sgRNA1 Donor5 + Cas9 + sgRNA5 Reference Conservaon Reference Conservaon 5’ Juncon 3’ Juncon Reference Conservaon Reference Conservaon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1000bp 1500bp 89.5 % 91.0 % 0.4 % 0.6 % 12.5 % 4.6 % Q1 Q2 Q3 Q4 b a - F-RCA + F-RCA Red ﬂuorescence intensity Green ﬂuorescence intensity Cas9 Donor1 Mgat1 sgRNA1 Mgat1 sgRNA5 - - - - + + - - + + - + - - + - Donor5 + + - - RCA selecon - - - - - + + - + + + + - + - + 89.5 % 91.0 % 0.4 % 0.6 % 12.5 % 4.6 % Q1 Q2 Q3 Q4 d e 5’ Juncon 3’ Juncon Donor1 + Cas9 + sgRNA1 Donor5 + Cas9 + sgRNA5 Reference Conservaon Reference Conservaon 5’ Juncon 3’ Juncon Reference Conservaon Reference Conservaon d - - - - - + + + - + + + M - - - - 2000bp 6000bp 5000bp Cas9 Donor1 Mgat1 sgRNA1 Mgat1 sgRNA5 Donor5 + + + + Pool Clone Pool Clone + + + + - + + + - - - - - + + + - - - - M Juncon PCR - + - + - + - + 1 2 3 4 5 6 7 8 f Relave Mgat1 copy number (sgRNA5 target site) 0.0 0.2 0.4 0.6 0.8 1.0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 CHO-S Relave mCherry copy number 0 1 2 3 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Relave Mgat1 copy number (sgRNA1 target site) 0.0 0.2 0.4 0.6 0.8 1.0 2 3 4 5 6 7 8 9 10 CHO-S Relave mCherry copy number 0 1 2 3 4 2 3 4 5 6 7 8 9 10 g f Relave Mgat1 copy number (sgRNA5 target site) 0.0 0.2 0.4 0.6 0.8 1.0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 CHO-S Relave Mgat1 copy number (sgRNA1 target site) 0.0 0.2 0.4 0.6 0.8 1.0 2 3 4 5 6 7 8 9 10 CHO-S g g g Figure 2 \| Targeted integration into Mgat1 locus using CRISPR/Cas9. Results It is likely that most cells belonging to red fluorescence positive/green fluorescence negative (Q4) population upon F-RCA-I staining correspond to mCherry expressors with NHEJ mutation in Mgat1 locus, and not the targeted integrants. Hence, we used RCA-I lectin selection in order to enrich for Mgat1-disrupted clones occurred by targeted integration. RCA-I treatment effectively induced cell growth arrest and subsequent cell death in wild type CHO-S cells, implying RCA-I induced cyto- toxicity (Supplementary Fig. 2b). 7 days post-RCA-I treatment, RCA-I resistant cells grew to confluency only in the stable pools transfected Application of targeted integration platform to the LdhA locus. We also tested the current integration strategy on LdhA locus. LdhA encodes the A subunit of lactate dehydrogenase that is responsible for interconversion of pyruvate and lactate, one of the main accumulated waste products during CHO cell culture. LdhA is therefore a pro- mising metabolic target gene for knockout in order to enhance culture performance. Five sgRNAs were designed for targeting exon 1, exon 3, exon 4, and exon 5 of the LdhA locus (Fig. 3a), followed by deep sequencing analysis to compare the indel efficiency (Fig. 3b). Indel frequencies were reported from 7.6% to 15.9%. Based on this, sgRNA2 target site was selected for homology arm design and integration site. Comparable to the previous loci tested, co- transfection of the donor plasmid with Cas9 and sgRNA plasmid resulted in precise targeted integration of donor DNA into the LdhA locus. It was only observed in the stable selected pool as determined by junction PCR analysis with expected DNA bases at the genome-donor boundaries (Fig. 3c, Lane 6 and 8, Fig. 3d). Amongst 61 isolated clonal cells, 10 clones were identified as both junction PCR positive (16.4%, resulting in targeting efficiency of 7.4%, Table 1). Results In contrast to relative copy number of COSMC and Mgat1 SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 4 www.nature.com/scientificreports Relave Mgat1 copy number (sgRNA5 target site) 0.0 0.2 0.4 0.6 0.8 1.0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 CHO-S Relave mCherry copy number 0 1 2 3 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Mgat1 locus: sgRNA5 // // sgRNA1 sgRNA2 sgRNA3 sgRNA4 a b c d - - - - - - + + - - + + 5’ juncon Cas9 Donor 5 Mgat1 sgRNA1 Transient - - - - M M Mgat1 sgRNA5 - - + + M - + - + - - + + - - - - Stable - - + - + - - + + 3’ juncon - - - M 5’ juncon - - - - + + + - - - + + - 3’ juncon + - - M 5’ juncon + - + - - + - + Donor 1 - - - - + + + + - - - - Stable [Sorted] ++ ++ +- +- ++ ++ +- +- - + + - + - + + - + - + + - + - + + - + + + - + + - M + + + + - + + - + + - - - - 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ e MiSeq Mgat1 sgRNA3 sgRNA2 sgRNA1 sgRNA4 0% 20% 40% 60% 80% 100% wt sequence Indel Replicate #1 Replicate #2 sgRNA3 sgRNA2 sgRNA5 sgRNA4 sgRNA5 sgRNA1 - F-RCA + F-RCA Red ﬂuorescence intensity Green ﬂuorescence intensity Cas9 Donor1 Mgat1 sgRNA1 Mgat1 sgRNA5 - - - - + + - - + + - + - - + - Donor5 + + - - RCA selecon - - - - - + + - + + + + - + - + 5’ Juncon 3’ Juncon Donor1 + Cas9 + sgRNA1 Donor5 + Cas9 + sgRNA5 Reference Conservaon Reference Conservaon 5’ Juncon 3’ Juncon Reference Conservaon Reference Conservaon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1000bp 1500bp - - - - - + + + - + + + M - - - - 2000bp 6000bp 5000bp Cas9 Donor1 Mgat1 sgRNA1 Mgat1 sgRNA5 Donor5 + + + + Pool Clone Pool Clone + + + + - + + + - - - - - + + + - - - - M Juncon PCR - + - + - + - + 1 2 3 4 5 6 7 8 Relave Mgat1 copy number (sgRNA1 target site) 0.0 0.2 0.4 0.6 0.8 1.0 2 3 4 5 6 7 8 9 10 CHO-S Relave mCherry copy number 0 1 2 3 4 2 3 4 5 6 7 8 9 10 89.5 % 91.0 % 0.4 % 0.6 % 12.5 % 4.6 % Q1 Q2 Q3 Q4 f g 2 \| Targeted integration into Mgat1 locus using CRISPR/Cas9. Results (a) Illustration of the five sgRNA target genomic sites in Mgat1 ncy in Mgat1 locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing s. The genomic regions covering sgRNA target sites were amplified, then subjected to Miseq analysis. The percentage of wt and ind ed in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samp 9/39 junction PCR on transiently transfected cells and stable cell pools. (11) Stable cells expressing both mCherry and Zsgreen1-D pressing only mCherry. (d) Sanger sequencing of the 59/39 junction PCR amplicons. Amplicons from the stable cell pools were purif ced after PCR amplification. M, 1 kb DNA ladder. (e) Population analysis of Mgat1 disrupted cells by F-RCA-I staining. Based cence intensity of stable cell pools, which was further selected with RCA-I or not, alteration of fluorescence intensity was analyzed g. Each scatter plot was divided by four quadrants, denoted by Q1 to Q4. Q3 and Q4 populations, marked by red squares, represent n th F-RCA-I, indicating functional knockout of Mgat1 locus. Numerals below the red squares show the percentage of Q3 and Q4. (f t PCR results of stable pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of for sgRNA1 target site; 1.9 kb for sgRNA5 target site) or targeted integration (5.6 kb for sgRNA1 target site; 5.5 kb for sgRNA5 e copy number of Mgat1 and mCherry regions in clonal cells, as described in Fig. 1(e). Results SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 5 www.nature.com/scientificreports LdhA locus: a b c d 1 2 3 4 5 6 7 // // sgRNA2 Transient Stable - + - + - - + + - - + + M M 5’ juncon Cas9 Donor LdhA sgRNA 1500bp 2000bp + - + - + + 3’ juncon - - M 5’ juncon + + + + sgRNA1 sgRNA3 sgRNA4 sgRNA5 Reference Conservaon 3’ juncon 5’ juncon MiSeq LdhA sgRNA3 sgRNA2 sgRNA1 sgRNA4 0% 20% 40% 60% 80% 100% wt sequence Indel Replicate #1 Replicate #2 sgRNA3 sgRNA2 sgRNA5 sgRNA4 sgRNA5 sgRNA1 1 2 3 4 5 6 7 8 e er mber 3 LdhA locus: a 1 2 3 4 5 6 7 // // sgRNA2 sgRNA1 sgRNA3 sgRNA4 sgRNA5 a sgRNA2 sgRNA1 locus, most LdhA targeted clonal cells (eight out of 10 clones) yielded a partial number of relative LdhA copy number, indicating heterozygous disruptions at the LdhA locus caused by monoallelic knock-ins (Fig. 3e, left panel). Unexpectedly, two clones (clone #4 and #5) had unmodified copy numbers of LdhA relative to CHO-S wild type cells, probably due to non-clonal cell population or chromosomal LdhA locus: b c d 1 2 3 4 5 6 7 // // Transient Stable - + - + - - + + - - + + M M 5’ juncon Cas9 Donor LdhA sgRNA 1500bp 2000bp + - + - + + 3’ juncon - - M 5’ juncon + + + + sgRNA3 Reference Conservaon 3’ juncon 5’ juncon MiSeq LdhA sgRNA3 sgRNA2 sgRNA1 sgRNA4 0% 20% 40% 60% 80% 100% wt sequence Indel Replicate #1 Replicate #2 sgRNA3 sgRNA2 sgRNA5 sgRNA4 sgRNA5 sgRNA1 1 2 3 4 5 6 7 8 e Relave LdhA copy number 0.0 0.5 1.0 2 3 4 5 6 7 8 9 10 CHO-S 1 Relave mCherry copy number 0 1 2 3 2 3 4 5 6 7 8 9 10 1 Figure 3 \| Targeted integration into LdhA locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in LdhA locus. (b) Indel frequency in LdhA locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNA. The genomic regions covering sgRNA target sites were amplified, then subjected to deep sequencing analysis using Miseq. Results (a) Illustration of the five sgRNA target genomic sites in Mgat1 locus. (b) Indel frequency in Mgat1 locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNAs. The genomic regions covering sgRNA target sites were amplified, then subjected to Miseq analysis. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. (c) Agarose gel of 59/39 junction PCR on transiently transfected cells and stable cell pools. (11) Stable cells expressing both mCherry and Zsgreen1-DR; (1-) stable cells expressing only mCherry. (d) Sanger sequencing of the 59/39 junction PCR amplicons. Amplicons from the stable cell pools were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Population analysis of Mgat1 disrupted cells by F-RCA-I staining. Based on red/green fluorescence intensity of stable cell pools, which was further selected with RCA-I or not, alteration of fluorescence intensity was analyzed upon F-RCA-I staining. Each scatter plot was divided by four quadrants, denoted by Q1 to Q4. Q3 and Q4 populations, marked by red squares, represent negative stained cells with F-RCA-I, indicating functional knockout of Mgat1 locus. Numerals below the red squares show the percentage of Q3 and Q4. (f) Agarose gel of out-out PCR results of stable pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (2.0 kb for sgRNA1 target site; 1.9 kb for sgRNA5 target site) or targeted integration (5.6 kb for sgRNA1 target site; 5.5 kb for sgRNA5 target site). (g) Relative copy number of Mgat1 and mCherry regions in clonal cells, as described in Fig. 1(e). (Top) sgRNA1 target site (Bottom) sgRNA5 target site. Results The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. Investigation of target specific knock-in in transiently transfected and stable cell pools analyzed by (c) Agarose gel of 59/39 junction PCR (d) Sanger sequencing of the 59/39 junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Relative copy number of LdhA and mCherry regions in clonal cells, as described in Fig. 1(e). REPORTS \| 5 8572 \| DOI 10 1038/ 08572 6 LdhA locus: sgRNA3 b MiSeq LdhA sgRNA3 sgRNA2 sgRNA1 sgRNA4 0% 20% 40% 60% 80% 100% wt sequence Indel Replicate #1 Replicate #2 sgRNA3 sgRNA2 sgRNA5 sgRNA4 sgRNA5 sgRNA1 b b wt sequence d Reference Conservaon 3’ juncon 5’ juncon c Transient Stable - + - + - - + + - - + + M M 5’ juncon Cas9 Donor LdhA sgRNA 1500bp 2000bp + - + - + + 3’ juncon - - M 5’ juncon + + + + 1 2 3 4 5 6 7 8 d d c 3’ juncon e Relave mCherry copy number 0 1 2 3 2 3 4 5 6 7 8 9 10 1 Relave LdhA copy number 0.0 0.5 1.0 2 3 4 5 6 7 8 9 10 CHO-S 1 Figure 3 \| Targeted integration into LdhA locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in LdhA locus. (b) Indel frequency in LdhA locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNA. The genomic regions covering sgRNA target sites were amplified, then subjected to deep sequencing analysis using Miseq. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. Investigation of target specific knock-in in transiently transfected and stable cell pools analyzed by (c) Agarose gel of 59/39 junction PCR (d) Sanger sequencing of the 59/39 junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Relative copy number of LdhA and mCherry regions in clonal cells, as described in Fig. 1(e). Results o LdhA locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in LdhA locus. (b) Indel (Fig. 3e, left panel). Unexpectedly, two clones (clone #4 and #5) had unmodified copy numbers of LdhA relative to CHO-S wild type cells, probably due to non-clonal cell population or chromosomal locus, most LdhA targeted clonal cells (eight out of 10 clones) yielded a partial number of relative LdhA copy number, indicating heterozygous disruptions at the LdhA locus caused by monoallelic knock-ins SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 6 On-target Oﬀ-target1 Target sequence GAATATGTGAGTGTGGATGGAGG GAAGCTCTGAGTGTGGATGGAGG GGAGGTATGAGTGTGGATGGAGG ACTAATGTGAGTGTGGATGGTGG ACGTAATTGAGTGTGGATGGCGG AATCACTTGAGTGTGGATGGGGG Oﬀ-target2 Oﬀ-target3 Oﬀ-target4 Oﬀ-target5 COSMC Mismatches (nt) 3 4 4 5 5 - Posion NW_003614471 (-) 528266 NW_003613631 (-) 2008240 NW_003616800 (+) 22442 NW_003613801 (-) 473486 NW_003613869 (+) 1436175 NW_003628455 (-) 847 0% 20% 40% 60% 80% 100% wt sequence Indel On-target Oﬀ-target1 Target sequence GCTGGGCACTGATACCGACAAGG GCTGGGCACTGATGCCGACAGGG GCTGGACACTGATGCCGACAAGG GTTGGGCACTGATGCCGACAAGG TCCTGGCACTGATATCGACACGG Oﬀ-target2 Oﬀ-target3 Oﬀ-target4 LdhA Mismatches (nt) 1 2 2 4 - Posion NW_003613933 (-) 768769 NW_003614158 (+) 1034506 NW_003616055 (+) 76807 NW_003613598 (-) 1611480 NW_003614690 (+) 181885 0% 20% 40% 60% 80% 100% On-target Oﬀ-target1 Target sequence GTGGAGTTGGAGCGGCAGCGGGG ACGGAGATGGAGAGGCAGCGGGG GAGGAAATGGAGTGGCAGCGGGG GCAGACTTGGAGCTGCAGCGTGG CAGAAGCTGGAGCAGCAGCGAGG Oﬀ-target2 Oﬀ-target3 Oﬀ-target4 Mgat1 sgRNA5 site Mismatches (nt) 4 4 4 5 - Posion NW_003613681 (-) 1678866 NW_003613686 (+) 2563608 NW_003614274 (+) 602970 NW_003614984 (+) 61961 NW_003614027 (+) 1191945 On-target [RCA+] 0% 20% 40% 60% 80% 100% On-target Oﬀ-target1 Target sequence GCTCACACCCTTACGGCCAAAGG GCAAGCTCCCTTACCGCCAAGGG GCACCAGCCCTTACTGCCAAGGG Oﬀ-target2 Mgat1 sgRNA1 site Mismatches (nt) 5 5 - Posion NW_003613771 (-) 915191 NW_003613798 (-) 376677 NW_003614027(-) 1192681 0% 20% 40% 60% 80% 100% On-target [RCA+] a b c d > 2800 x > 5600 x 132 x > 2800 x > 1400 x > 8000 x > 8000 x 206 x 9.3 x > 10000 x > 10000 x 49 x 8.4 x 32 x 1060 x Figure 4 \| Comparison of mutation rates at on-target and potential off-target sites for (a) COSMC sgRNA2, (b) Mgat1 sgRNA1, (c) Mgat1 sgRNA5, and (d) LdhA sgRNA2. Information on position of sequences consists of contig, strand, and position within the corresponding contig. Mismatched bases and PAM sequences are shown in red and blue, respectively. Genomic DNA extracted from stable cell pools transfected with Cas9, sgRNA, and donor plasmid was used as template for PCR. PCR amplicons spanning corresponding on-target sites and potential off-target sites were subjected to deep sequencing analysis using Miseq. Results Indel frequencies at off-target sites of Mgat1 sgRNA1/sgRNA5 site were based on genomic DNA extracted from RCA-I resistant cell pools. Fold differences in indel frequencies between on-target and off-target sites were presented next to the bar plot. COSMC a b d Figure 4 \| Comparison of mutation rates at on-target and potential off-target sites for (a) COSMC sgRNA2, (b) Mgat1 sgRNA1, (c) Mgat1 sgRNA5, and (d) LdhA sgRNA2. Information on position of sequences consists of contig, strand, and position within the corresponding contig. Mismatched bases and PAM sequences are shown in red and blue, respectively. Genomic DNA extracted from stable cell pools transfected with Cas9, sgRNA, and donor plasmid was used as template for PCR. PCR amplicons spanning corresponding on-target sites and potential off-target sites were subjected to deep sequencing analysis using Miseq. Indel frequencies at off-target sites of Mgat1 sgRNA1/sgRNA5 site were based on genomic DNA extracted from RCA-I resistant cell pools. Fold differences in indel frequencies between on-target and off-target sites were presented next to the bar plot. instability. Heterozygous modification at LdhA locus led to decrease in mRNA expression levels of LdhA by approximately 50%, resulting in reduction of protein expression levels as well (Supplementary Fig. 3). Notably, deep sequencing analysis of nonintegrated allele revealed evidence of in-frame deletions created in LdhA targeted clonal cells, which is unlikely to lead to a loss-of-function (Supplementary Fig. 4). As a result of heterozygous modification in LdhA locus, we could not obtain expected size of PCR products from out-out PCR due to existence of a residual allele. From analysis of the relative mCherry copy number, similar results of consistent copy number across LdhA target clonal cells were obtained (Fig. 3e, right panel). Targeted integration at the LdhA locus was achieved using the current platform, which confirmed that this method was also applicable at a diploid locus. generated indels at frequencies of 56.8% (Fig. 4a). A similar off- target tendency was observed for the Mgat1 sgRNA1, but unlike sgRNA1, Mgat1 sgRNA5 displayed off-target indel generation at a frequency of 10.7% at off-target 1 (Fig. 4b,c). RCA-I enrichment for Mgat1-disrupted cells drastically increased an indel frequency from 16.4% and 37.3% to 83.4% and 98.9% at sgRNA1 and sgRNA5 on- target sites (Fig. 4b,c), but did not induce additional off-target indel mutations (Supplementary Fig. 5). Three out of four off-target sites for the LdhA sgRNA displayed off-target activity (Fig. 4d). Results Targeted and random integrants isolated from the stable cell pool nsfected with Cas9, sgRNA, and donor plasmid targeting COSMC locus were cultivated to assess (a) RFU and (b) relative specific growth rate (m rage mean intensity of mCherry fluorescence measured by imaging cell cytometer was used for RFU. The specific growth rate was calculated from t of viable cell concentration against culture time from day 0 to day 3. Each measured value was normalized by that of the clone number 1. Center li w the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range fr 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n 5 41, 40 sample points. Each sample poin resents average value of measurements at 3–5 different passages during cultivation for a month. (CV) coefficient of variation. Figure 5 \| Effect of targeted integration on homogeneous expression of GOI. Targeted and random integrants isolated from the stable cell pool transfected with Cas9, sgRNA, and donor plasmid targeting COSMC locus were cultivated to assess (a) RFU and (b) relative specific growth rate (m). Average mean intensity of mCherry fluorescence measured by imaging cell cytometer was used for RFU. The specific growth rate was calculated from a plot of viable cell concentration against culture time from day 0 to day 3. Each measured value was normalized by that of the clone number 1. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n 5 41, 40 sample points. Each sample point represents average value of measurements at 3–5 different passages during cultivation for a month. (CV) coefficient of variation. Figure 5 \| Effect of targeted integration on homogeneous expression of GOI. Targeted and random integrants isolated from the stable cell pool transfected with Cas9, sgRNA, and donor plasmid targeting COSMC locus were cultivated to assess (a) RFU and (b) relative specific growth rate (m). Average mean intensity of mCherry fluorescence measured by imaging cell cytometer was used for RFU. The specific growth rate was calculated from a plot of viable cell concentration against culture time from day 0 to day 3. Results Sites harboring one- or two-base mismatches contained indel mutations at a frequency between 0.9% and 5.1%. Comparing the indel frequencies observed at the off-target sites with those at the on- target site, all on-target frequencies were 8.4-fold higher as a minimum. Upon enrichment for positive integrant pools by RCA-I selection, on-target to off-target frequency difference was greatly increased from 9.3-fold to .10,000-fold (Fig. 4b,c). Due to these on-target to off-target frequency differences, integration events at the potential off-target sites will be less likely to occur. PCR amp- lification of regions, covering off-target sites in mCherry positive/ ZsGreen1-DR negative targeted clones, showed no off-target integra- tions, which supports this hypothesis (Supplementary Fig. 6). The low mutation level of overall off-target sites supported the reliability of the use of CRISPR/Cas9 system for targeted integration. Off-target effects associated with CRISPR/Cas9 mediated targeted integration. To assess potential off-target mutations introduced by the CRISPR/Cas9 activity, we first identified the most probable potential off-target sites of each sgRNA target sequence within the CHO genome. Prediction of off-target sites was based on three criteria (see Methods section), followed by analysis of the NHEJ- mediated indel frequencies at the on-target and off-target sites in genomic DNA isolated from stable cell pools transfected with Cas9, sgRNA, and donor plasmid by deep sequencing (Fig. 4). A low level (, 0.5%) of off-target mutations were detected at all five putative off-target sites for the COSMC sgRNA, despite high levels of CRISPR/Cas9 activity at the corresponding on-target site, which Correlation between targeted integration and homogeneous expres- sion of GOI. To assess the effect of targeted integration on cell culture SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 7 formance, differences in expression of mCherry and growth rates ween targeted integrants and random integrants were analyzed. Cherry positive/ZsGreen1-DR negative cell population was isolated t bl ll l t f t d ith C 9 RNA2 d d regarded as random integrants regardless of 59/39 junction PCR resu Amongst positive targeted integrants, clones with complete disrupt at the COSMC locus and similar levels of relative mCherry co b l t d (Fi 1 41) C ll th d ure 5 \| Effect of targeted integration on homogeneous expression of GOI. Results 12.5% in total) only from the population of random integrants while cells were expanding, underscoring unstable expression from random integration (Supplementary Fig. 9). These data show that CRISPR and HDR mediated targeted integration allowed stable and reproducible transgene expression in rCHO cell lines without affecting cell growth. Fig. 5b). Interestingly, targeted integrants displayed more stable mCherry expression with 4.4-fold less variation than that of the random integrants (CV: 14.3% versus 63.3%; Fig. 5b), indicating more homogeneous and predictive expressions from site-specific integration clones. The variability in expression levels of random integrants was caused by the influence of both copy number and integration site (Supplementary Fig. 8). Some of random integrants with 2- or 3-fold higher copy number exhibited 2- or 3-fold higher expression levels relative to the representative targeted integration clone. On the other hand, various expression levels were observed in a few random integrants despite similar levels of copy number. Moreover, although the percentage of mCherry positive/ZsGreen1- DR negative cells was initially close to 100% in all clones investigated, it was notably decreased in some clones (five out of 40 clones, i.e. 12.5% in total) only from the population of random integrants while cells were expanding, underscoring unstable expression from random integration (Supplementary Fig. 9). These data show that CRISPR and HDR mediated targeted integration allowed stable and reproducible transgene expression in rCHO cell lines without affecting cell growth. 54.6% for control samples transfected with the donor plasmid only, transfection of multiple plasmids lowered the transfection efficiency considerably. Additionally, expression levels from separate constructs may not be tightly coupled. This potential limitation can be overcome by using the aforementioned single expression vector11 to drive both Cas9 and sgRNA or FACS enrichment to aid selection of transfected cells. As observed in the on-target indel efficiency in stable cells and the targeting efficiency, the higher on-target indel efficiency is likely to lead to higher HDR-mediated integration (Fig. 4, Table 1). Therefore, the aforementioned approaches will aid in the increase in current targeting efficiencies. Moreover, single-strand breaks (SSBs) created by single sgRNA-Cas9 nickases have reported to be capable of inducing homologous recombination in mammalian cells, albeit at reduced frequency compared with wild-type Cas925,26. In conjunction with improvements in targeting efficiency, targeted integration via SSBs-induced homologous recombination will also be an attractive strategy with regard to higher specificity and fidelity. Discussion Transfection efficiency, which was measured based on the fraction of mCherry positive/ZsGreen1-DR positive cells 72 hours post transfection, was in the range of 24.4–34.0% when three plasmids, including donor plasmid, Cas9, and sgRNA plasmid, were co-transfected. Compared with transfection efficiency of 33.0– Site-specific integration of large transgenes during rCHO cell line construction has been impeded by extremely low integration effi- ciencies and specific requirements, including construction of plat- form cell lines, which limit flexible targeting to any desired sites. Here we developed a method for targeted integration by combining the CRISPR/Cas9 system with a donor plasmid in CHO cells. The pro- cedure allowed precise HDR-mediated integration of large gene expression cassettes into desired sites with a targeting efficiency between 7.4–27.8% depending on target locus and CRISPR/Cas9 activity. With the previous targeted integration studies in CHO cells relying on the NHEJ pathway for integration of short (, 100 bp) oligonucleotides16 or linear transgenes17, our approach is liberated from the issues concerning orientation of transgene integration and imperfect ligation caused by degradation of donor ends by exonu- clease activity or end resection of the chromosome. Furthermore, we report additional benefits common to the CRISPR/Cas9 system such as high efficiency, robustness, simplicity, ease of design, and low cost. g y p y g Compared with the successful HDR-mediated integration of trans- genes in human cells using the CRISPR/Cas9 system, which was observed 72 hours after transfection14, we did not detect integration events in transiently expressed cells, but only in stable selected pools although transgene size and experimental conditions were different (Fig. 1b, 2c, and 3c). This result supports intrinsically lower levels of HDR-based transgenesis in CHO cells, which agrees with previous studies16,17. In particular, the Mgat1 locus had non-appreciable fre- quencies of integration before RCA-I enrichment, which showed the locus-to-locus variation in terms of integration frequency (Table 1). Although the CRISPR/Cas9 system generated indel mutations via NHEJ with high efficiency between 16.4–56.8%, the rate of HDR- mediated integration remained relative low (Fig. 4, Table 1). The present targeting efficiency is sufficient for the generation of rCHO cells when paired with drug or phenotypic selection, but requirements for selection process can limit the practical application of targeted integration at certain loci in an actual industrial production process. Results Previous studies have reported that the PAM-proximal 8–13 bp of the target region is crucial for target recognition and cleavage by Cas911,19, and within PAM-proximal region, a single-base mismatch can be acceptable to conserve activity. Furthermore, up to three to five mismatches in the PAM-distal region can be tolerated without impairing the ability to cleave DNA27-29. More recently, genome-wide mapping of Cas9 binding sites using catalytically inactive Cas9 and ChIP-seq analysis has revealed that a 5-nucleotide seed region next to PAM is required for Cas9 binding in vivo and in vitro30. Based on this, we selected potential off-target sites for each target site to meet these criteria, and evaluated off-target mutation rates (Fig. 4). Nine out of 15 potential off-target sites were not affected. In four off-target sites, we detected indels with a frequency of 0.5–1.3%, which was rare enough to allow high fidelity of sgRNAs targeting on-target sites. The last two off-target sites, however, contained detectable off-target cut- tings at a frequency of 5.1% and 10.7%. A recent paper has suggested that in addition to finding off-targets at sites with few mismatches compared with the target sequence, off-targets may also be found at sites with small inserts/deletions compared with the target sequence under certain circumstances31. This type of off-targets have not been considered in the current work, but guide RNA design in the future should aim to identify such additional potential off-target sites so that appropriate care may be taken. Off-target mutations may not be critical to rCHO cell line construction compared with human gene therapy. However, potential problems in connection with off-target mutations could cause unpredictable toxic effects. This latent obstacle could also be mitigated by using approaches to improve on-target specificity of Cas9, such as the double-nicking approach26,29, truncated guide RNAs32, or dimeric CRISPR RNA-guided FokI nucleases33. Discussion Site-specific integration of large transgenes during rCHO cell line construction has been impeded by extremely low integration effi- ciencies and specific requirements, including construction of plat- form cell lines, which limit flexible targeting to any desired sites. Here we developed a method for targeted integration by combining the CRISPR/Cas9 system with a donor plasmid in CHO cells. The pro- cedure allowed precise HDR-mediated integration of large gene expression cassettes into desired sites with a targeting efficiency between 7.4–27.8% depending on target locus and CRISPR/Cas9 activity. With the previous targeted integration studies in CHO cells relying on the NHEJ pathway for integration of short (, 100 bp) oligonucleotides16 or linear transgenes17, our approach is liberated from the issues concerning orientation of transgene integration and imperfect ligation caused by degradation of donor ends by exonu- clease activity or end resection of the chromosome. Furthermore, we report additional benefits common to the CRISPR/Cas9 system such as high efficiency, robustness, simplicity, ease of design, and low cost. Compared with the successful HDR-mediated integration of trans- genes in human cells using the CRISPR/Cas9 system, which was observed 72 hours after transfection14, we did not detect integration events in transiently expressed cells, but only in stable selected pools although transgene size and experimental conditions were different (Fig. 1b, 2c, and 3c). This result supports intrinsically lower levels of HDR-based transgenesis in CHO cells, which agrees with previous studies16,17. In particular, the Mgat1 locus had non-appreciable fre- quencies of integration before RCA-I enrichment, which showed the locus-to-locus variation in terms of integration frequency (Table 1). Although the CRISPR/Cas9 system generated indel mutations via NHEJ with high efficiency between 16.4–56.8%, the rate of HDR- mediated integration remained relative low (Fig. 4, Table 1). The present targeting efficiency is sufficient for the generation of rCHO cells when paired with drug or phenotypic selection, but requirements for selection process can limit the practical application of targeted integration at certain loci in an actual industrial production process. However, the current method has sufficient room for further improvement in targeting efficiency by implementing recent advancements, such as rational design of highly active sgRNAs23, enrichment of cells with genome editing events24, or construction of expression vectors bearing both Cas9 and the sgRNA11 since the CRISPR/Cas9 system is continuously undergoing optimizations to improve activity. Results Each measured value was normalized by that of the clone number 1. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n 5 41, 40 sample points. Each sample point represents average value of measurements at 3–5 different passages during cultivation for a month. (CV) coefficient of variation. performance, differences in expression of mCherry and growth rates between targeted integrants and random integrants were analyzed. mCherry positive/ZsGreen1-DR negative cell population was isolated from stable cell pool transfected with Cas9, sgRNA2, and donor plasmid targeting COSMC locus by limiting dilution, then classified as either targeted integrants or random integrants according to results from 59/39 junction PCR and out-out PCR (Supplementary Fig. 7). Both 59/39 junction PCR and out-out PCR positive clones were defined as targeted integrants, and out-out PCR negative clones were regarded as random integrants regardless of 59/39 junction PCR results. Amongst positive targeted integrants, clones with complete disruption at the COSMC locus and similar levels of relative mCherry copy number were selected (Fig. 1e, n541). Cell growth and expression level of GOI of the two populations was then compared. Both populations displayed similar variations in the relative specific growth rate (CV: 13.8% versus 12.4%; Fig. 5a). An average expression level of targeted integrants was slightly lower than that of random integrants (RFU average: 103 versus 140, p , 0.05; SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 8 Fig. 5b). Interestingly, targeted integrants displayed more stable mCherry expression with 4.4-fold less variation than that of the random integrants (CV: 14.3% versus 63.3%; Fig. 5b), indicating more homogeneous and predictive expressions from site-specific integration clones. The variability in expression levels of random integrants was caused by the influence of both copy number and integration site (Supplementary Fig. 8). Some of random integrants with 2- or 3-fold higher copy number exhibited 2- or 3-fold higher expression levels relative to the representative targeted integration clone. On the other hand, various expression levels were observed in a few random integrants despite similar levels of copy number. Moreover, although the percentage of mCherry positive/ZsGreen1- DR negative cells was initially close to 100% in all clones investigated, it was notably decreased in some clones (five out of 40 clones, i.e. Discussion A further refinement of existing Chinese hamster reference genome and all of the relevant sequence information of CHO host cell lines, together with new data on genome stability at the chromosomal level will facilitate identification of potential target sites as well as a better characterization of the current targeted integration strategy. Moreover, a recent study demonstrated that actively transcribed chromatin struc- ture was preferentially repaired by homologous recombination over NHEJ upon DSB35. This trait fits perfectly into the presented platform and will reinforce the targeting efficiency. Thus, this approach should be able to provide a valuable genetic tool to characterize different potential good integration sites in CHO cells, ultimately leading to a rational construction of production cell lines assuring sustainable and high expression levels. Illumina, Madison, WI) or GeneJET Genomic DNA Purification Kit depending on the purpose of experiment. For single cell clones, DNA was isolated by adding 20 mL of QuickExtract DNA extraction solution when cells were confluent in 96 well plates. The mixture was then incubated at 65uC for 15 min, followed by 5 min incubation at 98uC. Of this mixture, 1–2 mL were used as template for junction PCR and out-out PCR, as described below. For the other experiments, GeneJET Genomic DNA Purification Kit was used to extract DNA from cell pellets up to 5 3 106 cells according to manufacturer’s instructions. 59/39 junction PCR was carried out using DreamTaq DNA polymerase (Thermo Fisher Scientific) by touchdown PCR (95uC for 2 min; 103: 95uC for 30 s, 68uC–58uC (21uC/cycle) for 30 s, 72uC for 2 min; 203: 95uC for 30 s, 58uC for 30 s, 72uC for 2 min; 72uC for 5 min). Out-Out PCR was performed using Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) by touchdown PCR (98uC for 30 s; 203: 98uC for 10 s, 64uC–54uC (20.5uC/cycle) for 30 s, 72uC for 3 min; 303: 98uC for 10 s, 54uC for 30 s, 72uC for 3 min; 72uC for 10 min). PCR primers are listed in Supplementary Table 2. Deep sequencing analysis. Deep sequencing was performed on a MiSeq Benchtop Sequencer (Illumina, San Diego, CA). Libraries were prepared based on Illumina ‘16S Metagenomic Sequencing Library Preparation’ with some modifications. Methods Plasmid design and construction. The CHO codon optimized Cas9 expression vector and sgRNA expression vector applied in this study were constructed as described previously20. The CRISPy bioinformatics tool20 was applied for generating sgRNA target sequences (Supplementary Table 1). Subsequently, single stranded oligos comprising the variable region of the sgRNA (Supplementary Table 2) were synthesized, annealed, and mixed with the expression vector backbone harboring U6 promoter, scaffold RNA sequence, and termination sequence to generate sgRNA expression vectors. p Donor plasmids were constructed with uracil-specific excision reagent (USER) cloning method according to Ref. 36. The different parts of the plasmid were amp- lified with uracil-containing primers and proofreading polymerase PfuX7. 59 and 39 homology arms flanking each sgRNA target sequence were amplified from genomic DNA of CHO-S cells, which were extracted by GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA). EF-1a promoter, NeoR expression cassette (pSV40-NeoR–SV40 pA), and expression vector backbone parts were directly amplified from commercial expression vectors, but ZsGreen1-DR expression cassette (pCMV-ZsGreen1-DR-BGH pA) and mCherry-BGH pA parts were amp- lified from pre-assembled expression vectors that were constructed with USER cloning method using commercial expression vectors. Primer sequences and all plasmids used as PCR templates are listed in Supplementary Table 2 and 3, respect- ively. Following purification of PCR products by agarose gel separation and NucleoSpinH Gel and PCR Clean-up kit (Macherey-Nagel, Du¨ren, Germany), the purified 7 PCR fragments including both homology arms, EF-1a promoter, mCherry- BGH pA, NeoR expression cassette, ZsGreen1-DR expression cassette, and backbone were assembled with USER enzyme (New England Biolabs, Ipswich, MA) and transformed into E. coli Mach1 competent cells (Life Technologies, Leuven, Belgium). Plasmids were harvested from transformants selected on LB agar plates with ampi- cillin. All constructs were verified with sequencing and purified using NucleoBond Xtra Midi EF (Macherey-Nagel) according to manufacturer’s instructions. Batch culture. Exponentially growing cells were inoculated at a concentration of 3.0– 4.0 3 105 cells/mL into Polystyrene transparent square 96-half-deepwell microplates with Sandwich cover (Enzyscreen, Leiden, Netherlands) containing 250–300 mL of culture medium, and the culture plates were then incubated on the Multitron cell (INFORS HT, Bottmingen, Switzerland) at 350 rpm in humidified 5% CO2 at 37uC. Viable cell concentration was estimated using PrestoBlueH Cell Viability Reagent (Life Technologies) according to manufacturer’s instructions. Briefly, 10 mL of PrestoBlue reagent was directly added to 90 mL of the mixture of cells and media in 96 well plates. Ricinus Communis Agglutinin-I (RCA-I) selection and phenotypic analysis of Mgat1 knockout cells. Mgat1 knockout cells were enriched in culture medium with Ricinus Communis Agglutinin-I (RCA-I) selection and phenotypic analysis of Mgat1 knockout cells. Mgat1 knockout cells were enriched in culture medium with 5 mg/mL of RCA-I (Vector Laboratories, Peterborough, UK) using the stable cell pools. After 7 days of selection, cells were adapted to grow in suspension, followed by phenotypic analysis. Cells were stained with NucBlueH Live ReadyProbesH Reagent with and without fluorescein labeled RCA-I (F-RCA-I, Vector Laboratories) at final concentration of 20 mg/ml for 45 min at RT. Cells were then washed three times with fresh culture medium and medium was completely removed. Celigo Imaging Cell Cytometer was used to measure fluorescence using the mask 1 target 1 1 target 2 application as described earlier. Mgat1 knockout cells. Mgat1 knockout cells were enriched in culture medium with 5 mg/mL of RCA-I (Vector Laboratories, Peterborough, UK) using the stable cell pools. After 7 days of selection, cells were adapted to grow in suspension, followed by phenotypic analysis. Cells were stained with NucBlueH Live ReadyProbesH Reagent with and without fluorescein labeled RCA-I (F-RCA-I, Vector Laboratories) at final concentration of 20 mg/ml for 45 min at RT. Cells were then washed three times with Stable cell pools were generated by seeding cells in CELLSTARH 6 well Advanced TC plates (Greiner Bio-one, Frickenhausen, Germany) on day 3 followed by selection process under G418 (500 mg/mL; Sigma-Aldrich). During selection, medium was changed every 3–4 days. After 2 weeks of selection, cells were detached with TrypLE (Life Technologies) and adapted to grow in suspension in non-tissue treated plates or Erlenmeyer flask depending on cell concentrations. For clonal selection, limiting dilution step was followed using the stable cell pools, which were either bulk sorted for mCherry positive/ZsGreen1-DR negative cell population using a BD FACSJazz cell sorter (BD Biosciences, San Jose, CA) or not. 1 cell was seeded per well in 200 ul medium in 96 well plates. The generated colonies were analyzed by a Celigo Imaging Cell Cytometer (Nexcelom Bioscience, Lawrence, MA) applying the Direct cell counting application to check for single colonies. Only the wells with round shaped colonies expressing mCherry were selected for further analysis. fresh culture medium and medium was completely removed. Celigo Imaging Cell Cytometer was used to measure fluorescence using the mask 1 target 1 1 target 2 application as described earlier. Off-target prediction. Discussion However, the current method has sufficient room for further improvement in targeting efficiency by implementing recent advancements, such as rational design of highly active sgRNAs23, enrichment of cells with genome editing events24, or construction of expression vectors bearing both Cas9 and the sgRNA11 since the CRISPR/Cas9 system is continuously undergoing optimizations to improve activity. Transfection efficiency, which was measured based on the fraction of mCherry positive/ZsGreen1-DR positive cells 72 hours post transfection, was in the range of 24.4–34.0% when three plasmids, including donor plasmid, Cas9, and sgRNA plasmid, were co-transfected. Compared with transfection efficiency of 33.0– Cultivation of targeted integrants displayed more stable and highly reproducible expression levels when compared with random inte- grants. Although this result demonstrates the minimal variability in expression level, it is limited to the specific locus and expression cassette. The expression level can depend on the target locus and the cassette design, particularly relating to the promoter used and its susceptibility to silencing. Thus, additional studies in various loci with different transgene expression cassettes are warranted to con- firm the generality of this finding. Despite homogeneous transgene expression levels in targeted integrants, some of the random integ- ration clones exhibited 2- or 3-fold higher expression levels relative to representative targeted integration clone (Fig. 5b, Supplementary Fig. 8). These observations were due to random integration into highly active chromosomal regions with the CHO genome or integ- ration of several copies of the donor plasmid. Given that the ultimate goal for construction of production cell lines is rapid generation of high and stable producers, ensuring comparable quality for regula- tory approval, the selection of ideal transgene insertion sites is highly demanded. Ideal insertion sites, also called ‘Safe harbors’ or ‘Hot spots’, should allow high and stable transcription/expression levels SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 9 www.nature.com/scientificreports of the GOI without perturbing essential regulatory elements or dis- rupting genes which are responsible for the quality of the products. Previous extensive studies on the selection and identification of desirable target sites in human cells provide reliable guidelines to facilitate rapid identification of such ‘‘hot spots’’ in CHO cells13,34. Discussion Genomic regions flanking target genomic sites were amplified using Phusion Hot Start II HF Pfu polymerase (Thermo Fisher Scientific) by standard PCR (98uC for 30 s; 253: 98uC for 10 s, 59uC for 30 s, 72uC for 30 s; 72uC for 10 min), with primers containing overhang sequences compatible with Illumina Nextera XT indexing (forward primer overhang: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG, reverse primer overhang: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG). PCR primers are listed in Supplementary Table 2. After PCR amplification, amplicons were purified using AMPure XP beads (Beckman Coulter, Brea, CA). Illumina Nextera XT Index (Illumina # FC-131-1001) sequencing adapters were integrated into the amplicons by PCR (98uC for 3 min; 83: 95uC for 30 s, 55uC for 30 s, 72uC for 30 s; 72uC for 5 min). The final libraries were purified with AMPure XP beads before quantification. The purified libraries were quantified using the QubitH dsDNA BR Assay Kit (Life Technologies), and the product size within the library was verified by 2% agarose gel. Sequencing was performed as a 151 paired-end run. Sequence data was analyzed using custom python code that compared the sequence data with the expected wild-type sequence from the CHO genome20. In short, it first paired the paired end fastq sequence files. Then, it de-multiplexed the sequence reads based on 59 or 39 similarity to the expected wild-type sequence followed by detection of potential indels to categorize sequences as either wild type or indel-containing. The final product of these steps is a list for each target or off-target with numbers for how often we see the wild type and how often we see indels and the size of these indels. Methods The plates were incubated for . 30 minutes at 37uC, and then fluorescence was measured by microplate reader (Synergy Mx, BioTek Instruments, Inc., Winooski, VT). The resulting fluorescence intensity was used to determine viable cell concentration based on the standard curve created with CHO-S cells. On day 3 after a new passage, cells were incubated with NucBlueH Live ReadyProbesH Reagent (Life Technologies) for 20 minutes, and red and green fluorescence was measured by a Celigo Imaging Cell Cytometer using the mask 1 target 1 1 target 2 application. Cells were identified using the blue fluorescence channel as mask, and the red and green fluorescent channels were used as target 1 and target 2, respectively. For cytometer analysis, about 10,000–20,000 cells were used per sample. Cell culture, transfection, and stable cell line construction. CHO-S cells from Life Technologies were grown in CD CHO medium supplemented with 8 mM L- Glutamine (Life Technologies) and cultivated in a 125-mL Erlenmeyer flask (Sigma- Aldrich, St. Louis, MO) with a working volume of 30 mL. The cells were incubated at 37uC, 5% CO2 with 120 rpm shaking and passaged every 3 days. Cells were transfected with expression vectors encoding CHO codon optimized Cas9, sgRNA targeting the integration site and corresponding donor plasmid. For each sample, 3 3 106 cells were transfected with a total of 3.75 mg of DNA using FreeStyleTM MAX reagent together with OptiPRO SFM medium (Life Technologies) according to manufacturer’s recommendations. 16 hours post transfection, the samples were incubated at 30uC for 32 hours before transferred back to 37uC. Ricinus Communis Agglutinin-I (RCA-I) selection and phenotypic analysis of Mgat1 knockout cells. Mgat1 knockout cells were enriched in culture medium with 22, 1393–1398 (2004). 31. Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014). 3. Wilson, C., Bellen, H. J. & Gehring, W. J. 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Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001). 9. Xu, X. et al. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat. Biotechnol. 29, 735–741 (2011). 10. Carroll, D. Genome engineering with targetable nucleases. Annu. Rev. Biochem. 83, 409–439 (2014). Acknowledgments 11. Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819–823 (2013). g The authors thank Anna Koza for her assistance with the MiSeq experiments and Karen Kathrine Brøndum for her assistance with the FACS. This work was supported by the Novo Nordisk Foundation. g The authors thank Anna Koza for her assistance with the MiSeq experiments and Karen Kathrine Brøndum for her assistance with the FACS. This work was supported by the Novo Nordisk Foundation. g The authors thank Anna Koza for her assistance with the MiSeq experiments and Karen Kathrine Brøndum for her assistance with the FACS. This work was supported by the Novo Nordisk Foundation. 12. Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009). g g 13. Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat. Methods 8, 861–869 (2011). Author contributions 14. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013). J.S.L. and H.F.K. designed the experiments and wrote the manuscript. J.S.L., T.B.K., L.E.P. performed the experiments. J.S.L., L.E.P. and H.F.K. analyzed the data. T.B.K. and L.E.P. commented on the manuscript. 15. Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011). 16. Orlando, S. J. et al. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res. 38, e152 (2010). Ricinus Communis Agglutinin-I (RCA-I) selection and phenotypic analysis of Mgat1 knockout cells. Mgat1 knockout cells were enriched in culture medium with The primers were designed using PrimerQuest (Integrated DNA Technologies, Coralville, IA) against transgene encompassing EF-1a – mCherry, target genes including COSMC, Mgat1 (sgRNA1 and sgRNA5 target site), and LdhA, and reference gene, Vcl (Supplementary Table 2). All primers were validated empirically by melting curve analysis and agarose gel electrophoresis. Standard curves generated with 4-fold serial dilutions of genomic DNA samples over 5 grades showed a good linearity (r2 . 0.98) and acceptable amplification efficiencies between 90% and 110% for all primer pairs. Each PCR reaction included no template controls in every PCR running, and had 3 replicates with 2 times repetition. Using a delta-delta threshold cycle (DDCT) method37, relative copy number was estimated with respect to wild type CHO-S cells for target genes and representative clones for mCherry transgene. 23. Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9- mediated gene inactivation. Nat. Biotechnol. doi: 10.1038/nbt.3026. [Epub ahead of print] (2014). 24. Ramakrishna, S. et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat. Commun. 5, 3378 (2014). 25. Vriend, L. E., Jasin, M. & Krawczyk, P. M. Assaying Break and Nick-Induced Homologous Recombination in Mammalian Cells Using the DR-GFP Reporter and Cas9 Nucleases. Methods Enzymol. 546, 175–191 (2014). g g and Cas9 Nucleases. Methods Enzymol. 546, 175–191 (2014). 26. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013). g g p y 27. Fu, Y. et al. High-frequency off-target mutagenesis induced b 27. Fu, Y. et al. High-frequency off-target mutagenesis induced by C nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013). 27. Fu, Y. et al. High frequency off target mutagenesis induced by CRI nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013). 28. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013). 29. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013). 1. Noh, S. M., Sathyamurthy, M. & Lee, G. M. Development of recombinant Chinese hamster ovary cell lines for therapeutic protein production. Curr. Opin. Chem. Eng. 2, 391–397 (2013). 30. Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014). g 2. Wurm, F. M. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. Ricinus Communis Agglutinin-I (RCA-I) selection and phenotypic analysis of Mgat1 knockout cells. Mgat1 knockout cells were enriched in culture medium with Off-targets were predicted using custom python code that compared the targets with all other potential targets in the CHO genome. Off-targets were then scored based on the following criteria: i) The first 5 bp immediately adjacent to the PAM sequence: cutoff value of 0; ii) The number of mismatches in the first 13 bp: cutoff value of up to 1; iii) The total number of mismatches including number of mismatches in first 13 bp: cutoff value up to 5. Quantitative Real Time PCR (qRT-PCR) for copy number analysis. For relative determination of copy number of transgene, qRT-PCR was performed on genomic DNA samples using Brilliant III Ultra-Fast SYBRH Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA) on Mx3005P qPCR System (Agilent Technologies) Genomic DNA extraction and PCR amplification. Genomic DNA was extracted from the cell pellets using QuickExtract DNA extraction solution (Epicentre, SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 10 www.nature.com/scientificreports (ZFN)-mediated gene knockout of mannosyl (alpha-1,3-)-glycoprotein beta-1,2- N-acetylglucosaminyltransferase (Mgat1). J. Biotechnol. 167, 24–32 (2013). according to manufacturer’s instructions. Reaction mixtures contained SYBR Green QPCR master mix, 250 nM of forward and reverse primers, reference dye, and 20 ng of genomic DNA. Amplification was executed with the following conditions: 95uC for 10 min; 403: 95uC for 20 s, 60uC for 30 s. The primers were designed using PrimerQuest (Integrated DNA Technologies, Coralville, IA) against transgene encompassing EF-1a – mCherry, target genes including COSMC, Mgat1 (sgRNA1 and sgRNA5 target site), and LdhA, and reference gene, Vcl (Supplementary Table 2). All primers were validated empirically by melting curve analysis and agarose gel electrophoresis. Standard curves generated with 4-fold serial dilutions of genomic DNA samples over 5 grades showed a good linearity (r2 . 0.98) and acceptable amplification efficiencies between 90% and 110% for all primer pairs. Each PCR reaction included no template controls in every PCR running, and had 3 replicates with 2 times repetition. Using a delta-delta threshold cycle (DDCT) method37, relative copy number was estimated with respect to wild type CHO-S cells for target genes and representative clones for mCherry transgene. according to manufacturer’s instructions. Reaction mixtures contained SYBR Green QPCR master mix, 250 nM of forward and reverse primers, reference dye, and 20 ng of genomic DNA. Amplification was executed with the following conditions: 95uC for 10 min; 403: 95uC for 20 s, 60uC for 30 s. Additional information Supplementary information accompanies this paper at http://www.nature.com/ scientificreports Supplementary information accompanies this paper at http://www.nature.com/ scientificreports 17. Cristea, S. et al. In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration. Biotechnol. Bioeng. 110, 871–880 (2013). Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. 18. Pennisi, E. The CRISPR craze. Science 341, 833–836 (2013). How to cite this article: Lee, J.S., Kallehauge, T.B., Pedersen, L.E. & Kildegaard, H.F. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Sci. Rep. 5, 8572; DOI:10.1038/srep08572 (2015). 19. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). 20. Ronda, C. et al. Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol. Bioeng. 111, 1604–1616 (2014). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 21. Chen, W. & Stanley, P. Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I. Glycobiology 13, 43–50 (2003). 22. Sealover, N. R. et al. Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease 22. Sealover, N. R. et al. Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease SCIENTIFIC REPORTS \| 5 : 8572 \| DOI: 10.1038/srep08572 11
https://openalex.org/W937540053	https://econjournals.sgh.waw.pl/kwes/article/download/220/194	Polish	null	Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy	Kwartalnik Kolegium Ekonomiczno-Społecznego Studia i Prace	2,012	cc-by	11,826	Anna Zorska Anna Zorska 1 P.F. Drucker, T e Age of Discontinuity: Guidelines to Changing Our Society, Transaction Pubs, New Brunswick (U.S.A.) 1992, s. 263. 2 L.J. Jasiński, Sektor wiedzy w rozwoju gospodarki, Wydawnictwo Key Text, Warszawa 2009, s. 23. 3 W.M. Gaczek, Gospodarka oparta na wiedzy w regionach europejskich, PAN–KPZG, Warszawa 2009, s. 27. y j g p y y 3 W.M. Gaczek, Gospodarka oparta na wiedzy w regionach europejskich, PAN–KPZG, Warszawa 2009, s. 27. 2 L.J. Jasiński, Sektor wiedzy w rozwoju gospodarki, Wydawnictwo Key Text, Warszawa 2009, s. 23. y j g p y y Gaczek, Gospodarka oparta na wiedzy w regionach europejskich, PAN–KPZG, Warszawa 2009, 1 P.F. Drucker, T e Age of Discontinuity: Guidelines to Changing Our Society, Transaction Pubs, New Brunswick (U.S.A.) 1992, s. 263. Wprowadzenie Termin „gospodarka oparta na wiedzy” (GOW) przypisuje się P.F. Drucke- rowi, który wyeksponował znaczenie wiedzy jako czynnika o kluczowym znacze- niu dla wzrostu całej gospodarki. Jego zdaniem „Nie ziemia czy kapitał, lecz wie- dza będzie głównym czynnikiem produkcji w XXI wieku”.1 Podobnie twierdzi L.J. Jasiński: „Pojawiło się określenie gospodarka oparta na wiedzy na oznacze- nie etapu w rozwoju gospodarczym, w którym wiedza staje się decydującym czynnikiem wzrostu i rozwoju”.2 Inne ujęcie omawianego terminu przedstawia W.M. Gaczek, twierdząc, że „Gospodarkę opartą na wiedzy można określić jako two- rzenie warunków sprzyjających funkcjonowaniu podmiotów, które opierają swoją działalność na wiedzy’’.3 Kieruje to uwagę ku mechanizmom i różnym instrumentom tworzenia warunków sprzyjających działalności podmiotów zaangażowanych w kre- owanie wiedzy i przekształcanie jej w innowacje użyteczne gospodarczo i społecznie. W tworzeniu wiedzy zasadniczą rolę odgrywają prace badawczo-rozwojowe (B+R). Jednakże same wyniki badań naukowych stanowią dopiero początek procesu przekształcania nowej wiedzy w ekonomiczne rozwiązania przynoszące korzyści różnym podmiotom. Bardzo ważne są nie tylko wynalazki i pomysły, lecz również zdolności oraz warunki maksymalnego wykorzystania wiedzy w formie nowych lub znacząco ulepszonych rozwiązań – innowacji – w działalności przedsiębiorstw i innych podmiotów. Korzyści powstają szybciej, są większe i mają szerszy zasięg, jeśli w procesach innowacyjnych uczestniczy wiele podmiotów, działają w sprzyja- jących warunkach otoczenia, łączy je współpraca i powiązania biznesowe. Tworzące się systemy innowacyjności – na wszystkich poziomach działalności gospodarczej 28 Anna Zorska Anna Zorska – stają się bardzo ważnym czynnikiem wsparcia rozwoju gospodarki wiedzy. Wiele państw ukierunkowało politykę gospodarczą na wspieranie rozwoju GOW poprzez stymulujące oddziaływanie na procesy innowacyjne wielu różnych podmiotów. Chodzi o kompleksowe wspieranie sprawnego funkcjonowania narodowego systemu innowacyjności (NSI), gdyż same wydatki z budżetu państwa na działalność B+R to za mało. Dobre i ciekawe doświadczenia w zakresie promowania rozwoju GOW poprzez kompleksowe wsparcie systemu innowacyjności przez politykę państwa ma Finlandia. Niniejsze opracowanie ma na celu ukazanie przebiegu procesów i działania syste- mów innowacyjnych w powiązaniu z rozwojem GOW w krajowej gospodarce. Roz- ważania zostały ujęte w trzech częściach, przy czym w punkcie pierwszym przed- stawiono rozwój GOW i jej charakterystykę. Na szczególne uwzględnienie zasługuje koncepcja GOW w ujęciu Banku Światowego, eksponująca cztery obszary wsparcia gospodarki wiedzy, określone jako f lary jej rozwoju. W punkcie drugim został sze- rzej omówiony najważniejszy f lar GOW – narodowy system innowacyjności, z uka- zaniem jego złożoności oraz otwierania się na powiązania międzynarodowe. Wprowadzenie Punkt trzeci jest poświęcony funkcjonowaniu krajowego systemu innowacyjności w Fin- landii oraz przedstawieniu wpływu na jej gospodarkę zmian globalnych powiązań innowacyjnych i biznesowych w sektorze informacyjnym. Rozważania wskazują na narastanie ponadgranicznych powiązań NSI oraz współzależności systemów innowa- cyjnych na różnych poziomach działalności, co nie zawsze przynosi skutki korzystne dla gospodarki krajowej. 1. Rozwój gospodarki opartej na wiedzy i charakterystyka jej f larów Chociaż pojęcie gospodarki opartej na wiedzy powstało pod koniec XX wieku, to trudno zaprzeczyć stwierdzeniu, że wiedza zawsze była ważna w działalności gospodarczej. W szczególności ważne znaczenie miała wiedza techniczna i bizne- sowa, której zastosowanie pozwalało przedsiębiorstwom (lub innym podmiotom) zmieniać istniejące lub wprowadzać nowe produkty, ulepszać procesy produkcyjne, usprawniać działalność. W szerszym ujęciu chodzi o postęp naukowo-techniczny, z którego część osiągnięć mogła być wykorzystana – jako nowe technologie – w dzia- łalności przedsiębiorstw, umożliwiając zwiększenie produkcji, przychodów i zysków. Zatem tworzenie i zastosowanie wiedzy w biznesie polegało i polega na opracowaniu Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 29 i wdrażaniu nowych lub znacząco ulepszonych rozwiązań – czyli innowacji – które sprzyjają podnoszeniu efektywności i konkurencyjności przedsiębiorstw. Rozwój gospodarki opartej na wiedzy jest ściśle związany z rewolucją informa- cyjną oraz procesem globalizacji. Ze względów chronologicznych należy najpierw wskazać na znaczenie globalizacji, z którą jest związana liberalizacja działalno- ści gospodarczej i szersze otwarcie gospodarek na przepływy towarów, czynników wytwórczych oraz informacji. Ponadto wraz z intensyf kacją globalnej konkurencji silnie zwiększyło się zapotrzebowanie na informacje i wiedzę przedsiębiorstw kon- kurujących globalnie – korporacji transnarodowych (KTN). Co bardzo ważne, z ich udziałem (dzięki pracom B+R, pomnażaniu wiedzy w wielu f liach zagranicznych) i na ich potrzeby przyspieszeniu uległ ogólny postęp naukowo-techniczny, którego osiągnięcia KTN przekształcają w innowacje i wdrażają globalnie. Służy to unowo- cześnianiu produktów, procesów produkcji i zbytu, infrastruktury biznesu, metod organizacji i zarządzania przez korporacje, a także głębszym przemianom ich łań- cuchów wartości dodanej, różnych strategii, organizacji oraz ekspansji zagranicz- nej. Podnoszenie międzynarodowej konkurencyjności przedsiębiorstw i gospoda- rek poprzez stymulowanie innowacyjności stało się również dążeniem wielu państw, których polityka oddziałuje na podaż wiedzy i nowych technologii, na popyt przed- siębiorstw dla biznesowego wykorzystania wiedzy w postaci innowacji, jak też na warunki instytucjonalne temu sprzyjające.4 Rozwój gospodarki opartej na wiedzy został zdynamizowany przez informa- cjne i komunikacyjne technologie (IKT), ale w istocie jej siłę napędową stworzyły szersze procesy: dynamiczny wzrost nowego sektora informacyjnego, komercjali- zacja i szybki przepływ informacji oraz nowe sposoby komunikowania się, a także przyspieszenie tworzenia, stosowania i pomnażania wiedzy w biznesie. y g 5 Należy podkreślić, że tworzenie wiedzy dokonuje się dzięki wykorzystaniu danych i informacji, w połączeniu z potencjałem ludzkich umiejętności, pomysłów, zaangażowania i motywacji. Por. W.M. Grudzewski, I.K. Hejduk, Zarządzanie wiedzą w przedsiębiorstwie, Dif n, Warszawa 2004, s. 73. 4 W.E. Steinmueller, Economics of Technology Policy, w: Handbook of the Economics of Innovation, Vol. 2, red. H.H. Bronwyn, N. Rosenberg, Elsevier, Amsterdam 2010, s. 1192–1214. 6 A.K. Koźmiński, Zarządzanie w warunkach niepewności. Podręcznik dla zaawansowanych, Wydawnictwo Naukowe PWN, Warszawa 2004, s. 94–105, w tym wykres 4.5. 7 A. Zorska, Globalizacja a „nowa gospodarka”. Implikacje dla Polski, „Prace i Materiały IGŚ” 2004 nr 255, s. 27–30. 8 M. Castells, Galaktyka Internetu. Ref eksje nad Internetem, biznesem i społeczeństwem, Dom Wydawniczy Rebis, Poznań 2003, s. 92. 8 M. Castells, Galaktyka Internetu. Ref eksje nad Internetem, biznesem i społeczeństwem, Dom Wydawniczy Rebis, Poznań 2003, s. 92. y y 7 A. Zorska, Globalizacja a „nowa gospodarka”. Implikacje dla Polski, „Prace i Materiały IGŚ” 2004 nr 255, s. 27–30. 6 A.K. Koźmiński, Zarządzanie w warunkach niepewności. Podręcznik dla zaawansowanych, Wydawnictwo Naukowe PWN, Warszawa 2004, s. 94–105, w tym wykres 4.5. 1. Rozwój gospodarki opartej na wiedzy i charakterystyka jej f larów Doty- czyło to nowo powstających f rm oraz branż (głównie usługowych, opartych na IKT), jak również modernizujących się, tradycyjnych przedsiębiorstw wytwarza- jących bardziej nowoczesne produkty w istniejących branżach przemysłu i usług.5 Unikalna wiedza (techniczna, biznesowa, inna) mogąca tworzyć kluczowe kom- petencje, trwałe przewagi konkurencyjne oraz skuteczne strategie konkurencji przedsiębiorstw, stała się ich cennym i specyf cznym, niematerialnym zasobem o znaczeniu strategicznym. Stała się też zasadniczym zasobem służącym rozwijaniu 30 Anna Zorska Anna Zorska zdolności innowacyjnych f rm.6 Działalność przedsiębiorstw – których przewagi konkurencyjne i strategie konkurencji są oparte na systematycznym rozwijaniu i stosowaniu wiedzy – stanowi zasadniczą siłę napędową rozwoju gospodarki opartej na wiedzy. Biznesowe wykorzystanie informacji i wiedzy intensyf kuje się i kumuluje, stopniowo kształtując nową charakterystykę całego układu makroekonomicznego. W dalszym rozwoju GOW w poszczególnych krajach znaczącą rolę odgry- wają determinanty ogólne (technologiczne, ekonomiczne, instytucjonalne) oraz sektorowe (dotyczące telekomunikacji), których wpływ można określić jako bezpośredni i pośredni. Duże znaczenie i wpływ bezpośredni mają zasoby i zdolności wytwórcze oraz warunki instytucjonalne, które są angażowane lub tworzą ramy zaangażowania podmiotów kreujących i stosujących wiedzę w gospodarce. Zasadnicze znaczenie dla wzrostu GOW mają następujące determi- nanty: poziom rozwoju gospodarki, nauki, techniki i edukacji w kraju, rozwój sek- tora telekomunikacyjnego, innowacyjność przedsiębiorstw.7 Poziom rozwoju gospodarczego determinuje możliwości tworzenia i jakość podstawowych zasobów – wiedzy i technologii oraz kwalif kowanej i utalentowanej siły roboczej – niezbędnych dla rozwoju GOW. Ważne znaczenie ma nowoczesność i struktura sektora telekomunikacyjnego, ceny, jakość i dostępność świadczonych usług, regulacje ustawowe i administracyjne dotyczące sektorów telekomunikacyj- nego i informacyjnego. Istotne jest korzystanie z IKT oraz usług informacyjnych przez indywidualne osoby, ale kluczowe znaczenie dla gospodarki kraju ma szero- kie stosowanie ww. technologii oraz usług przez przedsiębiorstwa w celu rozwijania innowacyjności i biznesu. Rozszerzają się kontakty biznesowe z f rmami za granicą, rozwijają się przepływy informacji, wyrobów, usług, technologii, kadr, kapitału. Ze względu na charakterystykę sieci informacyjnych w gospodarce opartej na wie- dzy aktywizują się przepływy i powiązania biznesowe w skali globalnej. Jednocześnie z udziałem nowych f rm – również z krajów mniej rozwiniętych – narasta konku- rencja na rynku globalnym. Jak stwierdził M. Castells, rozwój GOW charakteryzuje się tym, że wiedza staje się czynnikiem przesądzającym o innowacyjności, produk- tywności i międzynarodowej konkurencyjności gospodarki narodowej.8 Ze względu na duże znaczenie dla wzrostu gospodarczego krajów i podno- szenia poziomu życia ludności zagadnieniem GOW zajęły się międzynarodowe Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 31 organizacje i instytucje. 9 T e Knowledge-based Economy, OECD, Paris 1996, s. 7. 10 Gospodarka oparta na wiedzy. Perspektywy Banku Światowego, red. A. Kukliński, Biuro Banku Światowego w Polsce i Komitet Badań Naukowych, Warszawa 2003, s. 14–15. 1. Rozwój gospodarki opartej na wiedzy i charakterystyka jej f larów Jedną z pierwszych była OECD, która już w połowie lat dziewięćdziesiątych XX wieku prowadziła badania, przygotowała opracowanie na ten temat, sformułowała def nicję. Zdaniem badaczy OECD istotą GOW jest bez- pośrednie włączenie do działalności gospodarczej tworzenia, rozprzestrzeniania się oraz stosowania wiedzy i informacji.9 Wiedza jest tworzona w wyniku działal- ności B+R, a rozprzestrzenia się kanałami przepływu wiedzy, które mają charak- ter ogólnodostępny (publiczny), organizacyjny (w przedsiębiorstwach), instytu- cjonalny oraz relacyjny (poprzez współpracę różnych podmiotów). Akumulacja i rozwijanie wiedzy w gospodarce dokonuje się w sposób ogólny i formalny – między ludźmi, poprzez działanie w zespołach, poprzez organizację (w f rmach) i jej relacje z innymi podmiotami. O rozwoju GOW w gospodarce danego kraju świadczy wyż- sza dynamika wzrostu inwestycji, produkcji i zatrudnienia w zaawansowanych tech- nologicznie branżach/sektorach oraz rosnąca ich produktywność. We współczesnej gospodarce światowej GOW powstała początkowo w krajach wysoko rozwiniętych (OECD), a rozszerza się ku tym krajom rozwijającym się i transformującym, które modernizują zasoby i zdolności wytwórcze oraz są w stanie rozwijać zaawansowane technologicznie branże przemysłu i usług, jak np. kraje azjatyckie. g y g j j j y Duży wkład w rozwinięcie koncepcji GOW, badanie gospodarki opartej na wie- dzy w różnych krajach i formułowanie zaleceń dotyczących jej rozwoju wniósł Bank Światowy. W ujęciu tej organizacji GOW jest to gospodarka powszechnie wykorzy- stująca zaawansowane technologie, których rozwój i praktyczne wykorzystanie wymaga wysokich kwalif kacji.10 Oprócz def nicji, bardzo istotnym składnikiem koncepcji GOW opracowanej przez Bank Światowy jest wskazanie czterech f larów gospodarki wiedzy, o których będzie mowa poniżej. W takim ujęciu – def nicji GOW i jej f larów – będą badane zagadnienia będące tematem opracowania. Na poziomie def nicyjnym ujęcie Banku Światowego jest szersze niż ujęcie OECD, gdyż akcentuje duże znaczenie wysokich kwalif kacji dla GOW, tzn. kapitału ludzkiego, który tworzą wysoko kwalif kowane i utalentowane kadry. W koncepcjach i badaniach Banku GOW jest ujmowana jako dynamiczny, kompleksowy i złożony system technologiczny, społeczny i polityczny, otwarty na zewnętrzne powiązania. Zasadniczym czynnikiem dynamizującym ten system jest postęp naukowo-tech- niczny, tworzona wiedza, nowe (wysokie) technologie oraz kwalif kacje, zdolności innowacyjne różnych podmiotów i systemów (w ramach f rm, regionów, gospoda- rek). Kompleksowość i złożoność dotyczy całego, wieloetapowego procesu tworze- nia, stosowania i pomnażania wiedzy przez wiele podmiotów – od indywidualnych 32 Anna Zorska Anna Zorska osób i pracowników poprzez wszystkie organizacje (f rmy) oraz instytucje na róż- nych poziomach działalności gospodarczej, społecznej i politycznej. p y p y 12 W niektórych publikacjach Banku Światowego f lar ten jest też określony jako „innowacje i wdrażanie technologii” (innovation and technological adoption). W niniejszej pracy przyjmuje się wersją zasadniczą, tzn. narodowy system innowacyjności (national innovation system). 11 Sam indeks GOW zawiera po trzy podstawowe zmienne na każdy f lar. 1. Rozwój gospodarki opartej na wiedzy i charakterystyka jej f larów Rozwój GOW wspierają przepływy informacji, wiedzy, technologii, innowacji w skali globalnej oraz różnego rodzaju ponadgraniczne powiązania podmiotów. Zaletą ujęcia GOW przez Bank Światowy jest wskazanie czterech kluczowych obszarów funkcjonalnych (key functional areas) wspierania rozwoju omawianej gospodarki, czyli w późniejszym ujęciu – jej f larów (pillars). Dla wszystkich czterech f larów ustalono 76 zmiennych na podstawie kryteriów merytorycznych i dostępności danych statystycznych. Możliwe stało się obliczenie jednego, syntetycznego wskaź- nika (knowledge economy index – KEI),11 a w konsekwencji stworzenie rankingu kra- jów przygotowanych do rozwoju GOW. Ustalono cztery następujące f lary GOW: 1) system bodźców ekonomicznych i ramy instytucjonalne, 2) edukacja i szkolenie, 2) edukacja i szkolenie, 3) infrastruktura informacyjna, 4) narodowy system innowacyjności.12 4) narodowy system innowacyjności.12 Pierwszy f lar dotyczy elementów otoczenia biznesowo-instytucjonalnego, które służą (w różnym stopniu) przepływom wiedzy i informacji na rynku, prowadzeniu prac B+R, inwestowaniu w IKT i biznes oparty na wiedzy, rozwijaniu przedsiębior- czości. Otoczenie sprzyja rozwojowi GOW, jeśli usuwane są zniekształcenia rynku w zakresie przepływu wiedzy, a jednocześnie konkurencja na rynku skłania do two- rzenia wiedzy i jej zastosowania w biznesie. Ponadto państwo może tworzyć dobre warunki dla działalności badawczej, inwestycyjnej i podejmowania nowych przed- sięwzięć (o wyższym stopniu ryzyka). Drugi f lar odnosi się do wspierania rozwoju GOW przez system edukacji i szko- leń, który powinien służyć przygotowaniu wysoko kwalif kowanych kadr – czyli kapitału ludzkiego – do tworzenia wiedzy, uczestnictwa w jej dyfuzji i wdrażaniu w przedsięwzięciach biznesowych oraz innych. Ponieważ obecnie na świecie ist- nieje niewystarczająca podaż i wysoki koszt kwalif kowanych kadr, tworzenie kapi- tału ludzkiego (jego podaż, jakość, cena) staje się istotnym warunkiem przyciągania inwestycji (również zagranicznych) do GOW w poszczególnych krajach. Trzeci f lar dotyczy infrastruktury informacyjnej, która ma służyć efektyw- nej komunikacji, szybkiemu i taniemu rozprzestrzenianiu się informacji, zarówno bezpośrednio w celach biznesowych, jak i innych (edukacja, administracja, usługi Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 33 publiczne). Rozbudowa czy modernizowanie sieci i urządzeń telekomunikacyjnych, niezawodność połączeń i sprawny serwis, utrzymanie cen usług na niskim poziomie, zapewnienie bezpieczeństwa gromadzenia danych i korzystania z sieci informacyj- nych itd. – to wszystko decyduje o możliwościach i kosztach prowadzenia działalno- ści różnych podmiotów z wykorzystaniem o informacji i wiedzy. Jest to istotne dla rozwijania międzynarodowych powiązań służących przepływom wiedzy. Czwarty f lar tworzony jest przez narodowy (krajowy) system innowacyjności.13 Służy on tworzeniu i dyfuzji nowej wiedzy oraz przekształcaniu jej w innowacje w danym kraju. 13 W literaturze polskiej używany jest też termin „system innowacyjny”. Wydaje się jednak, że jest to sformułowanie sugerujące raczej wprowadzanie zmian i nowości do systemu (co może i powinno się zdarzać, lecz nie musi dominować) niż jego zdolność wspierania zdolności innowacyjnych, czyli innowacyjności. 14 W.M. Grudzewski, I.K. Hejduk, Zarządzanie wiedzą..., op.cit., s. 15–18, w tym rys. 2. ) j g p yj y y 14 W.M. Grudzewski, I.K. Hejduk, Zarządzanie wiedzą..., op.cit., s. 15–18, w tym rys. 2. 1. Rozwój gospodarki opartej na wiedzy i charakterystyka jej f larów Działanie tego systemu opiera się na procesach innowacyjnych zachodzących w przedsiębiorstwach, na rozszerzaniu się procesów na inne pod- mioty w sektorach i regionach, jak też na kształtowaniu tych procesów – głównie przez politykę państwa – na poziomie krajowym. Co istotne, system ten musi być otwarty, aby było możliwe poznanie globalnego zasobu wiedzy, jego selekcja i legalny transfer określonych „porcji” wiedzy, ich adaptacja i absorpcja w krajowych f rmach. Jednocześnie chodzi o umożliwienie lepszego wykorzystania zasobu wiedzy stwo- rzonego w danym kraju na potrzeby podmiotów zagranicznych, co będzie sprzyjać rozwojowi GOW na świecie. Koncepcja czterech f larów GOW stworzona przez Bank Światowy zainspirowała polskich badaczy – prof. W.M. Grudzewskiego i prof. I.K. Hejduk – do opracowania rozszerzonej wersji oparcia GOW na sześciu f larach. Dwa „nowe” f lary odnoszą się do poziomu mezo- i mikroekonomicznego, czego nie uwzględniono w koncep- cji Banku. Tak więc w szerszej koncepcji uwzględniono również aspekt regionalny oraz zarządzanie wiedzą w organizacji. Wspomniani autorzy prezentują nieco inną kolejność f larów, a mianowicie: 1) system innowacyjności, 2) system edukacyjny, 3) system informacyjno-komunikacyjny, 4) zarządzanie wiedzą w organizacji, 5) aspekt regionalny, 6) otocznie instytucjonalno-biznesowe.14 Jeśli uwzględnić bez- pośredni charakter i siłę oddziaływania sześciu f larów na GOW, to modyf kacja wprowadzona przez polskich badaczy wydaje się słuszna. Warto pokrótce scharakteryzować istotę „nowych” f larów w ujęciu polskich auto- rów. Procesy związane z upowszechnianiem się nowej wiedzy, technologii, innowa- cji stopniowo rozprzestrzeniają się w całej gospodarce, ale w istocie ich największe znaczenie i oddziaływanie ekonomiczne kształtuje się dzięki wykorzystaniu nowych osiągnięć i różnego rodzaju rozwiązań w przedsiębiorstwach. To one wypracowały różne sposoby zarządzania wiedzą, która – stanowiąc ich strategiczny zasób – musi 34 Anna Zorska Anna Zorska być efektywnie wykorzystana i dalej rozwijana. Jak słusznie zauważają autorzy: „[...] kształtowanie gospodarki opartej na wiedzy powinno przede wszystkim odbywać się na poziomie poszczególnych organizacji”.15 Rozwój GOW stanowi swego rodzaju rozległy system powiązany na różnych poziomach. Innowacje często upowszech- niają się wśród przedsiębiorstw blisko położonych i działających na rynku lokalnym czy regionalnym. Działania stymulujące tworzenie, przepływy i wykorzystanie wie- dzy, technologii, innowacji w regionie są efektywne, a nawet przynoszą synergiczne efekty. Promowanie GOW na poziomie regionalnym dokonuje się głównie przez stworzenie regionalnego systemu innowacyjności, w czym duży udział ma polityka państwa na poziomie kraju i instytucji samorządowo-administracyjnych. Ibidem, s. 18. 16 Knowledge Economy Index (KEI). 2008 Rankings, T e World Bank, Washington, D.C. 2008, s. 1–11. 15 Ibidem, s. 18. 1. Rozwój gospodarki opartej na wiedzy i charakterystyka jej f larów W krajach UE strategia lizbońska i następne programy promowania postępu wiedzy, a także polityka rozwoju regionalnego i inne rodzaje polityki stwarzają możliwości wspiera- nia regionalnych systemów innowacyjności. Częściowe włączenie zagadnień ujętych przez f lary „regionalny” i „organizacyjny” wydaje się istotne dla pełniejszego prze- analizowania narodowych systemów innowacyjności jako f laru GOW we współ- czesnej gospodarce. Koncepcja gospodarki wiedzy, bazującej na czterech f larach, dała możliwość kwantyf kacji i porównania warunków dla rozwoju GOW w różnych krajach. Sze- roko zakrojone badania – obejmujące 140 krajów – przeprowadził Bank Światowy dwukrotnie, przetwarzając dane dla lat 1995 i 2008. Wyniki analizy statystycznej zostały ujęte w syntetycznych wskaźnikach GOW, a to pozwoliło stworzyć ranking krajów o najlepszym przygotowaniu do rozwoju GOW.16 Czołówkę rankingu kra- jów o najwyższych na świecie wskaźnikach GOW w 2008 roku stanowiło 20 kra- jów, wśród których dominuje Europa Zachodnia i Północna – 14 krajów, a mniej liczne są części innych kontynentów: Ameryki Północnej – 2 kraje, Azji Wschodniej – 2 kraje, Australii i Oceanii – 2 kraje. W rankingach GOW szczególną uwagę zwraca grupa krajów nordyckich. Każdy z tych krajów charakteryzuje się stosunkowo niedużą gospodarką (poło- żoną peryferyjnie w Europie), lecz dobrze wyposażoną w zaawansowane zasoby i zdolności oraz sprawny system bodźców do wdrażania wiedzy w gospodarce. W 2008 roku najwyższy wskaźnik GOW i pozycję nr 1 w rankingu osiągnęła Dania (9,58 pkt.), a niewiele mniej uzyskały inne kraje nordyckie: Szwecja (9,56), Finlan- dia (9,37) i Norwegia (9,32). Pośród krajów nordyckich na miejscu 4. znalazła się Holandia (9,38). Można stwierdzić, że w zasadzie kraje nordyckie posiadają dobrze Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 35 i równomiernie rozwinięte cztery f lary GOW, przy czym przewaga tej grupy nad innymi krajami jest widoczna w zakresie systemu edukacji. Do krajów o najsilniejszym f larze krajowego systemu innowacyjności należą kolejno: Szwajcaria, Szwecja, Finlandia, Dania, Holandia, USA, Kanada, Singapur, Izrael, Tajwan.17 Ze względu na skalę i dynamikę osiągnięć w tworzeniu warunków dla GOW, a także powiązanie korzyści ekonomicznych z rosnącym dobrobytem społeczeństwa Finlandia stała się przedmiotem analizy w punkcie 3. opracowania. Wśród krajów Europy Środkowej najwyżej sklasyf kowano Czechy (miejsce 27., 7,92 pkt.) i Węgry (miejsce 28., 7,88 pkt.). W obu przypadkach wysoko oceniono bodźce służące wspieraniu GOW, a w przypadku Węgier również system innowa- cyjności. Polska znalazła się na miejscu 35. 1. Rozwój gospodarki opartej na wiedzy i charakterystyka jej f larów (7,52 pkt.), przy czym najniższą ocenę uzyskał system innowacyjności, a najwyższą – system edukacji.18 Oceny Banku Światowego pokrywają się z wynikami innych badań dotyczących innowacyjności i warunków dla rozwoju GOW w Polsce. Dla ich poprawy jest więc wskazane pozna- nie doświadczeń w funkcjonowaniu NSI w innych krajach. Ibidem, s. 2, tab. 1. 19 A. Pomykalski, Zarządzanie innowacjami, Wydawnictwo Naukowe PWN, Warszawa–Łódź 2001, s. 17–18. 18 Ibidem, s. 2, tab. 1. 17 Ibidem, s. 3, tab. 2.. 2. Procesy i systemy innowacyjne we współczesnej gospodarce We współczesnym rozumieniu innowacji istotne jest nie tylko wdrożenie nowej lub ulepszonej metody działania, lecz szersze ujęcie działalności innowacyjnej jako procesu powstawania i wdrażania zmian. W istocie chodzi o całościowy proces innowacyjny obejmujący koncepcję teoretyczną (wynalazek lub po prostu pomysł), rozwiązanie techniczne (nową technologię) lub inne (np. metody organizacji i zarządzania), a także jego wdrożenie i dalsze rozprzestrzenianie się (dyfuzję).19 W szerszym ujęciu innowacje są rezultatem procesów technicznych, ekono- micznych, społecznych, politycznych oraz różnych zmian organizacyjnych czy instytucjonalnych, które w pewnej mierze mogą być kształtowane. W procesie innowacyjnym realizowane są kolejne, następujące po sobie fazy od powstania wynalazku lub pomysłu do jego wdrożenia produkcyjnego i komercja- lizacji (sprzedaży produktu na rynku). Chodzi zatem o cały zespół różnych działań 36 Anna Zorska Anna Zorska o charakterze wdrożeniowym, które są realizowane głównie w przedsiębiorstwach.20 Na przebieg procesów innowacyjnych w przedsiębiorstwach mogą świadomie oddziaływać różne podmioty (inne f rmy, ośrodki badawcze, państwo) w celu ich przyspieszenia, rozszerzenia, zwiększenia efektywności, generowania większych korzyści dla określonej grupy benef cjentów. Z drugiej strony, na omawiane procesy wpływają różnego rodzaju przemiany zewnętrzne – zwłaszcza techniczne i rynkowe – w globalnym otoczeniu innowacyjnych podmiotów. W czasie obecnej rewolucji informacyjnej dokonały się duże zmiany w proce- sach innowacyjnych przedsiębiorstw. Ma miejsce szerokie wykorzystanie IKT, dostęp do bardzo wielu informacji, szybkie komunikowanie się w czasie rzeczywistym, nowe metody organizacji i zarządzania w oparciu o sieć informacyjną. Nastąpiła frag- mentacja, specjalizacja i umiędzynarodowienie prac B+R, unowocześnienie metod badawczych (np. modelowanie i symulacje, z ograniczeniem eksperymentów), roz- wój zarządzania wiedzą, szerokie wykorzystanie kooperacji technologicznej. Przy- spieszeniu uległo tworzenie wiedzy i przekształcanie jej w innowacje, co dało przed- siębiorstwom większe możliwości ich wykorzystania w biznesie w celu wzmacniania konkurencyjności międzynarodowej. yj ę y j Prace B+R początkowo były realizowane we własnych, wyspecjalizowanych jednostkach (działach, ośrodkach) poszczególnych f rm, ale wraz z rozbudową ich organizacji stopniowo zostały włączone inne jednostki (działy, f lie) oraz koope- rujące podmioty zewnętrzne (ośrodki badawcze, instytuty akademickie, innowa- cyjne f rmy). W procesach innowacyjnych dużych f rm obecnie uczestniczy wiele jednostek i podmiotów zlokalizowanych w różnych krajach, które to podmioty tworzą układ elementów o określonej strukturze, powiązaniach i oddziaływaniach. Procesy innowacyjne f rm przekształciły się w złożone systemy innowacyjne z udzia- łem różnych podmiotów. Oznacza to, że w określonym układzie innowacyjnych jednostek f rm i innych podmiotów mają miejsce istotne relacje, powiązania oraz interakcje między nimi, w czego wyniku pobudzana jest ich innowacyjność. ą ą yj ą, , y , , 21 D. Surówka-Marszałek, Rola organizacji sieciowych w kreowaniu technologii innowacyjnych, w: Innowacyjne systemy, procesy i metody zarządzania międzynarodowego, red. M. Trocki, Of cyna Wydawnicza SGH, Warszawa 2008, s. 52. 20 Zarządzanie działalnością innowacyjną, red. L. Białoń, Wydawnictwo Placet, Warszawa 2010, s. 27. 2. Procesy i systemy innowacyjne we współczesnej gospodarce Współ- czesne systemy innowacyjne nie tworzą się w sposób hierarchiczny, lecz sieciowy, bazujący na zmiennym układzie elementów, ich relacji oraz – co najważniejsze – różnych interakcji.21 Systemy innowacyjne mogą więc rozwijać się w rozbudowa- nym układzie organizacyjnym jednej f rmy – jej jednostek i kooperujących podmio- tów, ale też rozwijają się w sieciach wielu f rm, w różnych układach przestrzennych. W przypadku KTN powstały rozbudowane, ponadgraniczne systemy innowacyjne, Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 37 w których na zasadach sieciowych działają – w sposób wyspecjalizowany, skoordy- nowany i zintegrowany – własne jednostki (f lie, ośrodki) oraz innowacyjne pod- mioty z różnych krajów.22 Systemowy charakter działalności innowacyjnej został dostrzeżony przez badaczy w latach osiemdziesiątych XX wieku i wykorzystany do tworzenia polityki innowa- cyjnej lub technologicznej państwa w latach dziewięćdziesiątych. Polityka państwa ukierunkowała się na rozwijanie zasobów, zdolności i warunków dla innowacyjności w danym kraju w celu podnoszenia zdolności badawczych i innowacyjnych krajo- wych podmiotów w trzech sferach: biznesu, badawczo-akademickiej oraz instytucji publicznych (państwowych). W szerszym ujęciu korzystne efekty innowacyjności w kraju mogą przyczynić się do wzrostu konkurencyjności przedsiębiorstw na ryn- kach krajowym i międzynarodowym. Dla gospodarki kraju będzie to oznaczać wyższą produktywność czynników produkcji, wysoką dynamikę produkcji i zbytu, zatrudnienie i wyższe dochody, a w konsekwencji podnoszenie poziomu życia ludno- ści. Pod koniec lat dziewięćdziesiątych promowanie innowacyjności przedsiębiorstw – w szerszym ujęciu rozwoju GOW i społeczeństwa informacyjnego – stało się też jednym z zadań polityki ekonomicznej w UE, głównie w ramach polityki wspierania rozwoju regionów. Instytucje i administracja państwowa – na poziomie narodowym i ponadnarodowym – stały się trzecią grupą podmiotów oddziałujących na system innowacyjności w kraju. – w szerszym ujęciu rozwoju GOW i społeczeństwa informacyjnego – stało się też jednym z zadań polityki ekonomicznej w UE, głównie w ramach polityki wspierania rozwoju regionów. Instytucje i administracja państwowa – na poziomie narodowym i ponadnarodowym – stały się trzecią grupą podmiotów oddziałujących na system innowacyjności w kraju. W różnych teoretycznych ujęciach narodowego systemu innowacyjności (NSI) od początku dominowało podejście instytucjonalne, a jednocześnie funkcjonalne – skupione na tworzeniu i dyfuzji wiedzy, technologii, innowacji. Koncepcja NSI została po raz pierwszy przedstawiona przez Ch. Freemana (1987) jako wynik jego badań nad systemem innowacyjnym w Japonii w okresie powojennym. 23 Cytuję za: Y.C. Chang, Systems of Innovation, Spatial Knowledge Links and the Firm’s Innovation Performance: Towards a National – Global Complementarity View, ,,Regional Studies” 2009 Vol. 43, Nr 9, s. 1207. 22 Różne modele heterogenicznych sieci badawczo-innowacyjnych KTN przedstawia A. Zorska w: Przemiany w organizacji działalności innowacyjnej korporacji transnarodowych, „Studia i Prace KES” 2008 nr 5, s. 91–100. 24 B. Lundvall, National Systems of Innovation. Towards a T eorem of Innovation and Interactive Learning, Pinter, London 1992, s. 1. 25 Ibid 3 4 26 Cytuję za: M. Szajt, Narodowy System Innowacji w Polsce na tle innych działających w Europie, w: Innowacyjność w skali makro i mikro, red. B. Kryk, K. Piech, Instytut Wiedzy i Innowacji, Warszawa 2009, s. 58. 27 Cytuję za: A. Filipetti, D. Archibugi, Innovation in Times of Crisis: National Systems of Innovation, Structure and Demand, ,,Research Policy” 2011 Nr 2, s. 180. 25 Ibidem, s. 3–4. 24 B. Lundvall, National Systems of Innovation. Towards a T eorem of Innovation and Interactive Learning, Pinter, London 1992, s. 1. 25 Ibidem, s. 3–4. 26 Cytuję za: M. Szajt, Narodowy System Innowacji w Polsce na tle innych działających w Europie, w: Innowacyjność w skali makro i mikro, red. B. Kryk, K. Piech, Instytut Wiedzy i Innowacji, Warszawa 2009, s. 58. 27 Cytuję za: A. Filipetti, D. Archibugi, Innovation in Times of Crisis: National Systems of Innovation, Structure and Demand, ,,Research Policy” 2011 Nr 2, s. 180. 2. Procesy i systemy innowacyjne we współczesnej gospodarce Zdaniem tego autora narodowy system innowacyjności jest to: „[...] sieć instytucji w sektorach publicznym i badawczym, których działalność i powiązania naśladują, importują, modyf kują i rozprzestrzeniają nowe technologie”.23 W tym ujęciu system innowa- cyjny zaczyna „pracować” poza aktem stworzenia wiedzy i jej pierwszego wdroże- nia do praktyki, przyczyniając się głównie do szeroko rozumianej dyfuzji innowacji pochodzenia krajowego i zagranicznego. Koncepcja Freemana zainspirowała innych badaczy, a w niedługim czasie została zmodyf kowana przez B. Lundvalla (1992), 38 Anna Zorska Anna Zorska który stwierdził, że „[...] system innowacji tworzony jest przez elementy i powiąza- nia, które współdziałają w produkcji, dyfuzji i wykorzystaniu nowej, ekonomicznie użytecznej wiedzy”.24 Tenże autor podkreślał uwarunkowania narodowe (poziom roz- woju gospodarki, nauki i techniki, kwalif kacji, przedsiębiorczości) oraz państwowe (instytucjonalne, administracyjne) w funkcjonowaniu NSI.25 Jedną z bardziej znanych def nicji NSI stworzył S. Metcalf (1995). Jego zdaniem narodowy system innowacyjny to „[...] kompleks wyodrębnionych instytucji, które wspólnie i indywidualnie wnoszą wkład do rozwoju i rozprzestrzeniania się (dyfuzji) nowej technologii i które tworzą zrąb, w ramach którego rządy formułują i realizują politykę mającą za zadanie oddziaływanie na procesy innowacyjne”.26 W def nicji tej kluczowe znaczenie mają kreujące wiedzę instytucje (podmioty, aktorzy) oraz stwo- rzona przez nie struktura czy system („zrąb” w cytowanej def nicji) oraz polityka oddziałująca w jego ramach na procesy innowacyjne. W istocie, państwo – poprzez różne instrumenty i mechanizmy – może mieć wpływ na zbiorowość (liczbę, cha- rakterystykę) innowacyjnych podmiotów, na ich układ i powiązania (strukturę), jak też częściowo na przebieg i efekty działalności innowacyjnej. W badaniach nad innowacyjnością gospodarek i przedsiębiorstw ważne miejsce zajmuje systemowy charakter działalności innowacyjnej. Przedsiębiorstwa prowadzą taką działalność z wykorzystaniem rozległych powiązań i oddziaływań (interakcji) z różnymi podmiotami poza własną organizacją. Działalność innowacyjna przedsię- biorstw i innych podmiotów toczy się w specyf cznych uwarunkowaniach instytu- cjonalnych i strukturalnych istniejących w danym kraju. Taką myśl wyraża def nicja szwedzkich autorów C. Edquista i B. Lundvalla, którzy stwierdzili, że ,,[...] narodowy system innowacyjny jest tworzony przez instytucje i struktury ekonomiczne kształ- tujące tempo i kierunki przemian technologicznych w społeczeństwie”.27 Oprócz instytucji (aktorów, podmiotów) autorzy uwzględniają struktury ekonomiczne, odnoszące się do wyposażenia kraju w zasoby czynników produkcji (do ich wystę- powania, struktury, zaawansowania, jakości), a także do instytucji państwowych, w tym administracji publicznej na różnych poziomach gospodarowania. p 29 Szerzej na ten temat patrz: A. Zorska, Korporacje transnarodowe. Przemiany, oddziaływania, wyzwania, PWE, Warszawa 2007, s. 252–266. 28 L. Soete, B. Verspangen, B.T. Weel, Systems of Innovation, w: Handbook of the Economics of Innovation, op.cit., s. 1167–1169. 2. Procesy i systemy innowacyjne we współczesnej gospodarce Narodowy system innowacyjności opiera się na sieci podmiotów – organizacji (przedsiębiorstw, instytucji badawczych i administracyjnych), których współpraca Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 39 może być korzystna dla wszystkich podmiotów, gdyż stwarza im możliwości wza- jemnego uczenia się oraz współdziałania, a także stworzenia kapitału społecznego aktywizującego ich pracę w danym systemie.28 Przyczynia się to do zwiększenia dyna- miki i efektywności procesów innowacyjnych w tym systemie, jak też synergicznych korzyści przypadających wszystkim podmiotom i społeczeństwu. Z dorobku badań nad narodowymi systemami innowacyjności wynika, że ich bardzo istotnymi cechami są wielowymiarowość i złożoność. NSI można analizo- wać – jak też kształtować – w czterech wymiarach, które przeplatają się i na siebie oddziałują, a w konsekwencji wzmacniają innowacyjność na podstawowym pozio- mie mikroekonomicznym. Są to następujące wymiary: organizacyjny, funkcjonalny, podmiotowy, przestrzenny. Wymiar organizacyjny odnosi się do działalności innowacyjnej przedsiębiorstw i innych podmiotów bezpośrednio zaangażowanych w tego rodzaju działalność. Organizacyjne kwestie mają największe znaczenie w przedsiębiorstwach o rozbu- dowanych strukturach, jakimi są KTN. Jak już wspomniano, w działalności inno- wacyjnej KTN uczestniczą przede wszystkim własne jednostki korporacji, którymi są własne ośrodki B+R, działy badawcze i biura techniczne w f liach produkcyjnych. Jednostki wyróżniające się innowacyjnością uzyskują status „centrum doskonało- ści” („center of excellence”), otrzymują też mandat rozprzestrzeniania nowych roz- wiązań i uruchamiają proces uczenia się wewnątrz danej organizacji. Należy pod- kreślić, że obecnie działalność innowacyjna KTN rozwija się w dużej mierze poza centralnymi laboratoriami w kraju macierzystym danej f rmy, głównie w zagranicz- nych f liach i ośrodkach.29 W ostatnich latach przechodzą one „twórczą przemianę”, co oznacza większą samodzielność f lii zagranicznych w rozwijaniu prac B+R, ich przekształcaniu w innowacje i własne przewagi konkurencyjne. Zagraniczne f lie i ośrodki otrzymały też więcej uprawnień do prowadzenia współpracy techno- logicznej z niezależnymi podmiotami, którymi mogą być ośrodki czy instytuty badawcze, uczelnie, innowacyjne f rmy krajowe. W rezultacie w KTN funkcjo- nują tzw. podwójne sieci badawczo-innowacyjne, co oznacza powiązanie dzia- łalności innowacyjnej własnych (zależnych) jednostek i niezależnych podmio- tów. Ich współpraca pozwala przyspieszyć procesy innowacyjne oraz obniżyć ich koszty, a także szerzej wdrażać innowacje w celu zminimalizowania ryzyka imitacji i erozji przewag konkurencyjnych. Tego rodzaju sieci są organizowane 40 Anna Zorska Anna Zorska Anna Zorska i kierowane przez wiodące KTN, które stają się „integratorem” działalności wielu podmiotów oraz „animatorem” łączących je powiązań sieciowych.30 W skali kraju istnieje zwykle wiele systemów innowacyjnych przedsiębiorstw, które nie muszą być odizolowane i niezależne. 31 Ekspertyzy i analizy dotyczące zagadnień transformacji wiedzy, konkurencyjności i innowacyjności gospodarki, PARP, Warszawa 2009, s. 115. 30 A.P. De Man, T e Network Economy, Strategy, Structure and Management, Edward Elgar, Cheltenham– –Northampton 2004, s. 9–12, 31–33. 2. Procesy i systemy innowacyjne we współczesnej gospodarce Odwrotnie, jeśli istnieją ich „punkty wspólne” – np. w regionalnych systemach innowacyjności – przepływ wiedzy i dyfu- zja innowacji dokonują się szybciej, szerzej i niższym kosztem, co może skutkować większą konkurencyjnością międzynarodową f rm. Ponadto NSI są też tworzone jako pewnego rodzaju makrostruktura organizacyjna posiadająca kilka szczebli (stopni), po których dokonuje się przechodzenie od krajowej strategii innowacyjnej (technolo- gicznej) do realizacji konkretnych projektów przez przedsiębiorstwa i inne podmioty. Wymiar funkcjonalny jest związany z pewnego rodzaju specjalizacją w zada- niach realizowanych przez poszczególne jednostki i podmioty, czy też inaczej – elementy systemu innowacyjności na różnych poziomach. Na poziomie przed- siębiorstwa (zwłaszcza KTN) polega to na tym, że dla sprawnego i efektywnego funkcjonowania systemu innowacyjności poszczególne zadania są przydzielane wyspecjalizowanym jednostkom. Ośrodki czy działy badawcze realizują prace B+R (zwykle ich określone fragmenty), działy rozwoju zasobów ludzkich kompletują i szkolą kadry, dział techniczno-inwestycyjny przygotowuje zmiany w produkcji itd. Podobnie jest na poziomie krajowym, gdzie funkcjonuje sfera badawczo-rozwojowa (publiczne ośrodki naukowe i badawcze), edukacyjna i akademicka (kształcenie kadr, częściowo badania), instytucjonalno-administracyjna (wspieranie przedsiębiorczo- ści, jej f nansowanie), a także sfera biznesowa, czyli przedsiębiorstwa produkcyjno- -handlowe.31 Rezultatem dobrego i współzależnego funkcjonowania różnych sfer jest stworzenie większego zasobu nowej wiedzy, bardziej efektywne jej przekształ- canie w innowacje, ich szeroka dyfuzja w gospodarce. Wymiar podmiotowy jest z tym związany, ale zasługuje na wyodrębnienie. Bardzo istotne są tu dwa aspekty: zbiorowość podmiotów i wysoki poziom reali- zacji działań przez różne sfery i tworzące je podmioty, jak też ewoluujące powią- zania tych podmiotów. O rozwoju i zaawansowaniu NSI świadczy liczba przedsię- biorstw – zwłaszcza KTN – zaangażowanych w działalność innowacyjną, nakłady na prace B+R, osiągnięcia patentowe i rynkowe, kadry badaczy, sieci badawcze, udział w projektach badawczo-innowacyjnych itp. Można również dokonać ilościowej cha- rakterystyki poziomu rozwoju i działalności innych sfer – badawczej, akademickiej, administracyjnej. Możliwe jest wyrażenie zdolności innowacyjnych podmiotów 41 Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy w gospodarce za pomocą syntetycznego wskaźnika i skonfrontowanie go z pozio- mem wskaźników innych krajów, jak zrobił to Bank Światowy, oceniając f lary GOW. Jednakże poza wskaźnikami o charakterze ilościowym pozostaje ważny obszar jako- ściowej charakterystyki i oceny NSI, która dotyczy relacji i powiązań różnych grup podmiotów. To one (relacje, powiązania) decydują o dynamice zmian w NSI, które są związane z tworzeniem i przepływem nowej wiedzy, uczeniem się nowych rozwiązań przez różne podmioty, szerokim wykorzystaniem innowacji w biznesie. 33 Jeśli nie zaznaczono inaczej, to chodzi o region jako część kraju, wyodrębnioną geograf cznie, ekonomicznie i administracyjnie. W innych przypadkach chodzi o region jako część kontynentu, zwykle obejmującą grupę krajów integrujących się ekonomicznie (jak np. UE). 32 A. Marszałek, Model ,,potrójnej pętli” a zmiany w gospodarce opartej na wiedzy, „Gospodarka Narodowa” 2010 nr 1–2, s. 133–135. 33 Jeśli nie zaznaczono inaczej, to chodzi o region jako część kraju, wyodrębnioną geograf cznie, ekonomicznie i administracyjnie. W innych przypadkach chodzi o region jako część kontynentu, zwykle obejmującą grupę krajów integrujących się ekonomicznie (jak np. UE). 32 A. Marszałek, Model ,,potrójnej pętli” a zmiany w gospodarce opartej na wiedzy, „Gospodarka Narodowa” 2010 nr 1–2, s. 133–135. 2. Procesy i systemy innowacyjne we współczesnej gospodarce Zmieniające się relacje i powiązania między trzema zasadniczymi grupami pod- miotów w NSI opisano za pomocą trzech modeli „potrójnej pętli”. W modelu I pań- stwo (i jego instytucje) kształtowało warunki dla działalności innowacyjnej dwóch sfer: biznesowej i badawczo-akademickiej. W modelu II grupy podmiotów z trzech sfer – państwowej, badawczo-akademickiej i biznesowej – są połączone różnymi rela- cjami, ale też ograniczone określonymi rolami w systemie innowacyjności. W modelu III sfera badawcza wysuwa się na pierwszy plan, tworząc infrastrukturę wiedzy, w której ramach obszary działań różnych podmiotów zachodzą na siebie, a wewnątrz wspólnego dla nich obszaru powstają organizacje hybrydowe.32 Przykładowo, mogą to być spółki zawiązane do realizacji projektu badawczo-innowacyjnego z udziałem różnych podmiotów, których przedstawiciele wspólnie zarządzają nowym przedsię- wzięciem i mobilizują inne podmioty czy osoby do współpracy. Wymiar przestrzenny systemów innowacyjnych nabrał znaczenia w konsekwen- cji procesów globalizacji oraz informatyzacji w sferze ekonomicznej, a zwłaszcza w działalności przedsiębiorstw. Z jednej strony, przedsiębiorstwa uzyskały większe możliwości tworzenia wiedzy i jej stosowania na rynkach różnych krajów dzięki libe- ralizacji dostępu dla zagranicznych f rm i kapitału, a także dzięki globalnym sieciom informacyjnym, przepływom informacji i wiedzy. Z drugiej strony, zaostrzenie kon- kurencji na rynku globalnym zmusiło przedsiębiorstwa do rozwijania nowej wie- dzy jako kluczowego zasobu dla podnoszenia konkurencyjności międzynarodowej. Presja na rozwijanie zasobu wiedzy i jego biznesowe zastosowanie skłania f rmy do wykorzystania wszystkich możliwości w tym zakresie, w otoczeniu lokalnym, regio- nalnym, krajowym i globalnym. Te pierwsze – w skali lokalnej – zwykle nie mają dużego potencjału i wyczerpują się szybko, toteż obszar poszukiwań przedsiębiorstw rozszerza się na region,33 a następnie na kraj. 42 Anna Zorska Anna Zorska Regionalny wymiar działalności innowacyjnej nabrał znaczenia i przyczynił się do rozwoju regionalnych systemów innowacyjności (RSI). Do tworzenia wiedzy w regionach nawiązuje def nicja tych systemów w ujęciu B. Asheima i L. 34 B.T. Asheim, L. Coenen, Knowledge Bases and Regional Innovation Systems: Comparing Nordic Clusters, „Research Policy“ 2005 Vol. 34, s. 1174. 35 Są to m.in.: Y.Ch. Chang, L. Cusmano, M.L. Mancusi, A. Morrison, S. Roper, Ph. Shapira, J. Youtie. 36 Szerzej na ten temat patrz: W.M. Gaczek, Gospodarka oparta..., op.cit., s. 76–101. 37 Y.C. Chang, Systems of Innovation..., op.cit., s. 1208, 1220. 2. Procesy i systemy innowacyjne we współczesnej gospodarce Coenena, którzy twierdzą, że RSI polega obecnie na interaktywnym tworzeniu i wykorzystaniu wiedzy, a przy tym stanowi subsystem, który jest powiązany z innymi systemami – krajowym, ponadnarodowym (UE) oraz globalnym – i może rozciągać się na różne sektory w gospodarce regionu.34 Również inni autorzy zwracają uwagę na przeplata- nie się i łączenie systemów innowacyjnych na różnych poziomach innowacyjności, a jednocześnie w przestrzeni o różnej skali.35 Ponadto znaczenia nabiera oddziaływa- nie na RSI (w skali kraju) przez instytucje na poziomie ponadnarodowym (regionu kontynentalnego), czego przykładem jest Unia Europejska oraz jej rozbudowana i ewoluująca polityka regionalna i innowacyjna.36 Systemy innowacyjne regionów i krajów nabierają charakteru ponadgranicznego, przede wszystkim ze względu na działalność badawczą i transfer wiedzy przez KTN. Korporacje dokonują geograf cznego rozproszenia prac B+R (ich globalizowania) w celu pozyskania brakującej im wiedzy, która może istnieć w globalnym otoczeniu. Ponadto możliwe jest wspólne, bardziej efektywne tworzenie wiedzy z partnerami zagranicznymi, posiadającymi komplementarne zdolności i duże doświadczenie w specjalistycznych badaniach. Poprzez różnego rodzaju powiązania tworzone dzięki KTN relacje pomiędzy systemami innowacyjności w wymiarze korporacyjnym, kra- jowym i globalnym układają się w sposób komplementarny i symbiotyczny.37 Prze- jawia się to w tworzeniu przez korporacje zagranicznych ośrodków B+R, rozwija- niu działalności innowacyjnej f lii zagranicznych oraz kooperacji technologicznej z partnerami w tych krajach, gdzie istnieją uzupełniające się, zaawansowane tech- nologicznie zasoby i zdolności. Pomiędzy jednostkami korporacyjnymi i krajowymi podmiotami tworzą się powiązania i sieci badawczo-innowacyjne. Są one poten- cjalnie korzystne dla wszystkich uczestników, ale ostateczny podział korzyści zależy od ich siły przetargowej, czyli w praktyce nierzadko od interesu KTN. Globalne przepływy i powiązania w dziedzinie technologii rozwijają się, a wpływ tego procesu na specjalizacje technologiczne w poszczególnych krajach pogłębia się. Może to prowadzić do podnoszenia poziomu i wzrostu znaczenia nowych osiągnięć technologicznych powstających w pewnych branżach gospodarki określonego kraju. Z drugiej strony, następuje „otwieranie się” narodowych systemów innowacyjnych Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 43 na powiązania z innymi systemami krajowymi w regionie (np. wśród krajów UE) oraz systemami globalnymi (tworzonymi i zarządzanymi przez KTN).38 Otwiera- nie się NSI polega na dopuszczeniu zagranicznych podmiotów do obecności i dzia- łalności (np. poprzez ulokowanie korporacyjnych ośrodków badawczych czy f lii) oraz do rozwijania przez nie powiązań badawczych w danym kraju. Jednocześnie mogą mieć miejsce analogiczne działania krajowych podmiotów za granicą, a także włączenie zagranicznych instytucji do wspierania systemów regionalnych czy kra- jowego. y j g y f p 41 Zdarza się tak, jeśli krajowy system innowacyjności jest stosunkowo słaby, a innowacyjne f rmy krajowe szukają nowych możliwości awansu technologicznego poprzez powiązania międzynarodowe. 38 Ibidem, s. 1219. p g f g g 40 Przedmiotem badań były dwa sektory globalne – obwodów scalonych i biotechnologii – oraz różne systemy innowacyjności, w jakich uczestniczyły przedsiębiorstwa z tych sektorów, zlokalizowane w Wielkiej Brytanii i na Tajwanie. Źródło: Y.C. Chang, Systems of Innovation..., op.cit., s. 1200 i dalsze. , 39 L. Cusmano, M.L. Mancusi, A. Morrison, Globalization of Production and Innovation: How Outsourcing is Reshaping an Advanced Manufacturing Area, ,,Regional Studies” 2010 Vol. 44, Nr 3, s. 249. 38 Ibidem, s. 1219. 39 L. Cusmano, M.L. Mancusi, A. Morrison, Globalization of Production and Innovation: How Outsourcing is Reshaping an Advanced Manufacturing Area, ,,Regional Studies” 2010 Vol. 44, Nr 3, s. 249. 40 Przedmiotem badań były dwa sektory globalne – obwodów scalonych i biotechnologii – oraz różne systemy innowacyjności, w jakich uczestniczyły przedsiębiorstwa z tych sektorów, zlokalizowane w Wielkiej Brytanii i na Tajwanie. Źródło: Y.C. Chang, Systems of Innovation..., op.cit., s. 1200 i dalsze. 41 Zdarza się tak, jeśli krajowy system innowacyjności jest stosunkowo słaby, a innowacyjne f rmy krajowe szukają nowych możliwości awansu technologicznego poprzez powiązania międzynarodowe. 2. Procesy i systemy innowacyjne we współczesnej gospodarce Jednakże wejście zagranicznych f rm oraz instytucji do krajowego systemu innowacyjnego może prowadzić do uszczuplenia jego zasobu nowej wiedzy i poten- cjalnych korzyści wdrożeniowych dla krajowych f rm poprzez przejęcie i wykorzy- stanie nowości przez obce podmioty, głównie KTN.39 Badania wykazały, że w wysoce umiędzynarodowionym sektorze przeplatają się systemy innowacyjności działające na różnych poziomach, co implikuje ich koeg- zystencję.40 Oznacza to, że f rmy mogą korzystać z lokalnych powiązań w tworzeniu wiedzy, jak też rozszerzać tego typu powiązania na poziomie krajowym i wyższym regionalnym (przypadek UE), a nawet globalnym (np. dokonując zakupu nowych technologii od KTN na innych kontynentach).41 Funkcjonowanie różnych syste- mów innowacyjności – w sposób symbiotyczny i komplementarny – nie jest rzad- kim zjawiskiem w sektorach globalnych. Dlatego oddziaływanie na relacje trzech systemów – regionalnego, krajowego i globalnego – przez instytucje państwowe lub ponadpaństwowe (UE) może mieć pozytywny wpływ na innowacyjność przedsię- biorstw w danym kraju czy integrującym się regionie. We współczesnej gospodarce systemy innowacyjne na różnych poziomach stają się coraz bardziej złożone i prze- platające się (intertwinned), a w konsekwencji narasta ich integrowanie i współza- leżność ponad granicami państw. 44 Anna Zorska 42 World Development Indicators 2011, T e World Bank, Washington, D.C. 2011, s. 32, tab. 1.1. 43 Ibidem s 238 tab 4 4 oraz s 246 tab 4 6 42 World Development Indicators 2011, T e World Bank, Washington, D.C. 2011, s. 32, tab. 1.1. p 43 Ibidem, s. 238, tab. 4.4 oraz s. 246, tab. 4.6. 42 World Development Indicators 2011, T e World Bank, Washington, D.C. 2011, s. 32, tab. 1.1. 43 Ibidem, s. 238, tab. 4.4 oraz s. 246, tab. 4.6. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Za wyborem Finlandii jako kraju wspierającego systemy innowacyjne i rozwój GOW przemawiają następujące względy wyróżniające ten kraj: • osiągnięcia w promowaniu rozwoju gospodarki wiedzy przede wszystkim w oparciu o dwa f lary: systemy innowacyjności oraz edukacji, • udane połączenie działania rynku oraz instytucji stworzonych i kierowanych przez państwo w ramach prowadzonej polityki technologicznej, • redystrybucja korzyści z rozwoju GOW na cele edukacyjne i społeczne, • skuteczne podniesienie międzynarodowej konkurencyjności gospodarki. Metody działania i doświadczenia Finlandii mogą być w pewnym stopniu uży- teczne dla innych państw tworzących NSI oraz programy rozwoju GOW, chociaż uwarunkowania ekonomiczne, społeczne i polityczne omawianego kraju są w dużym stopniu specyf czne. Finlandia jest krajem niedużym (powierzchnia wynosi 338 tys. km²) i niezbyt ludnym (liczy niewiele ponad 5 mln mieszkańców), ma surowy klimat i krajobraz wypełniony jeziorami. Jest położona peryferyjnie w Europie Północnej, a przy tym pozbawiona większych zasobów minerałów i paliw oraz gruntów ornych. Mimo to Finowie osiągnęli dość wysoki poziom PKB, wynoszący prawie 36 tys. dol. per capita w 2009 roku (z uwzględnieniem parytetu siły nabywczej walut).42 Na wyroby prze- mysłowe przypadało 81% wartości eksportu towarowego Finlandii w 2008 roku, w tym około 60% stanowiły wyroby wysoko zaawansowane technologicznie. Udział nowoczesnych usług (komputerowych, informacyjnych, telekomunikacyjnych, biz- nesowych) w f ńskim eksporcie usługowym wynosił prawie 76%.43 Swoimi osią- gnięciami kraj ten wyróżnia się, gdyż w niedługim czasie dołączył do grupy liderów rozwoju GOW na świecie, międzynarodowej konkurencyjności, uczestników glo- balizacji oraz promuje sprawiedliwość społeczną. Pod koniec lat osiemdziesiątych XX wieku w Finlandii został zapoczątkowany – pod wpływem doświadczeń Szwecji – rozwój gospodarki, który charakteryzował się orientacją na tworzenie i stosowanie wiedzy. Rozwinięcie działań w tym zakresie zostało jednak zahamowane przez wielki kryzys gospodarczy, który dotknął gospo- darkę f ńską na początku lat dziewięćdziesiątych wskutek utraty głównego, zagra- nicznego rynku zbytu w Związku Radzieckim. Polityczny i transformacyjny kryzys Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 45 w Rosji przyniósł drastyczny spadek importu wielu wyrobów f ńskich, co – przy niewielkim rynku krajowym – silnie odbiło się na ograniczeniu produkcji i zatrud- nienia w Finlandii. Odpowiedzią rządu i społeczeństwa tego kraju było wzmożenie wysiłków skupionych na przebudowie i unowocześnianiu gospodarki. g 45 W dokumentach rządu f ńskiego przyjęto sformułowanie „polityka technologiczna”, co w istocie odpowiada używanemu w literaturze tematu terminowi „polityka innowacyjna”. W dalszych rozważaniach terminy te są używane zamiennie, chociaż w kontekście Finlandii częściej pojawia się pierwsze sformułowanie. 44 Szerzej na ten temat w publikacji: Finland as a Knowledge Economy. Elements of Success and Lessons Learned, red. C.J. Dahlman, J. Routti, P. Ylä-Antilla, T e World Bank, Washington, D.C. 2007. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Opracowano nową strategię wzrostu gospodarczego, która charakteryzowała się wytyczeniem rozwoju kilku nowoczesnych branż, w sprzyjających warunkach ekonomicznych oraz instytucjonalnych, w oparciu o krajowy system innowacyjności i wiodącą rolę wybranych przedsiębiorstw.44 W Finlandii tworzenie wiedzy, nowych technologii oraz innowacji zostało ści- śle powiązane z rozwojem przemysłu elektronicznego i usług w zakresie oprogra- mowania. Co istotne, rozwój tych nowych gałęzi był początkowo ściśle powiązany z potrzebami modernizacyjnymi już istniejących w kraju przemysłów o charakterze surowcowym i przetwórczym (tzn. przemysłów: drzewnego, celulozowego, papierni- czego, spożywczego), dostarczając im nowoczesne urządzenia pomiarowe, automa- tykę przemysłową, urządzenia do prac biurowych i łączności. W warunkach otwie- rania gospodarki na współpracę i wymianę zagraniczną nasiliła się konkurencja f rm krajowych i zagranicznych, zwiększyło się umiędzynarodowienie f ńskiej gospodarki. Dla ułatwienia restrukturyzacji i modernizacji przedsiębiorstw krajowych oraz pod- niesienia ich konkurencyjności działania państwa zostały ukierunkowane na poli- tykę technologiczną45 i wspieranie innowacyjności przedsiębiorstw. Rząd f ński ograniczył tradycyjną, bieżącą interwencję w gospodarce, skierowaną na wspieranie efektywności f rm krajowych i kształtowanie aktualnie korzystnych warunków makroekonomicznych. Nastąpiło przejście rządu do działań bardziej długofalowych i strategicznych, ukierunkowanych na podnoszenie międzynarodo- wej konkurencyjności f rm, dzięki poprawie jakości i poziomu technologicznego czynników wytwórczych, stworzeniu instytucji wspierających transfer wiedzy do przedsiębiorstw i umacnianiu sprzyjających innowacyjności warunków w otocze- niu gospodarczym. W czasie wychodzenia z kryzysu gospodarczego (po rozluźnie- niu powiązań z Rosją) od roku 1994 polityka unowocześniania f ńskiej gospodarki i jej orientacji ku GOW stała się zasadniczym kierunkiem aktywności ekonomicznej państwa i ogólnonarodowym zadaniem społeczeństwa Finlandii. Główne kierunki polityki państwa f ńskiego wobec rozwoju GOW w zasadzie odpowiadają koncepcji f larów w ujęciu Banku Światowego, co zostało omówione 46 Anna Zorska Anna Zorska w punkcie 1. Z konieczności trzy f lary zostaną omówione skrótowo, a szerzej będzie przedstawiona charakterystyka i funkcjonowanie NSI. Należy podkreślić, że w przy- padku Finlandii siła f larów jest wyrównana, co umożliwia uzyskanie synergicznych korzyści z interakcji postępu w różnych dziedzinach wspierania rozwoju GOW. Pierwszy f lar odnosi się do krajowego systemu bodźców ekonomicznych oraz ram instytucjonalnych, służących wspieraniu rozwoju GOW. W Finlandii tworze- nie rynkowych i instytucjonalnych podstaw rozwoju GOW było związane z odbu- dową gospodarki po kryzysie oraz tworzeniem nowych powiązań biznesowych bez dominującej pozycji Rosji. 48 Finlandię wyprzedziły następujące kraje: Dania, Szwecja, Szwajcaria, USA i Singapur. Polska znajdowała się na miejscu 62., za wieloma krajami Trzeciego Świata. Global Information Technology Report 2007–2008. Fostering Innovation T rough Networked Readiness, World Economic Forum–INSEAD, Palgrave– –McMillan, Hounmills– Basingstoke 2008, s. XIX. 46 Szerzej na temat przemian rynkowych w gospodarce f ńskiej pisze J. Wierzbołowski, Fińska droga do społeczeństwa informacyjnego i gospodarki opartej na wiedzy, Instytut Łączności, Warszawa 2003, s. 15–46. 47 Ibidem, s. 62. f 47 Ibidem, s. 62. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Oznaczało to stymulowanie konkurencji prywatnych f rm, umacnianie krajowego rynku oraz stopniowe otwieranie go na dostęp i powiązania z różnymi uczestnikami rynku globalnego.46 Drugi f lar dotyczy systemu edukacji i szkoleń, a jego siła (poziom wskaźnika i pozycja na tle innych państw) stawia Finlandię wśród krajów najbardziej zaanga- żowanych w tej dziedzinie. Podstawową zasadą f ńskiego systemu edukacji jest rów- ność dostępu (pod każdym względem) do szkolnictwa na wszystkich poziomach, dla każdego obywatela. Realizację tej zasady umożliwia duża liczba placówek szkolnych i akademickich, wysoka jakość usług edukacyjnych oraz niskie koszty kształcenia, a w dużej części bezpłatna nauka. Potrzeby rozwijających się sektorów informacyj- nego i GOW miały wpływ na politykę edukacyjną rządu f ńskiego. Znacząco zwięk- szono możliwości kształcenia na poziomie akademickim w zakresie technologii kom- puterowych oraz informacyjnych, z czego skorzystali zwłaszcza młodzi ludzie. Obok Korei Południowej, Finlandia osiągnęła najwyższy na świecie udział nauk informa- cyjnych wśród absolwentów uczelni.47 Trzeci f lar GOW tworzy infrastruktura informacyjna, która jest wykorzystywana przez różnych odbiorców, poczynając od przedsiębiorstw przemysłowych i f rm usłu- gowych, przez wiele instytucji administracji państwowej, do indywidualnych użyt- kowników. Poziom rozwoju i dostępność infrastruktury informacyjnej w Finlandii należy do najwyższych w świecie. Świadczy o tym wysoki poziom wskaźnika przy- gotowania do pracy w sieci (network readiness index), który pozwolił sklasyf kować Finlandię na szóstym miejscu na świecie w okresie 2007–2008 (ostatnim, dla któ- rego są dostępne dane).48 Nowoczesność, jakość i niezawodność sieci informacyj- nej, duża podaż i różnorodność usług informacyjnych oraz sprzętu IKT, ich niskie Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 47 ceny, a także bezpieczeństwo użytkowania sieci – to główne cechy funkcjonowania sektora informacyjnego w omawianym kraju. W Finlandii podstawą infrastruktury IKT jest klaster informacyjny, w którym wiodącą rolę odgrywa f rma Nokia, nazywana też „f rmą f agową” ze względu na przewodzenie całej „f otylli” krajowych spółek z nią kooperujących. W szerszym uję- ciu klaster informacyjny tworzy około 6 tys. spółek, w tym około 300 f rm będących podwykonawcami dla korporacji Nokia. Działalność tych spółek obejmuje świad- czenie różnego rodzaju nowoczesnych usług opartych na IKT, a także produkcję i serwis urządzeń telekomunikacyjnych, głównie telefonów komórkowych. Bliskie powiązania łączą spółki informacyjne z innymi sektorami: bankowości elektronicz- nej, handlu, szkolnictwa, rozrywki, administracji itd. Historia f rmy Nokia odzwierciedla nie tylko rozwój sektora informacyjnego w Finlandii, lecz również modernizacji tradycyjnych gałęzi gospodarki tego kraju. p f yj p 50 W 2005 roku wskaźniki Korei Południowej i Irlandii – będących najbliższymi rywalami Finlandii – wynosiły do 13%. Por. ibidem, s. 23. 49 M. Castells, P. Himanen, Społeczeństwo informacyjne..., op.cit., s. 47–64. 50 W 2005 roku wskaźniki Korei Południowej i Irlandii – będących najbliższymi rywalami Finlandii – wynosiły do 13%. Por. ibidem, s. 23. 49 M. Castells, P. Himanen, Społeczeństwo informacyjne..., op.cit., s. 47–64. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Od początku XX wieku do połowy lat dziewięćdziesiątych Nokia była konglome- ratem wytwarzającym wyroby celulozowe, gumowe i kable, a następnie elektronikę użytkową i pierwsze f ńskie telefony komórkowe. Ostry kryzys wymusił radykalne zmiany w f rmie, które polegały na restrukturyzacji produkcji (ku telefonii komór- kowej), uzdrowieniu f nansów, a także przebudowie struktury organizacji (ku mode- lowi sieciowemu). Pod kilkoma względami przemiany dokonane w Nokii wyprze- dziły inne f rmy w sektorze telefonii komórkowej na świecie. Najważniejsza okazała się udana transformacja Nokii w f rmę o ponadgranicznej sieci ośrodków i f lii, która obejmowała prace B+R w 15 krajach, produkcję w 10, a sprzedaż telefonów na 130 rynkach zagranicznych. Pomimo wysokiego stopnia umiędzynarodowienia Nokia nadal pozostaje kluczowym przedsiębiorstwem w Finlandii. Jej rola polega przede wszystkim na tworzeniu i rozpowszechnianiu specjalistycznej wiedzy informacyj- nej, czyli na wiodącym uczestnictwie w f ńskim systemie innowacyjności (będzie o tym mowa poniżej).49 Dzięki zaangażowaniu Nokii oraz tysięcy innych spółek w produkcję wyro- bów i usług bazujących na IKT Finlandia stała się krajem wyspecjalizowanym w szeroko rozumianej produkcji (wyrobów i usług) opartej na technologiach infor- macyjnych, która stanowi około 16% wartości dodanej w sektorze przedsiębiorstw prywatnych.50 Szczególnie duży jest udział sektora IT w eksporcie, gdyż na jego dostawy przypadało około 60% wartości f ńskiego eksportu. Oznacza to najwyż- szą w świecie specjalizację kraju w wywozie sprzętu telekomunikacyjnego oraz naj- większą nadwyżkę w handlu zagranicznym, w dziedzinie obrotów wyrobami wyso- 48 Anna Zorska Anna Zorska kich technologii.51 Jednakże z drugiej strony jest to silne uzależnienie produkcji w f ńskim sektorze informacyjnym od zmian popytu na rynku globalnym oraz od rywalizacji ze strony wiodących f rm z innych krajów. Czwarty f lar odnosi się do narodowego systemu innowacyjności, który w każ- dym kraju ma wspierać tworzenie i rozprzestrzenianie się wiedzy, nowych technolo- gii, innowacji – ku ich produkcyjnemu wykorzystaniu w gospodarce danego kraju. Wykształcenie sprawnego systemu innowacyjnego (na różnych poziomach działa- nia) wymaga aktywności państwa, które musi uruchomić odpowiednie instrumenty wspierające działalność różnych podmiotów skupionych w NSI, które stymulują two- rzenie wiedzy, jej przekształcanie w innowacje oraz szeroką dyfuzję w przedsiębior- stwach z różnych gałęzi przemysłu i usług. Zdaniem specjalistów w Finlandii powstał jeden z najbardziej zaawansowanych, sprawnych i skutecznych systemów innowacyjności, a w jego stworzeniu i kształ- towaniu wiodącą rolę odgrywa państwo.52 Instytucją, która realizuje politykę pań- stwa w omawianym zakresie, jest Rada Nauki i Polityki Technologicznej (Science and Technology Council, STPC), kierowana przez premiera rząd f ńskiego. 51 Ibidem, s. 43. 52 W.M. Gaczek, Gospodarka oparta..., op.cit., s. 67. Podobne opinie wyrażają inni autorzy cytowani w tym punkcie opracowania. 51 Ibidem, s. 43. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Rozdziału środków na infrastrukturę badawczą i same prace B+R dokonuje się z uwzględnieniem możliwości i potrzeb róż- nych regionów oraz miast (infrastruktura, edukacja, ośrodki badawcze, parki tech- nologiczne itp.). W ostatnich latach wzrasta rola SITRY w dysponowaniu kapitałem wysokiego ryzyka, za pomocą którego f nansuje powstające, nowe f rmy. – odpowiadają za ustalenie głównych kierunków działalności B+R oraz dostosowa- nie polityki naukowej i edukacyjnej do potrzeb gospodarki, m.in. wskazując sektory o znaczeniu strategicznym w przyszłości. Finansowanie prowadzonych badań oraz ich przekształcania w użyteczne innowacje powierzono agencji SITRA (we współ- pracy z ekspertami), która f nansuje projekty technologiczne realizowane w koope- racji i sieciowych powiązaniach różnych podmiotów – ośrodków badawczych i aka- demickich oraz przedsiębiorstw. Nakłady na prace B+R osiągnęły wysoki poziom i relację do PKB, stanowiąc 3,6% jego wartości. Rozdziału środków na infrastrukturę badawczą i same prace B+R dokonuje się z uwzględnieniem możliwości i potrzeb róż- nych regionów oraz miast (infrastruktura, edukacja, ośrodki badawcze, parki tech- nologiczne itp.). W ostatnich latach wzrasta rola SITRY w dysponowaniu kapitałem wysokiego ryzyka, za pomocą którego f nansuje powstające, nowe f rmy. W systemie innowacyjności uczelnie pełnią dwie ważne role. Po pierwsze, dostar- czają innowacyjnym podmiotom kadr o wysokich kwalif kacjach i tworzą kapitał ludzki. Po drugie, są miejscem realizacji prac badawczych (głównie o charakterze podstawowym) oraz tworzenia wiedzy i nowych technologii, które zasilają procesy innowacyjne f rm. W Finlandii jest 21 uniwersytetów, 31 politechnik oraz 21 dużych instytutów badawczych. Akademickie dyplomy z nauk ścisłych posiada ponad 33% absolwentów uczelni w Finlandii, podczas gdy w innych krajach UE dwa razy mniej. Szczególny nacisk kładzie się na kształcenie kadr w zakresie wysoko zaawansowa- nej informatyki. Najbardziej znanymi f ńskimi f rmami o wysokiej innowacyjności są Nokia i Linux. W f ńskim systemie innowacyjnym Nokia odegrała rolę f rmy wiodącej, której rozwój technologiczny i sieciowe powiązania były w dużej mierze wspie- rane i f nansowane przez instytucje państwowe. Również Nokia silnie inwestowała w prace B+R, przeznaczając na ten cel około 10% przychodów, co uczyniło z niej wiodącego „innowatora” na tle f rm krajowych i zagranicznych. W Finlandii inno- wacyjność Nokii promieniowała na setki innych f rm, które z nią kooperowały w pracach badawczo-wdrożeniowych oraz produkcyjnych (obowiązkowo, co było zastrzeżone w projektach technologicznych). Nokia stała się centralnym węzłem sieci f rm w sektorze informacyjnym, a w szerszym ujęciu – klastra informacyjnego. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Co trzy lata Rada dokonuje przeglądu i oceny realizacji polityki technologicznej i wprowa- dza zmiany lub nowe wytyczne do strategii rozwoju nauki i techniki w kraju. STPC spełnia funkcje głównego koordynatora działań instytucji państwowych, urzędów administracji samorządowej, wyższych uczelni, ośrodków badawczych oraz innych podmiotów, których działalność składa się na rozbudowany, lecz sprawny f ński sys- tem innowacyjności. Schemat tego systemu przedstawia rysunek 1. Na rysunku 1 zwraca uwagę nie tylko wiodąca pozycja instytucji państwa – ogólnych oraz wyspecjalizowanych w polityce technologicznej – oraz ich powiązań i oddziaływań, ale przede wszystkim różne poziomy kształtowania innowacyjności. Poziomy te wskazują na rolę odgórnych działań, ale w istocie ważne znaczenie ma ich przeniesienie na działalność podmiotów prywatnych i publicznych (f rm, agencji, banków) na najniższym poziomie. Dokonuje się stopniowa operacjonalizacja decyzji rządowych, tzn. ich przełożenie na konkretne działania (poparte zasilaniem f nan- sowym) przez różne podmioty tworzące i wdrażające innowacje lub temu służące (np. banki). Pobudzanie zdolności innowacyjnych zostało dobrze powiązane z poli- tyką rozwoju regionów peryferyjnych, skutecznie stymulując ich rozwój w ramach RSI (będzie o tym mowa poniżej). Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 49 Rysunek 1. Narodowy system innowacyjności w Finlandii Źródło: W.M. Gaczek, Gospodarka oparta na wiedzy w regionach europejskich, PAN–KPZG, Warszawa 2009, s. 68, ryc. 3.4. Źródło: W.M. Gaczek, Gospodarka oparta na wiedzy w regionach europejskich, PAN–KPZG, Warszawa 2009, s. 68, ryc. 3.4. 50 Anna Zorska Anna Zorska W układzie instytucjonalnym wspierającym innowacyjność i GOW w Finlan- dii szczególnie ważną rolę odgrywają trzy instytucje: Akademia Fińska, Narodowa A j T h l ii (TEKES) Fiń ki N d F d dl B+R (SITRA) Ak W układzie instytucjonalnym wspierającym innowacyjność i GOW w Finlan dii szczególnie ważną rolę odgrywają trzy instytucje: Akademia Fińska, Narodowa Agencja Technologii (TEKES), Fiński Narodowy Fundusz dla B+R (SITRA). Aka- demia Fińska i TEKES – we współpracy z partnerami z różnych branż i regionów – odpowiadają za ustalenie głównych kierunków działalności B+R oraz dostosowa- nie polityki naukowej i edukacyjnej do potrzeb gospodarki, m.in. wskazując sektory o znaczeniu strategicznym w przyszłości. Finansowanie prowadzonych badań oraz ich przekształcania w użyteczne innowacje powierzono agencji SITRA (we współ- pracy z ekspertami), która f nansuje projekty technologiczne realizowane w koope- racji i sieciowych powiązaniach różnych podmiotów – ośrodków badawczych i aka- demickich oraz przedsiębiorstw. Nakłady na prace B+R osiągnęły wysoki poziom i relację do PKB, stanowiąc 3,6% jego wartości. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Zagraniczna ekspansja tej f rmy – zwłaszcza utworzenie f lii oraz ośrodków badaw- czych – przyczyniła się do rozwoju ponadgranicznych powiązań całego f ńskiego sektora informacyjnego oraz do zmiany sposobów działania wielu innych f rm. 51 Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy W ostatniej dekadzie, w polityce rządu narasta stopniowe przechodzenie od scentra- lizowanej koordynacji (przez instytucje państwowe) do zdecentralizowanego ekspe- rymentowania na różnych płaszczyznach biznesu, w ramach modelu otwarcia f rm na innowacje z różnych źródeł. Oznacza to przechodzenie od odgórnego pobudza- nia innowacyjności f rm, poprzez ich udział w rządowych programach, do oddol- nego rozwijania przez f rmy wiedzy, technologii oraz innowacji, których produkcyjne wykorzystanie (zorientowane na rynek globalny) wymaga wsparcia f nansowego ze strony państwowych instytucji. Jednocześnie w Finlandii postępuje przechodzenie przedsiębiorstw i gospodarki od specjalizacji przemysłowych ku nowoczesnym usłu- gom opartym na IKT, które są realizowane w ramach globalnych sieci biznesowych różnych korporacji. y p j W f ńskim NSI istotne miejsce zajmują regionalne systemy innowacyjności. RSI są usytuowane na poziomie instytucji wspierających działania w zakresie trans- feru technologii do przedsiębiorstw oraz szkolnictwa wyższego, w konkretnych regio- nach (np. peryferyjnych). Potrzeba aktywizacji innowacyjności różnych podmiotów na poziomie regionalnym wynika z dużego, przestrzennego zróżnicowania gospo- darki Finlandii. Dysproporcje w tym zakresie wyrażają się powstaniem trzech roz- winiętych obszarów metropolitalnych, którymi są Helsinki/Espoo/Vantaa, a także Tampere i Turku. Jednocześnie wyludniają się północne regiony kraju oraz inne lokalizacje mniej zurbanizowane, co może przyczyniać się do ich degradacji eko- nomicznej w dłuższym okresie. W celu przeciwdziałania takim niekorzystnym pro- cesom od lat sześćdziesiątych wdrażana jest inicjatywa rządowa tworzenia nowych uczelni na terenie całego kraju. W odległych od stolicy regionach powstały uczel- nie, np. uniwersytety w Oulu oraz Laponii, które stały się ważnymi ośrodkami wyż- szej edukacji, tworzenia wiedzy i przetwarzania informacji. Inną ważną inicjatywą było utworzenie parków technologicznych (powiązanych z uczelniami) dla przed- siębiorstw prywatnych z sektora informacyjnego. Polityka wspierania RSI ewoluuje od przygotowania i wdrażania rządowych projektów technologicznych ku wspiera- niu wspólnych inicjatyw podmiotów na poziomie lokalnym, podejmowanych przez lokalne społeczności i władze samorządowe. To one wykorzystują dużą część środ- ków f nansowych oferowanych w ramach unijnych projektów i programów ope- racyjnych. Ponadto rozwija się współpraca regionów, czego przykładem może być tworzenie wspólnych instytutów badawczych, uczelni, programów informacyjnych, jak np. program e-Tampere. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Działania rządu f ńskiego na rzecz rozwoju edukacji oraz wspierania działalności innowacyjnej w mniej rozwiniętych regionach kraju są wskazywane przez Bank Światowy i unijne instytucje innym rządom jako przykład dobrych inicjatyw na rzecz promowania systemów innowacyjności w regionach. 52 Anna Zorska Anna Zorska Rozwój NSI w Finlandii dokonuje się w sposób kompleksowy, z wykorzystaniem powiązań z innymi f larami GOW, a przy tym są widoczne przejawy komplemen- tarności f ńskich systemów innowacyjnych z globalną działalnością innowacyjną. Jest to szczególnie widoczne w działalności f rmy Nokia. W latach osiemdziesiątych restrukturyzacja Nokii dokonywała się poprzez zakupy technologii oraz współpracę technologiczną z f rmami amerykańskimi (m.in. Radio Shack, At&T), a także euro- pejskimi (Alcatel – Francja, AEG – Niemcy). W latach dziewięćdziesiątych Nokia postawiła na rozwój organiczny własnymi siłami, ale w sposób umiędzynarodo- wiony, przenosząc kilka ośrodków B+R do Azji (Indie, Chiny) i USA. W ostatnich latach ma miejsce powiązanie procesów innowacyjnych f rmy Nokia w kraju (też w jego regionach) i na innych kontynentach, gdzie działa kilkanaście ośrodków B+R tej korporacji. Pracują one zarówno nad technologiami o globalnym znaczeniu dla biznesu omawianej f rmy, jak i nad dostosowaniem technologii do potrzeb lokal- nych (np. telefony dla uboższych klientów w Indiach). Z drugiej strony, f ński NSI został zasilony przez f lie zagranicznych f rm (powstałe z przejęcia lokalnych spółek), które prowadzą działalność innowacyjną na rzecz obcych f rm macierzystych, ale też kooperują z f rmami krajowymi. Filie te zatrudniają f ńskich specjalistów i są włą- czone w różne projekty technologiczne, subsydiowane przez f ński rząd. Systemy innowacyjności w Finlandii są otwarte, powiązane międzynarodowo i włączone w globalną działalność innowacyjną. Ponadgraniczne powiązania krajowego systemu innowacyjnego mogą impliko- wać przenoszenie impulsów kryzysowych z zewnątrz. Zależność od rynku globalnego stała się przyczyną poważnych problemów Nokii od roku 2008. Omawiana f rma nie doceniła zmian globalnego popytu, który skierował się m.in. ku nowej wersji smartfona w wydaniu f rmy Apple – iPhone’a. Nie mając równie atrakcyjnego pro- duktu tej klasy, Nokia przegrywa rywalizację z f rmami Apple i Samsung. Jej udział w globalnym rynku smartfonów obniżył się z 40% do 15% w latach 2005–2010. W rezultacie porażki Nokia zwalnia pracowników i zamyka fabryki, a negatywne konsekwencje trudności f rmy odczuwa cała gospodarka f ńska. W 2009 roku wartość f ńskiego eksportu obniżyła się o 12,8%, a PKB o blisko 9%. W 2010 roku uzyskano dodatnie stopy wzrostu eksportu i PKB. 3. Rozwój GOW i charakterystyka systemu innowacyjności w Finlandii Jednakże porażka Nokii nie spowodowała zmiany planów rządu f ńskiego dotyczących rozwoju sektora informacyjnego o glo- balnych powiązaniach. Uważa się, że ważna jest droga „ucieczki do przodu” f ńskich f rm przed rywalami na rynku globalnym. Drogą tą jest zwiększanie innowacyjno- ści przedsiębiorstw f ńskich, doskonalenie narodowego systemu innowacyjnego i dalsze rozwijanie GOW. Silne zaangażowanie f rm w innowacyjne przedsięwzięcia zorientowane na rynek globalny zwiększyło ryzyko ekonomiczne dla przedsiębiorstw oraz pracowników Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy 53 i szerzej – dla społeczeństwa. W Finlandii oraz w innych krajach nordyckich insty- tucje państwowe – w różnym stopniu i w nieco odmienny sposób – tworzą swoistą „siatkę bezpieczeństwa”. Z jednej strony, polega to na tworzeniu nowych możliwości dla przeżywających kryzys f rm (udział w nowych projektach i przedsięwzięciach) oraz pracowników (np. doszkalanie czy zmiana kwalif kacji), z drugiej zaś – na opiece socjalnej. Działania te wpisują się w koncepcję „wspierającego państwa dobrobytu” („enabling welfare state”), które pomaga obywatelom, f rmom i regionom pokonać przeszkody rozwojowe, udźwignąć ryzyko niepowodzenia, zaangażować się w nowe kierunki działalności. Efektem takiej polityki są dużo mniejsze społeczne koszty przemian i różnice w poziomie życia ludności, a jednocześnie wyższa jakość życia społeczeństwa w Finlandii niż w innych krajach rozwiniętych. Podsumowanie O korzyściach ekonomicznych i społecznych płynących z rozwoju oraz wykorzy- stania wiedzy w gospodarce nie decyduje sam fakt jej istnienia, lecz umiejętne prze- kształcenie wiedzy w nowe lub znacząco ulepszone rozwiązania użyteczne dla przed- siębiorstw, czyli w innowacje. W gospodarce krajowej powstają większe korzyści, jeśli wiedza oraz innowacje są tworzone, stosowane i rozwijane przez wiele współpracują- cych przedsiębiorstw oraz innych podmiotów. Wynika to z przemian w działalności innowacyjnej przedsiębiorstw, zwłaszcza korporacji transnarodowych, dokonujących się w dobie globalizacji, informatyzacji i rozwoju GOW. W innowacyjności przed- siębiorstw umacnia się jej charakter procesowy, systemowy i kooperatywny, a przy tym postępuje rozwój ponadgranicznych powiązań w procesach innowacyjnych oraz wzrastają przepływy informacji, wiedzy, technologii, innowacji pomiędzy krajami. Promowanie rozwoju GOW może być korzystne dla gospodarki kraju, ale wymaga, aby zaistniały określone warunki technologiczne, ekonomiczne oraz insty- tucjonalne. Ustalił je Bank Światowy i rozpropagował jako cztery f lary GOW. Są to: 1) system bodźców i ramy instytucjonalne, 2) edukacja i szkolenia, 3) infrastruktura informacyjna, 4) narodowy system innowacyjności. Z doświadczeń różnych krajów wynika, że najważniejszym i najtrudniejszym do stworzenia w kraju jest f lar czwarty – narodowy system innowacyjności. Istotą NSI jest różnorodna działalność i uczest- nictwo wielu podmiotów, których procesy innowacyjne są powiązane i współzależne. Przebieg tych procesów może być kształtowany przez państwo poprzez tworzenie takich warunków w otoczeniu innowacyjnych podmiotów, które stymulują ich two- rzenie i dyfuzję wiedzy, technologii oraz innowacji. W kształtowaniu NSI uwzględnia 54 Anna Zorska Anna Zorska się różne wymiary takiego systemu: organizacyjny, funkcjonalny, podmiotowy i przestrzenny (zwłaszcza regionalny i globalny). W dobie globalizacji, regionalizacji i rewolucji informacyjnej coraz bardziej znaczące dla innowacyjności f rm i innych podmiotów stają się ich powiązania ponadgraniczne – w przestrzeni ponadnarodo- wej (np. UE), a nawet globalnej. Otwieranie się NSI na ponadgraniczne powiąza- nia i globalne współzależności w procesach innowacyjnych staje się ważnym, lecz dotychczas mniej zbadanym aspektem NSI. Finlandia jest krajem, który ukierunkował rozwój gospodarki na promowa- nie GOW dzięki stworzeniu odpowiednich dla niej warunków instytucjonalnych, ekonomicznych i technologicznych. Fiński narodowy system innowacyjności – w powiązaniu z innymi f larami gospodarki wiedzy – umożliwił rozwijanie zasobów wiedzy i kwalif kacji, wdrażanie IKT, innych technologii oraz innowacji w przedsię- biorstwach, a w konsekwencji zaktywizowanie i unowocześnienie gospodarki oraz podniesienie poziomu życia społeczeństwa. National innovation system as a pillar of the knowledge-based economy Growth of the knowledge-based economy has been inf uenced by economic and technological processes in the contemporary world, particularly globalization and information revolution. Research and development activities, knowledge, new technologies and innovations have assumed a crucial signif cance at all levels of economic activity and for that reason are supported by government policies. According to a World Bank concept growth of the knowledge-based economy can be stimulated by four groups of determinants – or so-called pillars – including: economic and institutional system of a given country, education and training, information infrastructure and national innovation system. Activity of the innova- tion system promotes creation and dif usion of new knowledge and its application as innovations by national enterprises in co-operation with universities, research centers and public institutions. T e activities of all entities can be stimulated by government innovation policies. T e problems of national innovativeness are discussed from theoretical and empirical viewpoints, focusing on experience of Finland which is one of the leading countries in world’s rankings of knowledge economy and national innovation systems. T e f nal conclusion underlines the fact that cross-border linkages of innovation systems within enterprises and countries can generate some threats to a national economy during global economic crisis. Podsumowanie Jak się wydaje, zbyt silne oparcie rozwoju NSI i GOW na odgórnym zarządzaniu rozbudowanym systemem i na działalności jednej f rmy – była i jest to Nokia – przyniosło nie najlepsze rezultaty w ostatnich latach. Silna pozycja w światowym sektorze IKT nie uchroniła Nokii przed pertur- bacjami w warunkach dużej zmienności popytu, kryzysu i bardzo silnej rywalizacji na rynku globalnym. Osłabienie Nokii w globalnej konkurencji poskutkowało prze- niesieniem impulsów światowego kryzysu 2008–2009 na gospodarkę małego kraju – Finlandii. Ponadgraniczne powiązania systemu innowacyjnego Nokii i f ńskiego NSI stały się kanałem transmisji kryzysowych impulsów w sektorze informacyjnym i gospodarce na świecie. Również te doświadczenia Finlandii mogą stanowić ważne – Finlandii. Ponadgraniczne powiązania systemu innowacyjnego Nokii i f ńskiego NSI stały się kanałem transmisji kryzysowych impulsów w sektorze informacyjnym i gospodarce na świecie. Również te doświadczenia Finlandii mogą stanowić ważne lekcje dla innych krajów. – Finlandii. Ponadgraniczne powiązania systemu innowacyjnego Nokii i f ńskiego NSI stały się kanałem transmisji kryzysowych impulsów w sektorze informacyjnym i gospodarce na świecie. Również te doświadczenia Finlandii mogą stanowić ważne lekcje dla innych krajów. Otwieranie się NSI i rozwój ponadgranicznych powiązań oznacza z jednej strony rozszerzanie się innowacyjnych systemów przedsiębiorstw lokalnych poza granice krajów macierzystych. Może to zwiększać innowacyjność tych przedsiębiorstw dzięki „zasileniu” ich aktywów niematerialnych i zdolności technologicznych przez wie- dzę oraz innowacje pochodzące od podmiotów zagranicznych. Jednakże z drugiej strony następuje przenikanie podmiotów obcego pochodzenia do NSI, co oznacza ich udział w wykorzystaniu nowej wiedzy powstającej w kraju goszczącym. Jeśli zostanie ona zaadaptowana i zastosowana w strategiach konkurencji obcych f rm, to właśnie im mogą przypaść większe korzyści z komercjalizacji tej wiedzy na rynku global- nym. Chociaż pisze się nieco eufemistycznie o koegzystencji i symbiozie systemów innowacyjności różnych f rm, regionów i krajów, to jednak dla wielu z nich może to oznaczać „wyciek” nowej wiedzy i korzyści na rzecz obcych podmiotów i rywali na rynku globalnym. Potrzebne są rozważne i ostrożne działania państwa, zmierzające do ochrony interesów krajowych podmiotów i społeczeństwa. 55 Narodowy system innowacyjności jako filar gospodarki opartej na wiedzy Le système national d’innovation comme un pillier de l’économie fondée sur la connaissance Les transformations économiques et technologiques dans le monde contempo- rain, en particulier la mondialisation et la révolution de l’information, ont con- tribué au développement de l’économie fondée sur la connaissance. Les activités de recherche et de développement, les connaissances, les nouvelles technologies et les innovations ont pris une importance cruciale à tous les niveaux de l’activité économique et pour cette raison elles sont prises en charge par les politiques gouvernementales. Selon un concept de la Banque mondiale, la croissance de l’économie du savoir peut être stimulée par quatre groupes de déterminants ou soi-disant piliers, y compris: le système économique et institutionnel d’un pays, l’éducation et la formation, l’infrastructure de l’information, le système national d’innovation. Le fonctionnement du système d’innovation favorise la création et la dif usion de nouvelles connaissances et son application comme des innovations par les entreprises nationales, en collaboration avec les universités, les centres 56 Anna Zorska de recherche et les institutions publiques. Les activités de toutes ces entités peu- vent être stimulées par la politique d’innovation du gouvernement. Le problème de capacité d’innovation nationale est largement discuté dans une perspective théorique et empirique. Un accent particulier est mis sur l’experience de la Fin- lande qui est l’un des pays les plus avancés dans les classements mondiaux de l’économie du savoir et des systèmes nationaux d’innovation. La conclusion f nale souligne le fait que les liens transfrontaliers de systèmes d’innovation entre les entreprises et les pays peuvent générer des menaces qui pèsent sur une économie nationale au cours de la crise économique mondiale.
https://openalex.org/W2142485337	https://www.research.ed.ac.uk/files/9087618/A_Systematic_Analysis_of_Host_Factors_Reveals_a_Med23_Interferon_Regulatory_Axis_against_Herpes_Simplex_Virus_Type_1_Replication.pdf	English	null	A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication	PLOS pathogens	2,013	cc-by	20,773	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Published In: PLoS Pathogens Publisher Rights Statement: Copyright: © 2013 Griffiths 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. Publisher Rights Statement: A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication Citation for published version: Griffiths, SJ, Koegl, M, Boutell, C, Zenner, HL, Crump, CM, Pica, F, Gonzalez, O, Friedel, CC, Barry, G, Martin, K, Craigon, MH, Chen, R, Kaza, LN, Fossum, E, Fazakerley, JK, Efstathiou, S, Volpi, A, Zimmer, R, Ghazal, P & Haas, J 2013, 'A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication', PLoS Pathogens, vol. 9, no. 8, e1003514. https://doi.org/10.1371/journal.ppat.1003514 Citation for published version: Griffiths, SJ, Koegl, M, Boutell, C, Zenner, HL, Crump, CM, Pica, F, Gonzalez, O, Friedel, CC, Barry, G, Martin, K, Craigon, MH, Chen, R, Kaza, LN, Fossum, E, Fazakerley, JK, Efstathiou, S, Volpi, A, Zimmer, R, Ghazal, P & Haas, J 2013, 'A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication', PLoS Pathogens, vol. 9, no. 8, e1003514. https://doi.org/10.1371/journal.ppat.1003514 Citation for published version: Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Edinburgh Research Explorer Edinburgh Research Explorer Abstract Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-l) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-l induction suggests this is the major transcription factor for IFN-l expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-l secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-l3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-l, provides evidence for the crucial role of IFN-l in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome. Received January 23, 2013; Accepted May 24, 2013; Published August 8, 2013 Copyright: 2013 Griffiths et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors declare no competing financial interests. This work was supported by grants provided by BayGene (Bayerisches Staatsministerium fu¨r Wissenschaft, Forschung und Kunst JH), DFG (SFB 576 JH), MRC (G0501453 JH), BBSRC (BB/D019621/1 PG; JKF), Scottish Enterprise (PG), the Wellcome Trust (WT086403MA, SE; WT066784 PG) and the European Union (AntiPathoGN). Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: juergen.haas@ed.ac.uk Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 A Systematic Analysis of Host Factors Reveals a Med23- Interferon-l Regulatory Axis against Herpes Simplex Virus Type 1 Replication Samantha J. Griffiths1, Manfred Koegl2, Chris Boutell3, Helen L. Zenner4, Colin M. Crump4, Francesca Pica5, Orland Gonzalez6, Caroline C. Friedel6, Gerald Barry7, Kim Martin1, Marie H. Craigon1, Rui Chen1, Lakshmi N. Kaza1, Even Fossum1, John K. Fazakerley7, Stacey Efstathiou4, Antonio Volpi5, Ralf Zimmer6, Peter Ghazal1,8, Ju¨ rgen Haas1,9* Samantha J. Griffiths1, Manfred Koegl2, Chris Boutell3, Helen L. Zenner4, Colin M. Crump4, Francesca Pica5, Orland Gonzalez6, Caroline C. Friedel6, Gerald Barry7, Kim Martin1, Marie H. Craigon1, Rui Chen1, Lakshmi N. Kaza1, Even Fossum1, John K. Fazakerley7, Stacey Efstathiou4, Antonio Volpi5, Ralf Zimmer6, Peter Ghazal1,8, Ju¨ rgen Haas1,9* 1 Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdom, 2 Preclinical Target Development and Genomics and Proteomics Core Facilities, German Cancer Research Center, Heidelberg, Germany, 3 MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom, 4 Division of Virology, Department of Pathology Cambridge University, Cambridge, United Kingdom, 5 University of Rome Tor Vergata, Rome, Italy, 6 Institute for Informatics, Ludwig- Maximilians Universita¨t Mu¨nchen, Mu¨nchen, Germany, 7 The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom, 8 Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, United Kingdom, 9 Max von Pettenkofer Institut, Ludwig-Maximilians Universita¨t Mu¨nchen, Mu¨nchen, Germany PLOS Pathogens \| www.plospathogens.org Citation: Griffiths SJ, Koegl M, Boutell C, Zenner HL, Crump CM, et al. (2013) A Systematic Analysis of Host Factors Reveals against Herpes Simplex Virus Type 1 Replication. PLoS Pathog 9(8): e1003514. doi:10.1371/journal.ppat.1003514 Editor: Shou-Jiang Gao, University of Southern California Keck School of Medicine, United States of America August 2013 \| Volume 9 \| Issue 8 \| e1003514 against Herpes Simplex Virus Type 1 Replication. PLoS Pathog 9(8): e1003514. doi:10.1371/journal.ppat.1003514 Editor: Shou-Jiang Gao, University of Southern California Keck School of Medicine, United States of America Author Summary Author Summary Herpes simplex virus type 1 (HSV-1) infects the vast majority of the global population. Whilst most people experience the relatively mild symptoms of cold sores, some individuals suffer more serious diseases like viral meningitis and encephalitis. HSV-1 is also becoming more common as a cause of genital herpes, traditionally associated with HSV-2 infection. Co-infection with HSV-2 is a major contributor to HIV transmission, so a better understanding of HSV-1/HSV-2 disease has wide implica- tions for global healthcare. After initial infection, all herpesviruses have the ability to remain dormant, and can awaken to cause a symptomatic infection at any stage. Whether the virus remains dormant or active is the result of a finely tuned balance between our immune system and evasion techniques developed by the virus. In this study we have found a new method by which the replication of the virus is counteracted. The cellular protein Med23 was found to actively induce an innate anti-viral immune response in the form of the Type III interferons (IFN- lambda), by binding IRF7, a key regulator of interferons, and modulating its activity. Interferon lambda is well known to be important in the control of Hepatitis C infection, and a genetic mutation correlating to an increase in interferon lambda levels is strongly linked to clearance of infection. Here we find the same association between this genetic mutation and the clinical severity of recurrent cases of HSV-1 infection (coldsores). These data identify a Med23-interferon lambda regulatory axis of innate immunity, show that interferon lambda plays a significant role in HSV-1 infection, and contribute to the expanding evidence for interferon lambda in disease control. Understanding the complex interplay between viral and host components is critical to the definition of herpesvirus infection and pathogenesis. As herpesviruses encode a large number of proteins, in contrast to small RNA viruses such as HIV and Influenza, many cellular processes may be directly affected by viral proteins, and whilst there exists a wealth of information on individual viral proteins, there remain large gaps in our understanding of the HSV-1 life cycle and its interaction with its host. Here, we present data from the first integrative and systematic screening approach to characterise the role of cellular proteins in the HSV-1 life cycle. A siRNA depletion screen identifies host factors (HFs) for HSV-1 replication Host factors (HFs) which positively or negatively regulate HSV-1 replication were identified by screening a druggable genome siRNA library (4 siRNAs per gene) targeting 7,237 human genes against a HSV-1 reporter virus expressing the enhanced green fluorescent protein (eGFP; HSV-1 strain C12) in the epithelial Hela cell line, due to their ease of transfection and susceptibility to HSV-1 infection [34]. To generate a robust and reliable dataset the screen was carried out three times in triplicate, with one replicate used in a cell viability assay to determine any cytotoxic effects of gene depletion and duplicates infected for the virus infection assay. The siRNA library was reverse- transfected into Hela cells before infecting with HSV-1 and monitoring virus growth kinetics as a measure of GFP-fluorescence (Figure 1b). By following virus growth over multiple rounds of replication, host proteins involved in all stages of the virus life cycle can be identified. Replication slopes during linear growth were normalized to controls (mock-transfected cells, and cells transfected with a siRNA unable to be processed by the RNA Silencing Complex, RSCF) and the mean of six replicates was calculated. siRNAs found to be cytotoxic (81 in total) were excluded from further analyses, and a hitlist of 358 containing the top 2.5% inhibitory and the top 2.5% enhancing HFs was generated (Table S1 in Text S2). The identified HSV-1 HFs were compared to datasets from published siRNA depletion screens aimed at identifying cellular factors affecting HIV-1 [7,8,9], West Nile Virus (WNV) [14], Hepatitis C Virus (HCV) [13], Dengue virus [15] and Influenza A virus [10,11,12]. Of our 358 HFs, 54 cellular proteins (15.1%) overlapped with these other virus screens (Influenza A, 29; HIV-1, 24; HCV, 6; WNV, 2; Dengue virus, 1) (Figure 1c; Table S2 in Text S2). 100 changes in the cellular proteome within the first 6h of infection with HSV-1 [5], and a recent analysis of virion- incorporated cellular proteins found that about 30% of these directly affected virus growth [6]. To systematically identify host factors (HFs) required for viral replication, RNAi screens have been performed with a range of different RNA and DNA viruses including HIV-1 [7,8,9], Influenza A virus [10,11,12], Hepatitis C virus [13], West Nile virus [14], Dengue virus [15], Enterovirus [16] and Vaccinia virus [17,18]. Author Summary A genome-scale RNAi knockdown screen to identify HFs functionally influencing HSV-1 replication was performed in parallel with a yeast two-hybrid (Y2H) protein interaction screen to simultaneously gain insight into potential mechanisms of action. Combined analyses confirmed the importance of known cellular proteins involved in processes such as cell cycle, proteins transport and gene expression important for virus replication. Furthermore, we identified a subunit of the Mediator multi-protein complex, Med23, as a key regulator of IFN-l induction, which appears to be of crucial significance for the control of HSV-1 both in vitro and in vivo. These data demonstrate the power of a combined screening strategy to investigate pathogen:host interactions and identify novel host factors and cellular pathway targets for the develop- ment of essential clinical interventions. Introduction understanding of HSV disease, and herpesviruses in general, has wide implications for global healthcare. Up to 90% of the global population is infected with the a- herpesvirus Herpes simplex virus type I (HSV-1). Whilst HSV-1 is largely responsible for outbreaks of vesicular oral skin lesions (fever blisters, or cold sores), it can also cause a variety of more severe diseases including encephalitis, meningitis and keratitis [1,2]. Furthermore, the frequency of association with genital lesions (previously associated mainly with HSV-2 infection) is increasing. As co-infection with HSV is a significant contributing factor to transmission of the Human Immunodeficiency Virus (HIV), our Like all herpesviruses, HSV-1 establishes lytic (epithelial cells) and asymptomatic latent infection (sensory neurons in trigeminal and sacral ganglia) which undergoes periodic reactivation [3]. The equilibrium between these two infection states requires a fine balance between innate and adaptive immune responses, and viral immune evasion mechanisms [4]. Whilst aspects of the HSV-1 replication cycle have been intensively investigated, there remain gaps in our understanding of the complexity of virus:host interactions. For example, a proteomics study identified over August 2013 \| Volume 9 \| Issue 8 \| e1003514 August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 1 Med23-IFN-l Regulation of HSV-1 Replication cycle regulation, nuclear transport and immune response appears to be conserved [33]. PLOS Pathogens \| www.plospathogens.org A siRNA depletion screen identifies host factors (HFs) for HSV-1 replication The overlap between the results of these studies is generally very low [19], reflecting either differences in biology, or different experimental set-ups, cutoff and selection criteria. In addition, microenvironmental effects might also play a role for the differences of the results [20]. Whilst loss-of-function siRNA screens provide functional information on specific genes, protein interaction studies can provide insight into the mechanism of action by identifying physical interaction partners between pathogen and host. Genome-scale virus-host protein interaction screens using the yeast-two-hybrid system have been performed for HCV [21], Influenza A virus [22], Epstein Barr virus (EBV) [23], Vaccinia virus [24,25], SARS coronavirus [26] and several non-human viruses [27]. Based on these genome-scale studies and individual interactions found by literature curation, several virus-host interaction databases have been created including the HIV-1, human protein interaction database at NCBI [28], VirHostNet [29], VirusMINT [30], PIG [31] and HPIDB [32]. Although there is little overlap between individual cellular interactors of different viruses, targeting of a number of cellular processes such as cell A genome-scale yeast two-hybrid protein interaction screen identifies novel viral protein interaction partners HSV-1 is currently known to encode at least 84 proteins, expressed sequentially under strict temporal regulation during August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 2 Med23-IFN-l Regulation of HSV-1 Replication Figure 1. Identification of HSV-1 host factors by RNAi and Y2H screens. (a) Strategy to identify host factors interacting with viral proteins or influencing HSV-1 replication. (b) RNAi perturbation screen by kinetic analysis of HSV-1 replication. Hela cells were reverse-transfected with siRNA SMARTpools (4 siRNAs per gene). After 48 h the siRNAs were tested for cytotoxicity (3 replicates) or the capacity to influence replication of the HSV-1 GFP reporter virus C12 (6 replicates) from 24 to 80 h post-infection. Virus replication slopes during the linear phase were calculated and normalized to mock-transfected cells. Replication slopes were then compared to replication upon knockdown of essential (ICP4, VP16) or non-essential (VP11/12) viral genes, a cellular receptor for HSV-1 (HVEM) or control RISC-free siRNA (RSCF). (c) Overlap between the HSV-1 HFs identified in this study with those published in HIV-1 [7,8,9], Hepatitis C Virus (HCV) [13] and Influenza A virus [10,11,12]. doi:10.1371/journal.ppat.1003514.g001 Figure 1. Identification of HSV-1 host factors by RNAi and Y2H screens. (a) Strategy to identify host factors interacting with viral proteins or influencing HSV-1 replication. A siRNA depletion screen identifies host factors (HFs) for HSV-1 replication (b) RNAi perturbation screen by kinetic analysis of HSV-1 replication. Hela cells were reverse-transfected with siRNA SMARTpools (4 siRNAs per gene). After 48 h the siRNAs were tested for cytotoxicity (3 replicates) or the capacity to influence replication of the HSV-1 GFP reporter virus C12 (6 replicates) from 24 to 80 h post-infection. Virus replication slopes during the linear phase were calculated and normalized to mock-transfected cells. Replication slopes were then compared to replication upon knockdown of essential (ICP4, VP16) or non-essential (VP11/12) viral genes, a cellular receptor for HSV-1 (HVEM) or control RISC-free siRNA (RSCF). (c) Overlap between the HSV-1 HFs identified in this study with those published in HIV-1 [7,8,9], Hepatitis C Virus (HCV) [13] and Influenza A virus [10,11,12]. doi:10.1371/journal.ppat.1003514.g001 infection. To gain further mechanistic insight into host factors involved in HSV-1 infection, in parallel to the siRNA depletion screen we carried out a yeast two-hybrid protein interaction screen to identify cellular interaction partners of viral proteins. We generated a collection of 107 partial and full-length HSV-1 cDNA constructs and tested them for interactions with proteins encoded by a library of 12,381 human cDNA clones [35]. 231 HSV-1- human protein interactions were detected once (low confidence), and 63 more than once (high-confidence)(Table S3 in Text S2). Using these high-confidence interactions, the previously reported HSV-1 interactome [36] was connected into a human interactome (62,310 published protein interactions) to generate a combined pathogen-host interactome (Figure S1a). Both degree centrality (which indicates the number of interactions a protein has, where high values represent highly interactive ‘hubs’) and betweenness centrality (which indicates the number of shortest paths between August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 3 Med23-IFN-l Regulation of HSV-1 Replication replication (Table S8 in Text S2). Pathways included those involved in gene expression, transcription, splicing and transla- tional regulation (RNAi screen), and protein transport, cell cycle, and transcriptional repressor activity (Y2H screen). A combined analysis of HFs from both screens found dominant functional categories centred on the regulation of transcription (RNA polymerase II-associated genes, splicing factors, transcription activation and the Mediator complex) (Figure S3c, d). The physiological relevance of some HFs and pathways was confirmed by further biological validation. Protein transport pathways (in the form of dynein microtubule networks) are exploited by HSV-1 early after infection to shuttle viral capsids to the nucleus. Validity and specificity of HSV-1 HFs y p y An extended literature and database search identified 599 cellular proteins that interact with or are involved in infection with human herpesviruses. The overlap between the high-confidence Y2H cellular interactors (63) and HFs (358) with this set was statistically significant (p = 0.008) (Figure 2a; Figure S1h; Table S5 in Text S2). From this combined analysis, a subset of HFs was chosen for further validation. Protein interactions were tested in a mammalian cell system by LUMIER pull-down assay [37]. Of the 45 interactions tested, 26 (57.8%) were confirmed, with 15 strongly positive (z-score .2) and 11 weakly positive (z- score 1–2) (Figure S1g). siRNA deconvolution (4 siRNAs per gene tested individually) was used to further validate 72 HFs (Figure 2b; Figure S2). The replication phenotype could be confirmed ($2 or more siRNAs gave the same or better replication slope than observed in the primary screen) in a high proportion (83.3%) of candidates, highlighting the reliability of the primary screen dataset. Quantitative RT-PCR analysis of mRNA expression levels found a minimum depletion of 60% (mean 88%) in a subset of 52 genes (data not shown; Table S6 in Text S2) confirming the observed effects on HSV-1 replication are genuine and not due to ‘off-target’ effects or insufficient gene knockdown. To further investigate the virus-specificity of our identified HFs, we tested this subset for their effect on the replication of an additional a-herpesvirus (Varicella-Zoster virus, VZV), the b- herpesvirus Cytomegalovirus (CMV), and a completely unrelated RNA virus, Semliki Forest Virus (SFV). None of the three proteins which enhanced HSV-1 replication upon knockdown had an effect on either VZV or CMV, and one (NR3C2) was even inhibitory for SFV (Figure 2c). Of the 64 siRNAs which inhibited HSV-1, 27 (42.2%) were also inhibitory for VZV, 60 for CMV (93.8%) and 23 (35.9%) for SFV replication (Table S7 in Text S2). Some functional groups (transcriptional regulators) were required by most viruses, but there were notable differences between other proteins. For example IFITM-1, previously identified as an inhibitor of Influenza A, Dengue virus and WNV [10], inhibited VZV yet had a positive effect on HSV-1 replication. These data suggest that whilst there are some HFs which are broad in their effects on virus replication, a large proportion are species-specific. A siRNA depletion screen identifies host factors (HFs) for HSV-1 replication These screens confirmed known interactions between dynein subunits and viral proteins, and identified additional previously unknown interactions (Text S1 and Figure S4a). Several dynein chain subunits were found to be essential for virus replication, whilst the moderate effect of depletion of other subunits demonstrated a level of functional redundancy in HSV-1 capsid transport (Figure S4b–e) [38,39]. Intrinsic anti-viral host defense mechanisms, in the context of cellular E2 ubiquitin ligases, were also investigated. The immediate-early viral protein ICP0, an E3 ubiquitin ligase, is crucial for blocking anti-viral defense mechanisms by degrading promyeloctic leukemia (PML) nuclear bodies (ND10 domains) in the presence of cellular E2-ubiquitin-conjugating enzymes (E2s). Our siRNA screen found multiple E2s were required for this, and suggests that HSV-1 ICP0 is promiscuous in its exploitation of E2s to mediate PML degradation and ensure successful infection (Text S1 and Figure S5). any pair of proteins passing through the protein considered) were significantly increased for HSV-1 interactors, particularly in the high-confidence network (Figure S1b–e). These data suggest HSV-1 proteins preferentially target highly connected central human proteins in the cellular interaction network, similar to other viruses [23]. Analysis of this interactome for HFs identified by RNAi found they were enriched in the fraction of cellular proteins that directly interact with viral proteins or that interact via one intermediate, in comparison to proteins that only interact via 2 or more intermediates (p = 0.036, Fisher’s exact test)(Figure S1f). A direct comparison of HSV-1 protein interaction partners and the siRNA screen HFs found 215 genes in common. Of those, ten (4.6%) were identified as a hit in both screens (Table S4 in Text S2), suggesting that these technologies identify complimentary yet not necessarily overlapping HFs. Med23 is an anti-viral component of the pro-viral Mediator complex Further, several Mediator subunits (Med25, 29, 17 and 8) are known to interact with the HSV-1 transactivator VP16 (UL48) and other herpesviral proteins [41] (Figure S6a). Consistently, the Mediator complex was found to be strongly required for HSV-1 replication, with depletion of the majority of subunits (Med 4, 6, 7, 8, 14, 16, 17, 21, 25, 26, 27 and 28) leading to a severe reduction in virus replication in the primary screen (Figure S6b) or in confirmatory deconvolution assays (Figure 3b). However, depletion of the Med23 subunit was striking in that it led to a significant enhancement of virus growth (Figure 3b). Flow cytometry quantification found that removal of Med23 not only increased the total number of infected cells (combination of GFPlo and GFPhi cells; 75.5% in comparison to 49.6% in mock-transfected cells) but also the copy number of virus genomes (GFPhi cells; 44.8% in comparison to 24.4% in mock- transfected cells) (Figure 3c). Gain-of-function experiments found overexpression of Med23 led to a corresponding inhibition of two strains of HSV-1 (Figure 3d; Figure S6c), confirming Med23 is a natural anti-viral component of the pro-viral Mediator complex. This anti-viral effect of Med23 was specific for HSV-1, as replication of VZV (a-herpesvirus), hCMV (b-herpesvirus), Vaccinia virus (DNA) and SFV (RNA virus) remained unaffected by Med23 depletion (Figure 3e). Combined bioinformatic analyses of protein interaction and siRNA depletion screens found a significant functional enrichment for proteins involved in transcription, and identified multi-protein complexes enriched for pro-viral HFs which strongly inhibited HSV-1 upon depletion, including the RNA-polymerase II, eIF3 and Mediator complexes (Figure 3a). The Mediator complex links the cellular transcription machinery (RNA polymerase II) to specific transcription factors, and the identification of many Mediator subunits as HFs in other viral siRNA depletion screens highlights its significant role in viral genome transcription [7,9,11,40] (Table S2 in Text S2). Further, several Mediator subunits (Med25, 29, 17 and 8) are known to interact with the HSV-1 transactivator VP16 (UL48) and other herpesviral proteins [41] (Figure S6a). Consistently, the Mediator complex was found to be strongly required for HSV-1 replication, with depletion of the majority of subunits (Med 4, 6, 7, 8, 14, 16, 17, 21, 25, 26, 27 and 28) leading to a severe reduction in virus replication in the primary screen (Figure S6b) or in confirmatory deconvolution assays (Figure 3b). Med23 is an anti-viral component of the pro-viral Mediator complex However, depletion of the Med23 subunit was striking in that it led to a significant enhancement of virus growth g g g (Figure 3b). Flow cytometry quantification found that removal of Med23 not only increased the total number of infected cells (combination of GFPlo and GFPhi cells; 75.5% in comparison to 49.6% in mock-transfected cells) but also the copy number of virus genomes (GFPhi cells; 44.8% in comparison to 24.4% in mock- transfected cells) (Figure 3c). Gain-of-function experiments found overexpression of Med23 led to a corresponding inhibition of two strains of HSV-1 (Figure 3d; Figure S6c), confirming Med23 is a natural anti-viral component of the pro-viral Mediator complex. This anti-viral effect of Med23 was specific for HSV-1, as replication of VZV (a-herpesvirus), hCMV (b-herpesvirus), Vaccinia virus (DNA) and SFV (RNA virus) remained unaffected by Med23 depletion (Figure 3e). PLOS Pathogens \| www.plospathogens.org Med23 is an anti-viral component of the pro-viral Mediator complex p Combined bioinformatic analyses of protein interaction and siRNA depletion screens found a significant functional enrichment for proteins involved in transcription, and identified multi-protein complexes enriched for pro-viral HFs which strongly inhibited HSV-1 upon depletion, including the RNA-polymerase II, eIF3 and Mediator complexes (Figure 3a). The Mediator complex links the cellular transcription machinery (RNA polymerase II) to specific transcription factors, and the identification of many Mediator subunits as HFs in other viral siRNA depletion screens highlights its significant role in viral genome transcription [7,9,11,40] (Table S2 in Text S2). Further, several Mediator subunits (Med25, 29, 17 and 8) are known to interact with the HSV-1 transactivator VP16 (UL48) and other herpesviral proteins [41] (Figure S6a). Consistently, the Mediator complex was found to be strongly required for HSV-1 replication, with depletion of the majority of subunits (Med 4, 6, 7, 8, 14, 16, 17, 21, 25, 26, 27 and 28) leading to a severe reduction in virus replication in the primary screen (Figure S6b) or in confirmatory deconvolution assays (Figure 3b). However, depletion of the Med23 subunit was striking in that it led to a significant enhancement of virus growth (Figure 3b). Flow cytometry quantification found that removal of Med23 not only increased the total number of infected cells (combination of GFPlo and GFPhi cells; 75.5% in comparison to 49.6% in mock-transfected cells) but also the copy number of virus genomes (GFPhi cells; 44.8% in comparison to 24.4% in mock- transfected cells) (Figure 3c). Gain-of-function experiments found overexpression of Med23 led to a corresponding inhibition of two strains of HSV-1 (Figure 3d; Figure S6c), confirming Med23 is a natural anti-viral component of the pro-viral Mediator complex. This anti-viral effect of Med23 was specific for HSV-1, as replication of VZV (a-herpesvirus), hCMV (b-herpesvirus), Vaccinia virus (DNA) and SFV (RNA virus) remained unaffected by Med23 depletion (Figure 3e). Combined bioinformatic analyses of protein interaction and siRNA depletion screens found a significant functional enrichment for proteins involved in transcription, and identified multi-protein complexes enriched for pro-viral HFs which strongly inhibited HSV-1 upon depletion, including the RNA-polymerase II, eIF3 and Mediator complexes (Figure 3a). The Mediator complex links the cellular transcription machinery (RNA polymerase II) to specific transcription factors, and the identification of many Mediator subunits as HFs in other viral siRNA depletion screens highlights its significant role in viral genome transcription [7,9,11,40] (Table S2 in Text S2). Med23-IFN-l Regulation of HSV-1 Replication PLOS Pathogens \| www.plospathogens.org Med23 inhibits HSV-1 replication by inducing a type III interferon (IFN-l) response Whilst co-expression of Med23 with IRF7 had no further effect on IFN-b expression, a synergistic induction of the IFN-l1 promoter and, to a lesser extent, the ISRE, was observed (IFN-l1 doubled to ,18-fold, p = 0.02) (Figure 5b). Interestingly, a Med23 mutant unable to induce immediate early gene expression via jun/fos (R617Q, or R611Q in Med23 transcript variant 1 used here) synergistically induced ISRE expression with IRF7, yet was unable to further enhance IRF7- mediated induction of IFN-l1 (data not shown). A similar synergistic effect of Med23 and IRF7 was seen at the protein level, where co-expression increased supernatant levels of IFN-l3 more than 2-fold those seen with Med23 or IRF7 alone (Figure S7c, d). Med23 could exert anti-viral effects either by having an inhibitory effect on viral transactivators or by interacting with and having a positive effect on an existing anti-viral factor. We first tested whether Med23 directly affects viral gene expression using luciferase reporters with HSV-1 promoters, however observed no inhibitory effect (data not shown). Since the Mediator complex and Med23 in particular is known to be involved in Jak/Stat- mediated interferon signaling [42], we used the lung epithelial cell line A549 and its Stat-1-deficient derivative A549-V [43] to determine if Med23 influences HSV-1 replication by modulating innate immunity. In the parental A549 cells the phenotype of HSV-1 replication was the same as that observed in Hela cells, where depletion of Med23 enhanced replication and over- expression inhibited virus growth. However, in the Stat1-deficient A549-V cells HSV-1 replication was unaffected by both depletion and over-expression of Med23 (Fig. 4a), indicating that Med23 requires an intact Jak/Stat signalling pathway to exert its anti-viral effects. To determine which interferon may be responsible for the anti- viral effects of Med23, A549 cells were depleted for Med23 and infected with HSV-1 following pre-stimulation with Type I (IFN-a or IFN-b), Type II (IFN-c) or Type III (the distinct IFN-l1 or the almost identical IFN-l2 and -l3, termed IFN-l2/3) interferons. Whilst treatment with IFN-a, -b and -c significantly decreased HSV-1 replication levels, the observed ,2-fold enhancement of HSV-1 replication following Med23 depletion was still seen. However, pre-treatment with the both IFN-l1 and IFN-l2/3 blocked the enhancing effect of Med23 depletion (Figure 4b). IFN-l gene expression is associated with the recurrence and severity of recurrent HSV-1 disease Successful disease and treatment outcome in Hepatitis C virus infection (demonstration of a sustained virologic response) is strongly associated with a single nucleotide polymorphism (SNP) in the IFN-l3 promoter (rs12979860; CC genotype over CT or TT) and higher plasma levels of IFN-l3 [49],[50]. Furthermore, IFN-l expression is impaired in a cohort of ethnically Italian individuals suffering recurrent HSV-1-related herpes labialis reactivation [51]. To determine if the clinical severity of HSV-1 disease is due to the observed deficiency in IFN-l expression, we screened a subset of the recurrent herpes labialis (HL) cohort and additional subjects for the IFN-l3 promoter polymorphism. Genotypic analysis found the presence of a T (CT or TT genotype) had a dose-dependent association with clinical severity, with the homozygous TT genotype being more prevalent as disease severity increases (Figure 6). In spite of the relatively small sample numbers in some clinical categories (Table 1), the association of a CT or TT genotype with the most severe recurrence of herpes labialis (H+) was statistically significant (p = 0.014; Fishers’s exact t-test). As the CC genotype is directly associated with increased IFN-l3 levels [51], these data highlight a previously unknown association between the frequency/severity of recurrence of herpes labialis, the CT/TT genotype and subsequent reduction in secretion of IFN-l3. It is of importance to investigate this genotype association with a larger cohort of HL patients, as well as those suffering with other HSV-1-related disease, in order to determine the role of IFN-l in the full spectrum of HSV-1 pathogenesis. HSV-1 HFs are involved in diverse cellular pathways and at multiple stages of the HSV-1 life cycle HSV-1 HFs are involved in diverse cellular pathways and at multiple stages of the HSV-1 life cycle Functional and pathway analysis of the 358 HSV-1 HFs identified in the siRNA depletion screen (Figure S3a), and of direct and indirect virus-host interactions with multiple interaction partners (Figure S3b), found a significant enrichment of a wide range of cellular processes involved in multiple stages of virus g y Functional and pathway analysis of the 358 HSV-1 HFs identified in the siRNA depletion screen (Figure S3a), and of direct and indirect virus-host interactions with multiple interaction partners (Figure S3b), found a significant enrichment of a wide range of cellular processes involved in multiple stages of virus August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 4 Med23-IFN-l Regulation of HSV-1 Replication g p Figure 2. Primary validation of HFs for HSV-1. (a) Overlap between HSV-1 HFs, cellular protein interactors of HSV-1 proteins identified by Y2H system, and published protein interactors of all human herpesviral proteins. (b) Validation of a subset of HFs by siRNA deconvolution. A subset of HFs Figure 2. Primary validation of HFs for HSV-1. (a) Overlap be system, and published protein interactors of all human herpesviral p en HSV-1 HFs cellular protein interactors of HSV-1 proteins identified by Y Figure 2. Primary validation of HFs for HSV-1. (a) Overlap between HSV-1 HFs, cellular protein interactors of HSV-1 proteins identified by Y2H system, and published protein interactors of all human herpesviral proteins. (b) Validation of a subset of HFs by siRNA deconvolution. A subset of HFs August 2013 \| Volume 9 \| Issue 8 \| e1003514 August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 5 Med23-IFN-l Regulation of HSV-1 Replication was selected for validation with deconvoluted siRNAs to confirm the phenotype observed in the primary screen. The effect of the four individual siRNAs (1–4) and a reconstituted SMARTpool (SP) were tested by reverse-transfecting into Hela cells before infecting after 48 h with HSV-1-eGFP (C12) and monitoring replication. Replication slopes were calculated and normalized as described, and compared to the primary screen slope (1u). A heat map of replication slopes was generated where red represents inhibition (replication slope ,0.5) and green represents enhancement (slope .1). The phenotype was considered validated if $2 siRNAs produced the same or better phenotype as the primary screen. (c) Virus specificity of HSV-1 HFs. Med23 inhibits HSV-1 replication by inducing a type III interferon (IFN-l) response Investigation into the effect of Med23 on interferon induction by qRT-PCR found that whilst Med23 over-expression induced IFN- b (,3-fold increase), induction of IFN-l1 and l2/3 was considerably and statistically significantly higher (,26-fold induc- tion; p = 0.003 and 0.002, respectively) (Figure 4c). This induction was specific, as levels of other cytokines and interfer- on-regulatory factors (IRFs) were unaffected by Med23 overex- pression (Figure S7a). Secretion of IFN-l2/3 protein was also increased in all cell lines tested, but most significantly to ,11-fold in A549 cells (Figure 4d), which is consistent with a recent report showing that type III interferons are the dominant type of IFNs expressed by primary airway epithelial cells [44]. Furthermore, qPCR analysis found depletion of Med23 inhibited the induction of IFN-l expression following HSV-1 infection of A549 cells in comparison to cells transfected with the RSCF siRNA control (Figure 4e). Together, these data suggest that IFN-l is responsible for the observed inhibitory effect of Med23 on HSV- 1 replication. HSV-1 HFs are involved in diverse cellular pathways and at multiple stages of the HSV-1 life cycle The effect of HF siRNA SMARTpools on the replication of VZV (a-herpesvirus), hCMV (b-herpesvirus) or Semliki Forest virus (SFV; RNA virus) was determined and compared to HSV-1. Normalized replication 626STDEV of the controls was considered inhibiting/enhancing. doi:10.1371/journal.ppat.1003514.g002 Med23 inhibits HSV-1 replication by inducing a type III interferon (IFN-l) response confirmatory co-immunoprecipitation experiments in mammalian cells with a panel of IRFs found that Med23 interacted with IRF4 and IRF7 (Figure 5a; Figure S7b). We also observed a weak interaction with IRF9, which may explain the previously observed effect of Med23 on Jak/Stat signalling [42]. To determine if this interaction had a functional effect, we looked at whether Med23 influenced IRF-mediated induction of IFN-l. In a luciferase reporter assay, neither IRF4 nor IRF9 led to a significant induction of the IFN-l1 promoter, either alone or in conjunction with Med23 (data not shown). IRF7 induced expression from the IFN-b and IFN-l1 promoters to similar levels (,7-fold and 9-fold higher than background, respectively), whilst the ISRE, induced by IRF7 and also present in the IRF7 promoter, was induced ,15-fold (Figure 5b). Whilst co-expression of Med23 with IRF7 had no further effect on IFN-b expression, a synergistic induction of the IFN-l1 promoter and, to a lesser extent, the ISRE, was observed (IFN-l1 doubled to ,18-fold, p = 0.02) (Figure 5b). Interestingly, a Med23 mutant unable to induce immediate early gene expression via jun/fos (R617Q, or R611Q in Med23 transcript variant 1 used here) synergistically induced ISRE expression with IRF7, yet was unable to further enhance IRF7- mediated induction of IFN-l1 (data not shown). A similar synergistic effect of Med23 and IRF7 was seen at the protein level, where co-expression increased supernatant levels of IFN-l3 more than 2-fold those seen with Med23 or IRF7 alone (Figure S7c, d). confirmatory co-immunoprecipitation experiments in mammalian cells with a panel of IRFs found that Med23 interacted with IRF4 and IRF7 (Figure 5a; Figure S7b). We also observed a weak interaction with IRF9, which may explain the previously observed effect of Med23 on Jak/Stat signalling [42]. To determine if this interaction had a functional effect, we looked at whether Med23 influenced IRF-mediated induction of IFN-l. In a luciferase reporter assay, neither IRF4 nor IRF9 led to a significant induction of the IFN-l1 promoter, either alone or in conjunction with Med23 (data not shown). IRF7 induced expression from the IFN-b and IFN-l1 promoters to similar levels (,7-fold and 9-fold higher than background, respectively), whilst the ISRE, induced by IRF7 and also present in the IRF7 promoter, was induced ,15-fold (Figure 5b). IFN-l is synergistically induced following a direct interaction between Med23 and IRF7 (c) Fluorescence microscopy and FACS analysis of Hela cells depleted of Med23. Hela cells transfected with either ICP4 or Med23 siRNA SMARTpools were infected with the recombinant HSV-1 GFP reporter virus C12 (MOI 1) and analysed by fluorescence microscopy and FACS analysis. Numbers indicate the percentage of cells in the uninfected, GFPlo or GFPhi populations. (d) Overexpression of Med23 inhibits HSV-1 C12-GFP and VP26-YFP. Hela or HEK cells overexpressing Med23 transiently (Hela) or stably (HEK) were infected with HSV-1 C12-eGFP or HSV-1 VP26-YFP at MOI 0.5. Replication slopes were monitored and normalized to control (pCR3)-transfected cells. Error bars represent the mean of at least three independent experiments. (e) Depletion of Med23 specifically affects HSV-1. Hela cells were depleted for Med23 with a siRNA SMARTPool and infected with HSV-1, Varicella zoster virus (VZV), human cytomegalovirus (hCMV), Vaccinia virus (VacV) or Semliki Forest Virus (SFV). Replication slopes (HSV-1, VZV, hCMV, VacV) or endpoint replication values (SFV) were calculated and normalized to controls. Error bars represent the mean of at least three independent experiments. d i 10 1371/j l t 1003514 003 doi:10.1371/journal.ppat.1003514.g003 of 83.3% was confirmed in deconvoluted siRNA experiments. This, combined with qPCR data demonstrating a minimum gene depletion of 69%, suggests that the functional phenotypes on virus replication are genuine, and not due to ‘off-target’ effects. The confirmation of such a high proportion of selected validation candidates, in spite of the potential technical drawbacks, highlights the reliability of our primary screen datasets, and thus provides an invaluable resource for the herpesvirology research community. Taken together, these data identify Med23 as a novel anti-viral factor which acts as a key regulator of IFN-l expression by interacting with and enhancing the activity of IRF7, a major transcription factor involved in innate immunity. Our observation of a link between the clinical severity of HSV-1 disease and, a CT/ TT genotype at a SNP known to regulate IFN-l3 secretion demonstrates the significance of IFN-l in the control of HSV-1 replication in vivo. Whilst this study provides no direct link between the IFN-l3 promoter polymorphism and Med23, these associa- tions of IFN-l with HSV-1 disease, combined with our observations that Med23 is required for the induction of IFN-l following HSV-1 infection, identifies for the first time a link between Med23 and IFN-l, provides a clinical context for Med23 regulation of IFN-l expression and underscores the potential biological significance of these data. IFN-l is synergistically induced following a direct interaction between Med23 and IRF7 As IFN-l expression is induced following activation of pathogen recognition receptors (PRRs) by virus infection [45,46,47,48], we tested whether Med23 induced IFN-l by directly interacting with an interferon-responsive transcription factor (IRF). Y2H and August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 6 Med23-IFN-l Regulation of HSV-1 Repli Med23-IFN-l Regulation of HSV-1 Replication Figure 3. Med23 is a novel anti-viral HF against HSV-1. (a) Multiprotein complexes involved in HSV-1 replication. The distribution of replicatio slopes of components from eight human protein complexes found to be critical to HSV-1 infection were ranked inhibiting (Rank 1, left) to enhancin PLOS Pathogens \| www.plospathogens.org 7 August 2013 \| Volume 9 \| Issue 8 \| e100351 Figure 3. Med23 is a novel anti-viral HF against HSV-1. (a) Multiprotein complexes involved in HSV-1 replication. The distribution of replication slopes of components from eight human protein complexes found to be critical to HSV-1 infection were ranked inhibiting (Rank 1, left) to enhancing Figure 3. Med23 is a novel anti-viral HF against HSV-1. (a) Multiprotein complexes involved in HSV-1 replication. The distribution of replication slopes of components from eight human protein complexes found to be critical to HSV-1 infection were ranked inhibiting (Rank 1, left) to enhancing PLOS P th \| l th 7 A t 2013 \| V l 9 \| I 8 \| 1003514 Figure 3. Med23 is a novel anti-viral HF against HSV-1. (a) Multiprotein complexes involved in HSV-1 replication. The distribution of replication slopes of components from eight human protein complexes found to be critical to HSV-1 infection were ranked inhibiting (Rank 1, left) to enhancing PLOS Pathogens \| www.plospathogens.org 7 August 2013 \| Volume 9 \| Issue 8 \| e1003514 August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org PLOS Pathogens \| www.plospathogens.org 7 Med23-IFN-l Regulation of HSV-1 Replication (Rank 7157, right). Arrow denotes Med23. (b) Deconvoluted siRNAs confirm role of Mediator subunits in HSV-1 replication. The effect of four individual siRNAs (1–4) and a reconstituted SMARTpool (SP) on a range of subunits of the Mediator complex were compared to the primary screen (P). Med25 was not present in the primary screen so has no comparative ‘P’. Hela cells were transfected and infected with HSV-1-eGFP. Replication slopes were calculated and normalized to controls. Error bars represent the mean of three independent experiments done in duplicate. IFN-l is synergistically induced following a direct interaction between Med23 and IRF7 One interesting outcome of this study was the surprisingly low overlap between hits identified using these different technologies. Of the 215 genes in common between the siRNA and cDNA libraries, only 10 (4.7%) were classified as a hit by both methods. This, however, is not unexpected, as even the overlap between studies using the same technology has been reported to be low. For example, the overlap between the three previously published HIV screens was only 7% [19]. Furthermore, the degree of functional redundancy within the siRNA library, and cellular pathways in general, the potential situation-specificity of virus-host interactions, and the possibility of indirect interactions between viral and host proteins, suggest that these methodologies detecting functional outcomes or physical interactions are linked, but complementary rather than confirmatory. The use of HSV-1 in a combined genome-scale screening approach has led to the identification of a regulatory axis in anti- viral innate immunity, and this important finding not only highlights the power of such combined genome-scale screening approaches to identify novel host candidates for anti-herpesvirus drug discovery, but provides an invaluable dataset to the herpesvirus and scientific community at large. The identified HFs were enriched for a range of cellular processes, such as transcription, gene expression, protein transport and cell cycle (Figure S2 and S3), and involved at different stages of viral infection. We investigated HFs involved in capsid transport and ubiquitination of antiviral intrinsic host defence factors in more detail. Incoming HSV-1 capsids are transported to nuclear pores via the microtubule-organizing centre (MTOC), mediated by capsid proteins VP26 (UL35) and UL46 binding to the dynein light chains DYNLT1 (Tctex1) and DYNLT3 (rp3) [54,55]. Our Y2H screen confirmed the known interaction between the capsid protein VP26 and the dynein light chain DYNLT3 (Text S1 and Figure S4). Combined with the siRNA screen data, which found depletion of multiple light chain subunits had moderate anti-viral effects on HSV-1, these data confirm propositions of redundancy in the capsid transport process which ensures successful infection in the event of viral mutations [38,39], and provide further evidence that HSV-1 has evolved to be highly promiscuous in its exploitation of cellular pathways to its advantage. PLOS Pathogens \| www.plospathogens.org Discussion By nature of their scale, high-throughput screening technologies have limitations. RNAi technology is limited by technical issues such as off-target effects, where an alternative gene to the intended target is degraded, and insufficient gene knockdown. Similarly, Y2H protein interaction screens can generate both false-positive interactions, due to ‘sticky’ proteins and auto-activation of the reporter gene used, and false-negative interactions. Whereas the number of false positives can be considerably reduced by stringent screening and selection criteria, the low sensitivity of the Y2H assay, which detects 20–30% of known interactions, is inherent to the system and can only be marginally improved. This poor sensitivity is caused by factors such as structural restraints of the Y2H bait and prey fusion proteins, a lack of or existence of distinct protein modification in yeast cells, and cellular localization signals in bait and prey proteins preventing nuclear import [52]. However, as all other high-throughput methods for measuring binary protein interactions possess a similarly low sensitivity, but are considerably more laborious and expensive, the Y2H system is still the most commonly used technology [53]. To overcome the intrinsic host defence, HSV-1 induces a proteasome-dependent degradation of anti-viral promyelocytic leukemia (PML) nuclear bodies (ND10 domains) by the RING- finger ubiquitin ligase ICP0 expressed during early infection [56]. In vitro, ICP0 is a biochemically active E3 ubiquitin ligase in the presence of E2 ubiquitin conjugating enzymes (E2s) [UBE2D1 (UbcH5a) and UBE2E1 (UbcH6)] [57], but which E2s are used during infection has remained unclear. We identified 20 cellular E2s that are able to influence HSV-1 replication (Text S1 and Figure S5). Depletion of UBE2D1-4, UBE2E1-3 and UBE2N In this study we have exploited a combined genome-wide screening approach to investigate HSV-1 replication and interac- tion with its host. This identified 358 functional HFs modulating HSV-1 replication, and 63 cellular interaction partners. In validation experiments, 57.8% of the interactions were confirmed by co-immunoprecipitation assays in mammalian cells, and of the 358 functional HFs identified in the siRNA screen, the phenotype August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 8 Med23-IFN-l Regulation of HSV-1 Replication Figure 4. Med23 inhibits HSV-1 by inducing a Type III interferon (IFN-l) response. (a) Med23 depletion or over-expression has no effect i A549-V cells deficient in Jak/Stat signalling. Discussion Error bars represent the mean of technical replicates and is representative of multiple experiments. * = p-value 0.003; ** = p-value 0.002 (unpaired t- tests for unequal variances). (d) Overexpression of Med23 induces IFN-l secretion. A range of cell types were transfected with pCR3 or Med23, supernatant harvested 120 h post-transfection and IFN-l3 levels measured by ELISA. Chart shows the mean and standard deviation of duplicates over two experiments. (e) siRNA depletion of Med23 inhibits the induction of IFN-l2/3 following HSV-1 infection. A549 cells were transfected with control (RSCF) or Med23-specific siRNA (Med23 KD) before infecting with HSV-1-eGFP C12 (MOI 0.5). RNA was harvested 0 or 8 h post-infection and IFN-l2/3 mRNA levels measured by qRT-PCR. Expression was normalized as above, and calibrated to RSCF-transfected cells at 0 h post-infection. Error bars represent the mean of technical replicates and is representative of multiple experiments. doi:10.1371/journal.ppat.1003514.g004 Figure 5. Med23 induces IFN-l by interacting with the transcription factor IRF7. (a) Med23 directly interacts with IRFs. Med23 was overexpressed in HEK cells as a myc-tagged fusion protein individually with a range of HA-tagged IRFs. Protein amounts were quantified and equal amounts (325 mg) were immunoprecipitated (IP) with anti-HA or anti-myc antibody before western blot analysis and staining with anti-HA (WB) to confirm protein expression (IP; anti-HA IP) and identify interaction partners (Co-IP; anti-myc IP) (b) Med23 synergistically induces IRF7-responsive promoters. A549 cells were transfected with IFN-l1-, IFN-b- or ISRE-responsive luciferase reporter constructs with IRF7 alone or in addition to Med23. Promoter activity was determined by measurement of Firefly luciferase activity 33 hr post-transfection, and normalized to Renilla luciferase and pCR3- transfected cells. Error bars represent the mean of at least three independent experiments. Statistical significance of the synergistic increase in IFN-l induction by Med23 with IRF7 over IFN-b induction was determined by unpaired t-tests for unequal variances. * = p-value 0.02. doi:10.1371/journal.ppat.1003514.g005 Figure 5. Med23 induces IFN-l by interacting with the transcription factor IRF7. (a) Med23 directly interacts with IRFs. Med23 was overexpressed in HEK cells as a myc-tagged fusion protein individually with a range of HA-tagged IRFs. Protein amounts were quantified and equal amounts (325 mg) were immunoprecipitated (IP) with anti-HA or anti-myc antibody before western blot analysis and staining with anti-HA (WB) to confirm protein expression (IP; anti-HA IP) and identify interaction partners (Co-IP; anti-myc IP) (b) Med23 synergistically induces IRF7-responsive promoters. slopes calculated and normalized to controls (RSCF siRNA or pCR3). Error bars represent the mean of at least three independent experiments. (b) Pre- treatment with Type III interferons prevents Med23-mediated enhancement of HSV-1 replication. A549 cells were mock-transfected or transfected with Med23 siRNA. After 48 h cells were untreated or pre-treated with 50 ng/ml IFN-a, IFN-b, IFN-c or 100 ng/ml IFN-l1 or IFN-l2/3 for 6 h, before infecting with HSV-1-eGFP C12. Replication was monitored and slopes calculated and normalized to unstimulated, mock-transfected cells. (c) Overexpression of Med23 preferentially induces type III interferons. pCR3 or Med23 were overexpressed in A549 cells and induction of type I (IFN-b) and type III (IFN-l1, IFN-l2/3) was measured by qRT-PCR. mRNA levels were normalized to HPRT and calibrated to mock transfected cells (control). Error bars represent the mean of technical replicates and is representative of multiple experiments. * = p-value 0.003; ** = p-value 0.002 (unpaired t- tests for unequal variances). (d) Overexpression of Med23 induces IFN-l secretion. A range of cell types were transfected with pCR3 or Med23, supernatant harvested 120 h post-transfection and IFN-l3 levels measured by ELISA. Chart shows the mean and standard deviation of duplicates over two experiments. (e) siRNA depletion of Med23 inhibits the induction of IFN-l2/3 following HSV-1 infection. A549 cells were transfected with control (RSCF) or Med23-specific siRNA (Med23 KD) before infecting with HSV-1-eGFP C12 (MOI 0.5). RNA was harvested 0 or 8 h post-infection and IFN-l2/3 mRNA levels measured by qRT-PCR. Expression was normalized as above, and calibrated to RSCF-transfected cells at 0 h post-infection. Error bars represent the mean of technical replicates and is representative of multiple experiments. doi:10.1371/journal.ppat.1003514.g004 Discussion A549 cells and derivative A549-V cells were transfected with Med23 siRNA SMARTpool (Med23 KD) or pCR3-Med23 overexpression plasmid (Med23+) 48 h (siRNA) or 24 h (pCR3) before infection with HSV-1-eGFP C12. Replication was monitored, an PLOS Pathogens \| www.plospathogens.org 9 August 2013 \| Volume 9 \| Issue 8 \| e100351 Figure 4. Med23 inhibits HSV-1 by inducing a Type III interferon (IFN-l) response. (a) Med23 depletion or over-expression has no effect in A549-V cells deficient in Jak/Stat signalling. A549 cells and derivative A549-V cells were transfected with Med23 siRNA SMARTpool (Med23 KD) or a pCR3-Med23 overexpression plasmid (Med23+) 48 h (siRNA) or 24 h (pCR3) before infection with HSV-1-eGFP C12. Replication was monitored, and Figure 4. Med23 inhibits HSV-1 by inducing a Type III interferon (IFN-l) response. (a) Med23 depletion or over-expression has no effect in A549-V cells deficient in Jak/Stat signalling. A549 cells and derivative A549-V cells were transfected with Med23 siRNA SMARTpool (Med23 KD) or a pCR3-Med23 overexpression plasmid (Med23+) 48 h (siRNA) or 24 h (pCR3) before infection with HSV-1-eGFP C12. Replication was monitored, and Figure 4. Med23 inhibits HSV-1 by inducing a Type III interferon (IFN-l) response. (a) Med23 depletion or over-expression has no effect in A549-V cells deficient in Jak/Stat signalling. A549 cells and derivative A549-V cells were transfected with Med23 siRNA SMARTpool (Med23 KD) or a pCR3-Med23 overexpression plasmid (Med23+) 48 h (siRNA) or 24 h (pCR3) before infection with HSV-1-eGFP C12. Replication was monitored, and PLOS Pathogens \| www.plospathogens.org August 2013 \| Volume 9 \| Issue 8 \| e1003514 August 2013 \| Volume 9 \| Issue 8 \| e1003514 9 Med23-IFN-l Regulation of HSV-1 Replication slopes calculated and normalized to controls (RSCF siRNA or pCR3). Error bars represent the mean of at least three independent experiments. (b) Pre- treatment with Type III interferons prevents Med23-mediated enhancement of HSV-1 replication. A549 cells were mock-transfected or transfected with Med23 siRNA. After 48 h cells were untreated or pre-treated with 50 ng/ml IFN-a, IFN-b, IFN-c or 100 ng/ml IFN-l1 or IFN-l2/3 for 6 h, before infecting with HSV-1-eGFP C12. Replication was monitored and slopes calculated and normalized to unstimulated, mock-transfected cells. (c) Overexpression of Med23 preferentially induces type III interferons. pCR3 or Med23 were overexpressed in A549 cells and induction of type I (IFN-b) and type III (IFN-l1, IFN-l2/3) was measured by qRT-PCR. mRNA levels were normalized to HPRT and calibrated to mock transfected cells (control). Med23-IFN-l Regulation of HSV-1 Replication Discussion This induction was mediated via a direct interaction with the transcription factor IRF7, which resulted in a synergistic increase in IFN-l expression. Med23 was unable, however, to further enhance IRF7-induced levels of IFN-b, suggesting an additional level of complexity to the regulation of interferon signalling. Interestingly, the inhibitory effect of Med23 was specific to HSV-1, with replication of a range of other viruses including Vaccinia virus and Semliki Forest Virus being unaffected by Med23 depletion. As Vaccinia virus is resistant to IFN-l anti-viral activity [62], this observation further highlights the importance of IFN-l, as opposed to IFN-b, in the anti-viral effect of Med23. The R617Q mutation in Med23 (R611Q in Med23 transcript variant One of the multi-protein complexes affecting HSV-1 replication was the Mediator complex, a large (.30 subunits) complex which links specific transcription factors to the RNA polymerase II transcription machinery [40]. As the requirement of Mediator subunits in the replication of herpes and other viruses is already well-known [7,9,11,41], it was striking that depletion of the Med23 subunit exerted the opposite phenotype and led to a strong increase in virus growth. The Mediator is composed of four distinct modules termed the head, middle, tail and kinase domains, which provide the Mediator with some degree of active control over transcription [59]. As individual subunits of this large complex interact with and exert functional effects via specific transcription factors, it is not unexpected that the observed anti- viral effects were specific to Med23 [60]. Within the Mediator, Med23 forms a tight sub-complex with Med24 and Med16 n of the IL28B genetic polymorphism rs12979860 with recurrence and severity of oral HSV-1 labialis. Table 1. Association of the IL28B genetic polymorphism rs12979860 with recurrence and severity of oral HSV-1 labialis. Genotype Clinical statusa nb Genderc nb Age range Median age CC CT TT NR 21 M 5 26–49 39 4 1 0 F 16 27–52 36 10 5 1 L 18 M 6 22–61 43 4 1 1 F 12 23–60 37 8 2 2 H 9 M 2 37–52 44.5 2 0 0 F 7 31–54 42 1 4 2 H+ 9 M 3 35–46 35 1 1 1 F 5 25–55 38 0 3 2 aSN, HSV-1 IgG seronegative; NR, non-recurrent; L, low recurrence; H, high recurrence; H+, high recurrence and clinical severity. See Methods for details. bn, number of patients. cM, male; F, female. Discussion A549 cells were transfected with IFN-l1-, IFN-b- or ISRE-responsive luciferase reporter constructs with IRF7 alone or in addition to Med23. Promoter activity was determined by measurement of Firefly luciferase activity 33 hr post-transfection, and normalized to Renilla luciferase and pCR3- transfected cells. Error bars represent the mean of at least three independent experiments. Statistical significance of the synergistic increase in IFN-l induction by Med23 with IRF7 over IFN-b induction was determined by unpaired t-tests for unequal variances. * = p-value 0.02. doi:10.1371/journal.ppat.1003514.g005 August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 10 Med23-IFN-l Regulation of HSV-1 Replication Figure 6. Occurrence and severity of clinical HSV-1 reactivation is associated with IFN-l expression. The frequency of genotype of the single nucleotide polymorphism rs12979860 in the IFN-l3 promoter region in HSV-1 individuals with a range of frequency and severity oral herpes labialis was determined by PCR. NR, non-recurrent; L, low recurrence; H, high recurrence; H+ high and clinically severe recurrence. Significance of genotype association was determined by Fisher’s exact test, comparing the frequency of the CC, CT or TT genotype in the NR vs the L, H or H+ clinical groups. * = p-value 0.014. See also Table 1 and Methods for further details. doi:10.1371/journal.ppat.1003514.g006 Figure 6. Occurrence and severity of clinical HSV-1 reactivation is associated with IFN-l expression. The frequency of genotype of the single nucleotide polymorphism rs12979860 in the IFN-l3 promoter region in HSV-1 individuals with a range of frequency and severity oral herpes labialis was determined by PCR. NR, non-recurrent; L, low recurrence; H, high recurrence; H+ high and clinically severe recurrence. Significance of genotype association was determined by Fisher’s exact test, comparing the frequency of the CC, CT or TT genotype in the NR vs the L, H or H+ clinical groups. * = p-value 0.014. See also Table 1 and Methods for further details. doi:10.1371/journal.ppat.1003514.g006 [60,61]. The increase in virus replication observed upon depletion of Med24 may be caused by the destabilisation of the structure of this sub-complex (Figure S6b). significantly increased the number of PML-positive cells post- infection, in an ICP0-dependent manner, indicating that ICP0 can use multiple E2s to degrade PML [57,58]. Investigations into the mechanism of action revealed Med23 inhibits HSV-1 replication by preferentially inducing a type III interferon response (IFN-l) at the mRNA and protein level. pat.1003514.t001 siRNA screen siRNA SMAR siRNA screen siRNA SMARTpools (4 siRNAs per gene) at 0.3 mM were dispensed in 10 ml volumes using a Rapidplate384 liquid handler (Qiagen) into triplicate black 384-well plates (Corning), sealed with adhesive seals (ThermoFisher) and plastic lids. Plates were stored at 280uC until needed (minimum 24 h, maximum 48 h). On the day of transfection, assay plates were thawed at room temperature and 10 ml transfection reagent (Dharmafect 1, Dharmacon), diluted in Hank’s buffered saline solution (HBSS, ThermoFisher) to give a final concentration of 0.1%, was added using a Multidrop 384 (ThermoFisher). Plates were incubated for 20 min at room temperature to allow formation of transfection complexes. During complex formation, low-passage (p20–22) Hela cells (ECACC) from ,50% confluent flasks were washed in PBS and trypsinised in Trypsin-EDTA (Lonza) before diluting in phenol red-free, antibiotic-free transfection medium (DMEM/F-12 1:1/5% FCS with 15 mM Hepes and L-glu; Gibco). Cells were counted and 36103 cells in 40 ml were added to each well using the Multidrop 384. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2. To infect, media was removed from plates by inversion, and 10 ml media (as for transfection, but containing penicillin-streptomycin; Lonza) or virus (HSV-1-eGFP strain C12, diluted to MOI 0.5 in infection media) [34] was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before 50 ml infection media was added and plates returned to the incubator. Replication was monitored as a function of eGFP fluorescence from 24 h to 80 h post-infection using the POLAR- star OPTIMA plate reader (BMG Labtech). Virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. There is mounting evidence for a role of the IFN-l family in the regulation of virus pathogenesis [45], particularly in the case of Hepatitis C infection where a polymorphism in the promoter region of IFN-l3 (IL-28B; polymorphism rs12979860), which correlates with plasma levels of IFN-l3 [50], is associated with disease and treatment outcome [49]. Individuals with recurrent HSV-1 reactivation have been shown to be deficient in IFN-l expression [51], and here we found the similar association between the IFN-l3 promoter polymorphism and ethnically Italian patients suffering recurrent and severe reactivations of HSV-1-related oral herpes outbreaks, albeit with a small sample group (n = 58). Yeast two-hybrid protein interaction screen The HSV-1 clone collection was cloned by recombinatorial (GATEWAYTM, Invitrogen) and conventional cloning into the bait vector pGBKT7, and screened against a library pooled from 12,381 MGC clones [35] in the pGADT7 prey vector using a semi- automated Y2H assay [77]. Interacting prey cDNAs were identified by sequential blasting of RefSeq, ENSEMBL and Unigene databases. BLAST hits with identical parameters (score, expectation value, length of alignment) were considered indistinguishable and counted separately. A high-confidence dataset was generated from interaction pairs isolated at least twice, or where the bait interacted with two highly related, non-promiscuous preys. In summary, this study provides a comprehensive and robust analysis of HFs that influence HSV-1 replication in vitro, which will benefit many future studies on HSV-1. The identification of Med23 as a crucial cellular component for IFN-l expression, and evidence for the significant role of type III IFN in the innate immune control of HSV-1 in vitro and in vivo, demonstrates the power of combined, genome-scale studies to identify physiologi- cally important HFs for virus pathogenesis. Future studies will clarify the role of genetic variations in both Med23 and IFN-l in HSV-1-related diseases, such as meningitis, keratitis and orola- bial/genital reactivations. siRNA screen siRNA SMAR Furthermore, sporadic mutations and genetic polymorphisms in innate immune receptor and signalling molecules that lead to the induction of type I and III IFNs have also been shown to be associated with Herpes Encephalitis [65], as well as oral and genital Herpes [66,67]. HSV infection controlled by a complex, interconnected and highly regulated network of cytokines expressed by innate immune cells. Type I IFNs mainly produced by HSV-infected keratinocytes [68] and pDCs [69] inhibit the spread from neurons to epithelial cells and between epithelial cells [70], similar to IFN-c. Type III IFNs are also able to directly inhibit HSV-1 infection in primary neurons, astrocytes, macrophages and dendritic cells [71,72]. IFN-c levels produced by peripheral blood CD4+ T-cells correlate with the frequency of HSV-1 reactivation [73]. IFN-l is able to induce expression of both itself and the type I IFNs, and a similar effect has also been observed for type I IFNs which induce both type I and III IFNs [71,72]. Type III IFNs are mainly expressed by myeloid dendritic cells (mDC) and monocyte-derived macrophages [74], and signal through the heterodimeric IL10RB/IL28RA receptor complex whose expression is largely restricted to cells of epithelial origin and plasmacytoid dendritic cells (pDC), in contrast to the broadly expressed type I IFN receptor (IFN-aR1/2) [75,76]. Since primary HSV-1 infection and reactivation affects skin and mucosa in the majority of cases, IFN-l may play a much greater role in the control of HSV-1 pathogenesis, likely in a complex network of coregulated type I and II IFNS, than previously thought. We hypothesize that HSV-infected DCs at the site of the lesion (such as skin Langerhans DCs whose role in IFN-l production is currently unknown, or intruding myeloid DCs) in individuals with the rs12979860 T/T or C/T haplotype express reduced levels of type III IFNs, and, in consequence, of type I IFNs, which leads to a reduced inhibition of local HSV-1 replication and the occurrence of fresh skin lesions. However, the relative contribution of IFN-l1 and l2/3 to the interferon- mediated control of HSV-1 replication in vivo, and indeed the role of Med23 in this, remains to be seen. Discussion doi:10.1371/journal.ppat.1003514.t001 PLOS Pathogens \| www.plospathogens.org 11 August 2013 \| Volume 9 \| Issue 8 \| e1003514 Table 1. Association of the IL28B genetic polymorphism rs12979860 with recurrence and severity of o aSN, HSV-1 IgG seronegative; NR, non-recurrent; L, low recurrence; H, high recurrence; H+, high recurrence and clinical severity. See Methods for details. bn, number of patients. cM l F f l p cM, male; F, female. doi:10.1371/journal.ppat.1003514.t001 August 2013 \| Volume 9 \| Issue 8 \| e1003514 Med23-IFN-l Regulation of HSV-1 Replication 1, used here) was unable to enhance IRF7-induced IFN-l expression. This mutation causes hereditary dementia [63], and the failure to induce IFN-l and thereby control HSV-1 in the brain may be a potential cofactor for the development of dementia, similar to Alzheimer’s disease [64]. Cell viability assay Cells were transfected as described above, and the cytotoxicity of siRNAs was determined using the CellTiter Blue (CTB, Promega) reagent, which gives a fluorescent or absorbance signal relative to the number of live cells. Briefly, 5 ml CTB was added per well using the Multidrop 384. Plates were incubated at 37uC in a humidified incubator with 5% CO2 for 2 h before measuring fluorescence (POLARstar OPTIMA plate reader). Readings were normalized to viability of mock-transfected cells, per plate, and mean cell viability over three replicates was calculated. Distribu- tion analysis of cell viability values identified median viability as 60%, and values ,60% were considered cytotoxic. PLOS Pathogens \| www.plospathogens.org Determination of virus specificity Plates were incubated at 37uC for 1 h before media (as for transfection media) was added manually to increase the volume to 100 ml per well. Plates were incubated at 37uC for 24 h before cells were lysed using a passive lysis buffer and Renilla luciferase levels measured with a microplate reader (Promega) using a dual luciferase reporter assay kit (Promega). Luciferase activity, which is representative of virus genome replication, was normalized to mock-transfected cells and mean luciferase activity from six replicates used for subsequent data analyses. p q y d) SFV replication assays. Selected siRNA SMARTpools were diluted to 0.5 mM in 16siRNA buffer and dispensed in 10 ml volumes in black 96-well plates (Corning). To this, 10 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.15% was manually added. Following a 20 min incubation to enable complex formation, 46104 Hela cells in 80 ml transfection media (DMEM/5% FCS/L-glu) were seeded on to the complexes bringing the final volume of transfection to 100 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion, and 25 ml media (as for transfection, but containing penicillin-streptomycin; Lonza) or virus (SFV4(3H)- Rluc [89] diluted in phosphate buffered saline (PBS)/0.75% bovine serum albumin to an MOI 0.01) was manually added. Plates were incubated at 37uC for 1 h before media (as for transfection media) was added manually to increase the volume to 100 ml per well. Plates were incubated at 37uC for 24 h before cells were lysed using a passive lysis buffer and Renilla luciferase levels measured with a microplate reader (Promega) using a dual luciferase reporter assay kit (Promega). Luciferase activity, which is representative of virus genome replication, was normalized to mock-transfected cells and mean luciferase activity from six replicates used for subsequent data analyses. a) HSV-1 replication assays. Selected siRNA SMARTpools were diluted to 0.3 mM in 16siRNA buffer and dispensed in black 96-well plates (Corning). To this 10 ml Dharmafect 1, diluted in HBSS to a final concentration of 0.15%, was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 16104 Hela cells in 80 ml transfection media were seeded on to the complexes bringing the final volume of transfection to 100 ml in each well. Med23-IFN-l Regulation of HSV-1 Replication To infect, media was removed from plates by inversion, and 10 ml media (as for transfection, but containing penicillin-streptomycin) or virus hCMV-GFP (strain AD169) [88], diluted to MOI 0.5 in infection media, was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before 50 ml infection media was added and plates returned to the incubator prior to monitoring virus replication. Replication was monitored as a function of eGFP fluorescence from 24 h to 80 h post-infection using the POLARstar OPTIMA plate reader (BMG Labtech). Virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. Med23-IFN-l Regulation of HSV-1 Replication Virus growth was measured in 3 h intervals as a function of eGFP fluorescence from 44 to 72 h post-infection. Virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. p q y c) hCMV replication assays. Selected siRNA SMARTpools were diluted to 0.5 mM in 16siRNA buffer and dispensed in 10 ml volumes in black 384-well plates (Corning). To this, 10 ml Dharmafect, 1 diluted in HBSS to a final concentration of 0.2%, was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 36103 MRC-5 (ATCC, CCL-171TM) in 40 ml growth medium (phenol red-free DMEM/ 10%FBS/L-glutamine/1% non-essential amino acids were seeded on to the complexes bringing the final volume of transfection to 60 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion, and 10 ml media (as for transfection, but containing penicillin-streptomycin) or virus hCMV-GFP (strain AD169) [88], diluted to MOI 0.5 in infection media, was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before 50 ml infection media was added and plates returned to the incubator prior to monitoring virus replication. Replication was monitored as a function of eGFP fluorescence from 24 h to 80 h post-infection using the POLARstar OPTIMA plate reader (BMG Labtech). Virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. c) hCMV replication assays. Selected siRNA SMARTpools were diluted to 0.5 mM in 16siRNA buffer and dispensed in 10 ml volumes in black 384-well plates (Corning). To this, 10 ml Dharmafect, 1 diluted in HBSS to a final concentration of 0.2%, was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 36103 MRC-5 (ATCC, CCL-171TM) in 40 ml growth medium (phenol red-free DMEM/ 10%FBS/L-glutamine/1% non-essential amino acids were seeded on to the complexes bringing the final volume of transfection to 60 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. siRNA SMARTpool deconvolution for HSV-1 replication assay validation The HSV-1 replication phenotype observed in the primary screen was validated for a subset of candidates by deconvoluting the assay SMARTpools. The four individual siRNAs targeting different regions of each gene, as well as a reconstituted SMARTpool, were diluted to 0.3 mM in 16siRNA buffer and dispensed to black 384- well plates. Transfection and infection was carried out as described above. Replication slopes were calculated and normalized as described, and a phenotype was considered validated if two or more of the four siRNAs resulted in the same, or better, phenotype. Determination of virus specificity For inter-viral comparison, siRNAs were considered inhibitory or enhancing if normalized replication was 626STDEV of the controls p q y d) SFV replication assays. Selected siRNA SMARTpools were diluted to 0.5 mM in 16siRNA buffer and dispensed in 10 ml volumes in black 96-well plates (Corning). To this, 10 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.15% was manually added. Following a 20 min incubation to enable complex formation, 46104 Hela cells in 80 ml transfection media (DMEM/5% FCS/L-glu) were seeded on to the complexes bringing the final volume of transfection to 100 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion, and 25 ml media (as for transfection, but containing penicillin-streptomycin; Lonza) or virus (SFV4(3H)- Rluc [89] diluted in phosphate buffered saline (PBS)/0.75% bovine serum albumin to an MOI 0.01) was manually added. Plates were incubated at 37uC for 1 h before media (as for transfection media) was added manually to increase the volume to 100 ml per well. Plates were incubated at 37uC for 24 h before cells were lysed using a passive lysis buffer and Renilla luciferase levels measured with a microplate reader (Promega) using a dual luciferase reporter assay kit (Promega). Luciferase activity, which is representative of virus genome replication, was normalized to mock-transfected cells and mean luciferase activity from six replicates used for subsequent data analyses. p q y d) SFV replication assays. Selected siRNA SMARTpools were diluted to 0.5 mM in 16siRNA buffer and dispensed in 10 ml volumes in black 96-well plates (Corning). To this, 10 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.15% was manually added. Following a 20 min incubation to enable complex formation, 46104 Hela cells in 80 ml transfection media (DMEM/5% FCS/L-glu) were seeded on to the complexes bringing the final volume of transfection to 100 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion, and 25 ml media (as for transfection, but containing penicillin-streptomycin; Lonza) or virus (SFV4(3H)- Rluc [89] diluted in phosphate buffered saline (PBS)/0.75% bovine serum albumin to an MOI 0.01) was manually added. Bioinformatic analyses Interactions between HSV-1 and human proteins were connected to a network of human protein-protein interactions (a August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 12 Med23-IFN-l Regulation of HSV-1 Replication Med23-IFN-l Regulation of HSV-1 Replication total of 62,310) taken from the databases HPRD [78](Release 9), BioGRID [79], DIP [80], MINT [30] and IntAct (downloaded May 18th 2010). A high-confidence interaction set (9,829 interactions) was compiled from interactions identified in at least two studies. Betweenness centrality (g(v) of a protein v was calculated as g(v) = gs{v{t(sst(v)/sst), where sst is the total number of shortest paths from protein s to protein t, and sst(v) is the number of those shortest paths that contain v. Betweenness centrality was normalized by dividing by the total number of protein pairs in the network. Enrichment for functional annota- tions from gene ontology (GO) [81], KEGG [82,83], REAC- TOME [84,85], and BIOCARTA was performed using DAVID [86]. Data on known human protein complexes was retrieved from the CORUM database, and complexes with subunits showing consistently stronger effects (inhibiting or enhancing) than expected by chance were detected using Wilcoxon’s rank-sum test. Genes included in the RNAi screen were ranked by their distance from the median knockdown, with the most inhibiting and enhancing genes being ranked highest. FDR was used for multiple testing correction. media (EMEM/10% FCS/1% non-essential amino acids) were seeded on to the complexes bringing the final volume of transfection to 60 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion and ,25 colony forming units of VZV-eGFP-infected MeWo cells (vaccine strain Oka) [87] diluted in MeWo growth media were seeded on to the complexes using the Multidrop 384. Virus growth was measured in 3 h intervals as a function of eGFP fluorescence from 44 to 72 h post-infection. Virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. media (EMEM/10% FCS/1% non-essential amino acids) were seeded on to the complexes bringing the final volume of transfection to 60 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion and ,25 colony forming units of VZV-eGFP-infected MeWo cells (vaccine strain Oka) [87] diluted in MeWo growth media were seeded on to the complexes using the Multidrop 384. Transient and stable over-expression of Med23 Transient and stable over expression of Med23 For transient over-expression, 1.56104 HEK293 cells were seeded in black 96-well plates. The following day, cells were transfected with 100 ng pCR3-Med23 using LipofectamineTM LTX (Invitrogen) and incubated for 48 h before infection with the recombinant HSV-1 reporter viruses C12 and VP26-YFP at MOI 0.5. Replication growth curves were monitored, and endpoint replication (as determined by fluorescence) was normalized to untransfected cells. For stable expression, Hela cells were transduced with pLenti-Med23, generated using the ViraPowerTM Lentiviral Expression System (Invitrogen), as per manufacturers’ instructions. Stable cells were infected, and replication monitored, as above. For confirmation of overexpression, RNA was extracted (QIAGEN RNA-easy kit) and expression quantified by one-step RT-qPCR (Thermofisher), with gene-specific primers (Table S9 in Text S2), and probes from the Universal Probe Library (Roche). Expression levels were normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase 1 (HPRT) and calibrated to mock-transfected cells. qPCR was carried out in duplicate for each sample, and normalized expression levels averaged. The VP26-YFP reporter virus was generated from a KOS BAC kindly provided by David Leib using the Red-mediated recombination system, where the first 4 amino acids of VP26 were replaced with YFP [91,92,93]. Effect of Med23 in Stat1-deficient A549-V cells Effect of Med23 in Stat1-deficient A549-V cells The effect of Med23 over-expression and depletion was investigated in interferon-deficient cells. The human alveolar epithelial cell line A549, and A549-V, a Stat1-deficient derivative cell line stably expressing the V protein from Simian virus 5 [43], were seeded at 26104 cells per well in a 96-well plate, and transfected with Med23 siRNA or pCR3-Med23 and infected as described for Hela cells. Flow cytometry of HSV-1-eGFP infected cells y y Select siRNAs SMARTpools were diluted to 500 nM in HBSS and 100 ml was incubated with 100 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.15% in individual wells of a 12- well plate. After 20 min incubation, 26105 Hela cells in 800 ml transfection medium were added. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection by removing medium and adding 500 ml HSV-1-eGFP at a MOI of 1. After incubation for 1 h at 37uC, virus was removed and replaced with 2 ml growth medium. After 48 h, medium was removed, cells rinsed in PBS and dislodged by trypsinisation. Cells were washed in PBS and pelleted by centrifugation for 10 min at 199 g. Superna- tant was removed and cells fixed in 4% paraformaldehyde before analysing for eGFP expression by flow cytometry (FACS DiVa, BD Biosciences) using the CellQuest software package. qPCR for siRNA knockdown Hela cells were transfected with selected SMARTpool siRNAs in 96-well plates, in triplicate, as described. After 48 h transfection, medium was removed, cells rinsed in PBS and lysed in 100 ml TRIZOL (Invitrogen). Triplicate wells were combined, and RNA extracted by standard phenol:chloroform extraction methods. mRNA levels were determined by TaqMan qPCR, using the one-step RT- qPCR kit (Thermofisher), with gene-specific primers (Table S9 in Text S2), and probes from the Universal Probe Library (Roche). Expression levels normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase 1 (HPRT) and calibrated to mock-transfected cells. qPCR was carried out in duplicate for each sample, and the mean of normalized expression levels calculated. LUMIER pull-down assay for validating Y2H protein interactions Proteins were transiently expressed in HEK293 cells as hybrid proteins with the Staphylococcus aureus protein A tag or Renilla reniformis luciferase fused to their amino termini. 20 ng of each expression construct were transfected into 16104 HEK293 cells using 0.05 ml of lipofectamine 2000 (Invitrogen) in 96-well plates. After 40 h, medium was removed and cells were lysed on ice in 10 ml of ice-cold lysis buffer (20 mM Tris pH 7.5, 250 mM NaCl, 1% TritonX-100, 10 mM EDTA, 10 mM DTT, Protease Inhibitor Cocktail (Roche), Phosphatase Inhibitor Cocktail (Roche), Benzonase (Novagen) 25 units per ml final concentration) containing sheep-anti-rabbit IgG- coated magnetic beads (Invitrogen, Dynabeads M280, 2 mg/ml final concentration). Lysates were incubated on ice for 15 minutes. 100 ml of wash buffer (PBS, 1 mM DTT) were added per well, and 10% of the diluted lysate was removed to determine the luciferase activity present in each sample before washing. The remaining sample was washed 6 times in wash buffer in a Tecan Hydroflex plate washer. Luciferase activity was measured in the lysate as well as in washed beads. Negative controls were wells transfected with the plasmid expressing the luciferase fusion protein and a vector expressing two copies of protein A. For each sample, four values were measured: the luciferase present in 10% of the sample before washing (‘‘input’’), the luciferase activity present on the beads after washing (‘‘bound’’), and the same values for the negative controls (‘‘input nc’’, and ‘‘bound nc’’). Normalized interaction signals were calculated as follows: Log(bound)/log(input) – log(bound nc)/ log(input nc). Normalized interaction signals were z-transformed by subtracting the mean and dividing by the standard deviation. The mean and standard deviation were calculated from large datasets of protein pairs which were not expected to interact, i.e. from negative reference sets. Med23-IFN-l Regulation of HSV-1 Replication penicillin-streptomycin) or 15 ml media containing Vaccinia virus strain WR with eGFP-tagged A5 protein [90], diluted to MOI 0.05, was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before 50 ml of media was added to each well, the plates inverted to remove the media and virus, and a final volume of 50 ml of media added to the plates before they were returned to the incubator. Replication was calculated as a function of eGFP fluorescence at 48 h post-infection using the POLARstar OPTI- MA plate reader (BMG Labtech). Virus replication was normal- ized to mock transfected wells on individual assay plates, and the mean replication from eight replicates used for subsequent data analyses. HBSS to a final concentration of 0.15%. After 20 min incubation, 36104 Hela cells in 320 ml transfection medium were added, mixed with the transfection complexes and transferred to 8-well glass bottomed chamber slides (Becton Dickinson). Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection by removing medium and adding 100 ml HSV-1-eGFP at a MOI of 1. After incubation for 1 h at 37uC, virus was removed and replaced with 500 ml growth medium. Images were acquired 48 h post-infection. Determination of virus specificity Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion, and 25 ml media (as for transfection, but containing penicillin- streptomycin; Lonza) or virus (strain C12, diluted to MOI 0.5 in infection media) was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before virus was removed by plate inversion and 100 ml infection media was added. Plates were returned to the incubator before replication was monitored as a function of eGFP fluorescence from 24 h to 80 h post-infection. Virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. b) VZV replication assays. Selected siRNA SMARTpools were diluted to 0.3 mM in 16siRNA buffer and dispensed in black 384-well plates (Corning). To this, 10 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.07% was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 2.56103 MeWo cells (ATCC, HTB-65TM) in 40 ml b) VZV replication assays. Selected siRNA SMARTpools were diluted to 0.3 mM in 16siRNA buffer and dispensed in black 384-well plates (Corning). To this, 10 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.07% was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 2.56103 MeWo cells (ATCC, HTB-65TM) in 40 ml e) Vaccinia virus replication assays. Hela cells were transfected as described in primary siRNA screen. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO2 before infection. To infect, media was removed from plates by inversion, and 15 ml media (as for transfection, but containing August 2013 \| Volume 9 \| Issue 8 \| e1003514 13 PLOS Pathogens \| www.plospathogens.org Med23-IFN-l Regulation of HSV-1 Replication Interferon stimulation assays A549 cells were transfected in triplicate in black 96-well plates, as described. After incubation at 37uC for 24 h, cells were serum- starved for 24 h by growing in low serum (1%) medium, before cells were left untreated or stimulated with 50 ng/ml IFN-a, IFN- b or IFN-c, or 100 ng/ml IFN-l1 or IFN-l2/3 in serum-reduced medium. After 6 h cells were infected with HSV-1 C12 diluted to MOI 0.5 in IFN-containing serum-reduced medium. After 1 h incubation, virus was removed and media replaced with 100 ml serum-reduced growth medium containing no interferon, IFN-a/- b/-c at 50 ng/ml, or IFN-l1/-l2/3 at 100 ng/ml. Replication was monitored as described and normalized to replication in mock-transfected, unstimulated cells. HSV-1 microscopy qRT-PCR analysis of interferon and cytokine induction A549 cells were transfected with 100 ng pCR3 or Med23 overexpression plasmids, or RSCF or Med23 SMARTpool siRNA Selected siRNAs SMARTpools were diluted to 500 nM in HBSS and 40 ml was incubated with 40 ml Dharmafect 1 diluted in PLOS Pathogens \| www.plospathogens.org August 2013 \| Volume 9 \| Issue 8 \| e1003514 August 2013 \| Volume 9 \| Issue 8 \| e1003514 14 Med23-IFN-l Regulation of HSV-1 Replication (50 nM) in duplicates, in 96-well plates as described. RNA was harvested 20 h post-transfection, and mRNA expression levels quantified by qRT-PCR, as described. inhibitors. Debris was removed by centrifugation, and protein quantified with a BCA protein assay kit (Thermo scientific, UK) as per manufacturers’ instructions. Equal protein quantities (650 mg per sample) were pre-cleared by continuous mixing with 50 ml pre- equilibrated Protein G Sepharose beads for 1 h at 4uC. Samples were centrifuged and supernatants halved before overnight mixing incubation with beads pre-coated with 1 mg a-HA (Roche, UK) or a-c-Myc (Santa Cruz, UK) at 4uC. Beads with precipitated proteins were washed three times with ice-cold NP40 buffer, resuspended in 26 SDS protein sample buffer and boiled for 10 min before SDS-PAGE separation on two 8% polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes overnight at 4uC before blocking with 5% milk in TBS-Tween then incubating with either a-HA or a-myc antibody (diluted 1:1000 in 5% milk/TBS-Tween). Membranes were washed in TBS-Tween before incubation with HRP-conjugated secondary antibody (diluted 1:3000 in 5% milk/TBS-Tween). After further washes, proteins were detected by incubation with ECL Western blotting detection system, exposing to X-ray film and developing films in an OPTIMAX X-Ray film processor. IFN-l1 luciferase reporter assay A point mutation (R611Q) was introduced into Med23 (Transcript Variant 1) by PCR with specific primers (see Table S9 in Text S2) and the clone verified by sequence analysis. A549 cells were co-transfected with 60 ng of pCR3, pCR3-Med23 or pCR3-R611Q, 60 ng of pCR3-IRF7, 20 ng of IFN-b-, IFN-l1- or ISRE-responsive luciferase reporter constructs and 10 ng pRL- TK, a Renilla Luciferase transfection control, in antibiotic-free low serum (1%) medium. After 33 h cells were lysed, and firefly (promoter reporter construct) and Renilla (transfection control) luciferase activity was measured (Dual-Luciferase Reporter Kit, Promega). Relative luminescence activity was normalized to Renilla (as a transfection control). IFN-l ELISA Induction of IFN-l by Med23 was determined in a range of cell types by seeding cells in 96-well plates to be ,80% confluent the next day. Cells were transfected in duplicates with 100 ng pCR3 or pCR3-Med23 using Lipofectamine LTX with Plus reagent (Invitrogen), in antibiotic-free medium. IFN-l levels quantified 96–120 h post-transfection. The synergistic effect of Med23 and IRFs on IFN-l induction was determined by co-transfection of pCR3 or pCR3-Med23 (50 ng) with pCR3-IRF7 (50 ng) in A549 cells, with Lipofectamine LTX with Plus reagent. The effect of Med23 depletion on IFN-l induction was determined by transfection of A549 cells in 96-well plates with 50 nM RSCF or Med23 siRNA. IFN-l was quantified 120 h post-transfection. IFN-l protein expression was quantified in supernatants using a commercial IFN-l DuoSet ELISA kit (R and D Systems). b) Yeast two-hybrid assays. Haploid yeast strains AH109 and Y187 were transformed with 1 mg prey (pGADT7 or pGADT7-IRF1, -IRF2, -IRF3, -IRF4, -IRF5, -IRF7 or -IRF9) or bait (pGBKT7 or pGBKT7-Med23) plasmid DNA, respective- ly, and grown overnight in synthetic defined (SD) medium lacking either leucine (-L; Prey) or tryptophan (-W; Bait) before prey- and bait-expressing haploid yeast cells were mated overnight in SD- LW/5% YPDA medium. Haploids were selected in SD-LW for 48 h before transferring to triple-knockout, histidine-deficient SD- LWH liquid medium containing 3-aminotriazol (3-AT) and 4- methylumbelliferyl-b-D-galactopyranoside (4-MuX) for 5–7 days. Interactions were tested in quadruplicates, detected by growth of colonies on SD-LWH agar with 3-AT, and quantified by measurement of fluorescence released from a-galactosidase cleavage of 4-MuX upon protein interaction. Relative fluorescence (RFU) was normalized to negative control interaction (empty pGADT7 mated with empty pGBKT7). Genotypic analysis of the IFN-l3 promoter Ethnically Italian subjects with or without a history of recurrent Herpes labialis (HL) gave written voluntary informed consent and were enrolled in this study at the University of Rome Tor Vergata with the approval of the Ethical Committee at the University of Rome Tor Vergata. All subjects were interviewed by medically trained investigators using an appropriate questionnaire and agreed to provide saliva and/or blood samples. Data and blood sample collection was carried out as previously described [51]. A total of 58 healthy immunocompetent individuals (17 men and 41 women) age 22–61 years (overall median age 38.5 yr; male median age 40 yr, range 22–61; female median age 38, range 23– 60) participated in the study. None of the patients presented with an active lesion at the time of or in the 3 weeks preceding saliva sample collection. For the purpose of this study, patients were characterized into no recurrence of HL (NR), low recurrence (L; 1–3 HL episodes/yr, with a maximum extension of 1 cm, mild symptoms and healing time ,7 days), high recurrence (H; 4 or more HL episodes/yr, extension of lesions more than 1 cm), very high recurrence (H+; more than 4 HL episodes/yr, extension of lesions .3 cm and/or involving nose or cheek beyond the lip, more severe and long lasting associated symptoms including itch, burning, paresthesias and/or neuralgia, with healing times .7 days and who required antiviral therapy). The NR group consisted of 21 individuals (16 women [median age (range) 36 (27–520] and 5 men [median age (range) 39 (26–49)]. The L consisted of 18 PLOS Pathogens \| www.plospathogens.org (TIF) Figure S2 Validation of HF identified by RNAi. HFs identified in the HSV-1 perturbation screen were validated with deconvoluted siRNAs and qPCR: (a) chromoboxes, (b) homeo- boxes, (c) general transcription factors, (d) nuclear receptors, (e) proteasome family members, (f) topoisomerases, (g) mediator complex subunits, (h) proteins involved in vesicle transport, (i) integrins, (j) Y2H interactors, (k) ubiquitin E2 ligases, (l) vesicle transport – further candidates, (m) interferon-stimulated mem- brane proteins and (n) others. HSV replication is presented as normalized replication slope, and is the mean of six individual assay points. Error bars represent standard deviation of the six data points. Deconvoluted siRNAs which had a sequence different to that in the original screen are highlighted in red. Genes for which the primary screen phenotype was not confirmed in the deconvolution assay are shown in bold text. (TIF) Below lists the GeneID numbers for genes and proteins mentioned within the text of this manuscript: IFITM1 (8519); IFN-a (cluster; 3438); IFN-b (3456); IFN-c (3458); IFN-l1 (282618); IFN-l2 (282616); IFN-l3(282617); IL10RB (3588); IL28RA (163702); IRF4 (3662); IRF7 (3665); IRF9 (10379); MED4 (29079); MED6 (10001); MED7 (9443); MED8 (112950); MED14 (9282); MED16 (10025); MED17 (9440); MED21 (9412); MED23 (9439); MED25 (81857); MED26 (9441); MED27 (9442); MED28 (80306); MED29 (55588); NR3C2 (4306). Supporting Information Text S1 Text for supporting figures. Descriptive text for Figures S4 and S5, additional methods for Figures S4 and S5, and appropriate references. Figure S3 HSV-1 HFs are involved in diverse cellular pathways and at multiple stages of the HSV life cycle. Enrichment of protein functions among the HSV-1 HFs. The enrichment for gene ontology (GO) terms and KEGG, BIO- CARTA or REACTOME pathway annotations among the HSV- 1 HFs identified by (a) RNAi and (b) Y2H assay was performed using DAVID bioinformatics software. (c) Direct interactions between human and HSV-1 proteins. The protein-protein interactions (PPIs) depicted are from the high confidence Y2H data set and from literature curation. Circles and diamonds correspond to human and HSV-1 proteins, respectively. Human proteins detected in the Y2H screen are drawn with red borders. Human genes that showed the strongest effects in the RNAi screen are colored yellow (extreme 10%) and orange (extreme 5%). (d) Highly interconnected regions in the human interaction network composed of HFs. Highly interconnected regions in the human interaction network composed of HFs. We assembled a human interaction network using data from the major PPI databases. A subnetwork consisting of HFs was then defined by limiting the network to the HFs detected in the Y2H screen, the RNAi (extreme 5%) screen, and the literature curation. Highly interconnected regions in the subnetwork were sought out using the MCODE algorithm. The top six scoring regions are shown. Proteins displayed in red correspond to HFs that are known only from the literature, and those in green are those that were detected in either of the screens performed in this study. The three boolean values beside each gene symbol represent whether the HF is Text S2 Supporting tables. Table S1 – 358 Top 2.5% inhibitory and enhancing genes. Table S2 – Overlap of primary hit list with RNAi screens in other viruses. Table S3 – HFs identified by Y2H. Table S4 – Known interactors of HSV-1. Table S5 – Overlap between HSV-1 siRNA and Y2H screens. Table S6 –Validation of primary screen phenotypes by siRNA SMARTpool deconvolution and quantitative RT-PCR. Table S7 – Specificity of identified HFs to HSV-1 replication. Table S8 – Functional and pathway analysis of siRNA HFs. Table S9 – Primers and probes for qPCR assays. (DOCX) Figure S1 Pathogen-host interactome analysis of RNAi and Y2H HFs. (a) Combined HSV-1-human interactomes with intraviral, virus-host and high-confidence host-host interactions. Med23-IFN-l Regulation of HSV-1 Replication individuals (12 women [median age (range) 37 years (23–60)] and 6 men [median age (range) 43 years (22–61)]). The H group consisted of 9 individuals (7 women [median age (range) 42 years (31–54)] and 2 men [median age (range) 44.5 years (37–52)]). The H+ group consisted of 8 individuals (5 women [median age (range) 38 years (28–55)] and 3 men [median age (range) 35 years (35– 46)]). Saliva samples (,3 ml) were obtained from each subject after an overnight fast and after rinsing the mouth twice with water, split into two aliquots and frozen at 220uC. Samples were anonymised and stored with dual code labels before shipping on dry ice to the University of Edinburgh for DNA extraction. Saliva and PBMC samples were thawed and DNA extracted using a QIAamp DNA Blood Mini Kit (Qiagen) as per manufacturer’s instructions, quantified using a NanoDrop and IL28B genotype determined by melt-curve analysis PCR on a LightCycler480 (Roche) using the LightMixH Kit IL28B (TIB Molbiol) as per manufacturer’s instructions. Significance of genotype association was determined by Fisher’s exact test, comparing the frequency of the CC, CT or TT genotype in the NR group versus the L (p- value = 1), H (p-value = 0.12) or H+ (p-value = 0.015) clinical groups. comparison of cellular interactors versus all proteins in human networks, where betweenness indicates the number of shortest paths between a protein pair, passing through the protein of interest. Statistically significant differences between viral targets and remaining proteins are denoted by . (f) Distribution of HFs in the virus-host interactomes. Level 1 proteins are direct interactors with HSV-1 proteins, level 2 their interactors and so on. (g) Validation of HSV-1-host Y2H interactors. A subset of protein interactions identified in the HSV-1-host Y2H screen were validated using the LUMIER pull-down assay in a mammalian cell system. Strength of interaction was determined by Z-score, where a score 1 to 2 represents a weak interaction and score .2 represents a strong interaction. (h) Distribution of direct HSV-1 targets in the RNAi screen. The proteins directly targeted by HSV-1 were taken from the Y2H data set and from the literature curation. An enrichment of literature-derived targets could be observed in the top 5% most inhibiting knockdowns (2.4-fold enrichment; p = 0.008), but the same could not be observed for the Y2H-detected direct targets. Med23 interaction with IRF7 Potential interactions between Med23 and the IRFs were determined by yeast two-hybrid analysis and confirmed by co- immunoprecipitation (Co-IP) in mammalian cells. a) Co-immunoprecipitation. Co-immunoprecipitation was performed using the epitope-tagged plasmids pGBKT7 (Myc epitope) and pGADT7 (HA epitope) containing the T7 promoter, and recombinant vaccinia virus vTF-7 expressing the T7 RNA polymerase (NIH AIDS repository). 56106 HEK293 cells were seeded in 10 cm dishes and the following day infected with vTF-7 (MOI 10) for 1 h before transfecting 10 mg each of empty bait (pGBKT7) or Med23-Bait, and IRF-Prey (pGADT7) vectors with Lipofectamine (Invitrogen). After 48 h cells were lysed on ice for 30 min in NP40 buffer, containing protease and phosphatase PLOS Pathogens \| www.plospathogens.org August 2013 \| Volume 9 \| Issue 8 \| e1003514 15 Med23-IFN-l Regulation of HSV-1 Replication Med23-IFN-l Regulation of HSV-1 Replication Med23-IFN-l Regulation of HSV-1 Replication comparison of cellular interactors versus all proteins in human networks, where betweenness indicates the number of shortest paths between a protein pair, passing through the protein of interest. Statistically significant differences between viral targets and remaining proteins are denoted by . (f) Distribution of HFs in the virus-host interactomes. Level 1 proteins are direct interactors with HSV-1 proteins, level 2 their interactors and so on. (g) Validation of HSV-1-host Y2H interactors. A subset of protein interactions identified in the HSV-1-host Y2H screen were validated using the LUMIER pull-down assay in a mammalian cell system. Strength of interaction was determined by Z-score, where a score 1 to 2 represents a weak interaction and score .2 represents a strong interaction. (h) Distribution of direct HSV-1 targets in the RNAi screen. The proteins directly targeted by HSV-1 were taken from the Y2H data set and from the literature curation. An enrichment of literature-derived targets could be observed in the top 5% most inhibiting knockdowns (2.4-fold enrichment; p = 0.008), but the same could not be observed for the Y2H-detected direct targets. Acknowledgments We are grateful to Tomozumi Imamichi (National Cancer Institute, Bethesda), Rick Randall (University of St Andrews) and Geoffrey L. Smith (University of Cambridge) for providing reagents, as well as George Sorensen, Tobias Bergmann, Gabriella Siszler, Frank Schwarz, Melanie Ott, Verena Raschbichler, Hannah Striebinger, Philippa Beard and Susanne Bailer for assistance. Figure S6 Med23 is an anti-viral component of the largely pro-viral multi-protein Mediator complex. (a) Diagrammatic summary of the role of Mediator complex subunits in virus replication. Subunits are coloured according to whether HSV-1 replication was unchanged (grey), inhibited (top 5%, red; top 10%, orange) or enhanced upon gene knockdown (top 5%, Supporting Information Protein levels of ICP0 and VP16 in cells depleted of DYNC1H1, DYNC1I2 or DYNC2LI1 were quantified with an Odyssey Imager and normalized to protein levels in control transfected cells (Non-targeting siRNA). (TIF) Figure S7 Med23 inhibits HSV-1 by directly interacting with IRF7 to induce a Type III, IFN-l interferon response. (a) Med23 specifically induces interferons. A549 cells were transfected with pCR3 or Med23 and induction of a panel of cytokines and interferons determined by qRT-PCR. Expression was normalized to HPRT and calibrated to pCR3-transfected cells. Error bars represent the standard deviation of technical replicates. (b) Med23 directly interacts with IRF7. Haploid yeast expressing Med23-bait or IRF-prey constructs were mated in nutrition-deficient Media containing 4-MuX. Interactions were detected by measurement of fluorescence released by a-galacto- sidase cleavage of 4-MuX upon protein interaction. Relative fluorescence (RFU) was normalized to the negative control (2, empty bait mated with empty prey constructs). Error bars represent the standard deviation of technical quadruplicates. +, known interactors Myc and Max. (c) Med23 and IRF7 synergistically induce IFN-l secretion. A549 cells were transfected with pCR3, Med23, IRF7 or Med23 with IRF7 and supernatant was harvested after 96 h to measure IFN-l3 levels (pg/ml) by ELISA. Error bars represent the standard deviation of biological replicates, and the chart is representative of multiple experiments. (d) Quantification of Med23 and IRF7 following overexpression. A549 cells were transfected with pCR3, Med23, IRF7 or Med23 with IRF7 and mRNA expression levels of Med23 or IRF7 quantified by qRT-PCR 96 h post-transfection. Error bars represent the standard deviation of biological replicates, and the chart is representative of multiple experiments. Figure S5 E2 ubiquitin conjugating enzymes in HSV-1 immune evasion. (a) Depletion of E2 ubiquitin ligases inhibits PML degradation following HSV-1 infection. Hela cells mounted on coverslips were depleted for a range of E2 ubiquitin ligases for 24 h before infecting with HSV-1 17+. Cells were fixed and stained for ICP0 (green) and PML (red), analysed by confocal microscopy and PML-positive cells were counted (5 fields of view per coverslip) and expressed as a mean percentage of PML- positive cells remaining (3 independent experiments). Error bars represent the standard deviation over 3 independent experiments. (b) Inhibition of PML degradation by E2s is ICP0-dependent. Hela cells were seeded on coverslips, transfected as above and infected with an ICP0 RING-finger deletion mutant (FXE). Remaining PML-positive cells were quantified as above. Supporting Information The role of dynein microtubule transport components in HSV-1 replication was analysed by comparing the replication slope of HSV-1-eGFP (C12)-infected cells depleted for a range of dynein chains from the primary siRNA perturbation screen. Error bars represent the mean of three independent experiments done in duplicate. (c) Depletion of dyneins inhibits virus particle release. The effect of dynein chain depletion on HSV-1 particle release was determined by quantifying virus titer in supernatants of Hela cells depleted of DYNC1H1 (heavy chain), DYNC1I2 (intermediate chain) and DYNC2LI1 (light intermedi- ate chains) in a high multiplicity (MOI 5; HSV-1 KOS) growth assay. Titers were compared to control transfected cells (NT, non- targeting siRNA). (d) Depletion of dynein chains prevents immediate-early gene expression. Immediate-early (ICP0) and late (VP16) viral protein expression in cells depleted of DYNC1H1, DYNC1I2 or DYNC2LI1 was analysed and quantified by Western blot. Levels were compared to control transfected cells (NT, non-targeting siRNA). (e) Quantification of ICP0 and VP16 protein expression. Protein levels of ICP0 and VP16 in cells depleted of DYNC1H1, DYNC1I2 or DYNC2LI1 were quantified with an Odyssey Imager and normalized to protein levels in control transfected cells (Non-targeting siRNA). (TIF) Figure S4 HFs involved in viral entry and capsid transport. (a) Diagrammatic summary of the role of dynein chains in HSV-1 infection. (b) Microtubule transport is required for HSV-1 infection. The role of dynein microtubule transport components in HSV-1 replication was analysed by comparing the replication slope of HSV-1-eGFP (C12)-infected cells depleted for a range of dynein chains from the primary siRNA perturbation screen. Error bars represent the mean of three independent experiments done in duplicate. (c) Depletion of dyneins inhibits virus particle release. The effect of dynein chain depletion on HSV-1 particle release was determined by quantifying virus titer in supernatants of Hela cells depleted of DYNC1H1 (heavy chain), DYNC1I2 (intermediate chain) and DYNC2LI1 (light intermedi- ate chains) in a high multiplicity (MOI 5; HSV-1 KOS) growth assay. Titers were compared to control transfected cells (NT, non- targeting siRNA). (d) Depletion of dynein chains prevents immediate-early gene expression. Immediate-early (ICP0) and late (VP16) viral protein expression in cells depleted of DYNC1H1, DYNC1I2 or DYNC2LI1 was analysed and quantified by Western blot. Levels were compared to control transfected cells (NT, non-targeting siRNA). (e) Quantification of ICP0 and VP16 protein expression. Supporting Information (c) Immunofluorescence staining for PML bodies in cells transfected with control siRNA not incorporated into the RISC complex (RSCF) or cells depleted of the E2 ubiquitin conjugating enzymes E2D1 or E2L3. The arrows highlight cells that have wt PML levels remaining in them whilst containing wt ICP0. (TIF) Supporting Information Green, viral proteins; Pink, direct (level 1) interactors (Y2H screen); Blue, level 1 interactors (Y2H/RNAi screens); Cyan, host protein-protein (level 2) interactors (Y2H screen); Yellow, Level 2 interactors (Y2H/RNAi screens). Due to the large size of the human network, and in order to discern viral proteins, only the first two levels were plotted (inset: all levels). Combined HSV-1- human interactome. Inset, all levels. (b) Intraviral and virus-host degree (# interactions) distribution of HSV-1 proteins. (c) Correlation between intraviral and virus-host degrees in HSV-1. (d) Degree comparison of cellular interactors versus all proteins in human networks, where the degree indicates the number of interactions a particular protein has. (e) Betweenness centrality August 2013 \| Volume 9 \| Issue 8 \| e1003514 PLOS Pathogens \| www.plospathogens.org 16 Med23-IFN-l Regulation of HSV-1 Replication present in the Y2H screen, the RNAi screen (extreme 5%), or the literature curated set, in that order. Dominant functional categories could be observed in each of the regions, including transcription (e.g., Mediator complex, RNA polymerase II and associated genes), translation initiation, splicing, and intracellular transport. present in the Y2H screen, the RNAi screen (extreme 5%), or the literature curated set, in that order. Dominant functional categories could be observed in each of the regions, including transcription (e.g., Mediator complex, RNA polymerase II and associated genes), translation initiation, splicing, and intracellular transport. (TIF) light green; top 10%, dark green). Subunits not included are white; herpesvirus proteins reported to target Mediator subunits are violet; Mediator subunits detected in other viral RNAi screens are highlighted by coloured diamonds. (b) Mediator complex subunits influence HSV-1 replication. Individual subunits of the Mediator complex and associated proteins were depleted by siRNA knockdown and infected with HSV-1 C12 (MOI 0.5). Replication was monitored over multiple rounds and the slope of replication over the linear phase was calculated and normalized to controls (mock-transfected cells). Grey, no significant effect; red, strongly pro-viral; green, strongly anti-viral. Error bars represent the mean of six replicates. 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https://openalex.org/W4319312410	https://ejurnalunsam.id/index.php/jmas/article/download/5083/3117	Indonesian	null	DETERMINAN PERTUMBUHAN LABA PADA PERUSAHAAN SUBSEKTOR PERTANIAN	Jurnal Mahasiswa Akuntansi Samudra	2,022	cc-by	5,362	JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) Volume 3 Nomor 1, FEBRUARI 2022. HALAMAN 17 -30 e- ISSN : 27970086 p- ISSN : 27970434 JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) Volume 3 Nomor 1, FEBRUARI 2022. HALAMAN 17 -30 e- ISSN : 27970086 p- ISSN : 27970434 Abstract The existence of Islamic banks regarding the products and services offered is still poorly known and understood by the people of Aek Tampang Village, South Padangsidimpuan District and some consider that Islamic banks are the same as conventional banks. This study aims to analyze the effect of marketing strategy and knowledge on the decision to become a customer. The population of this research is the Aek Tampang community, South Padangsidimpuan District. By using the Slovin formula, so the number of samples is 99 people. Data was collected by means of interviews and distributing questionnaires. Data analysis was carried out with the help of the SPSS version 23 application. The results of this study found that marketing strategy had no effect on the decision to become a customer of a sharia bank and knowledge had an influence on the decision to become a customer. Keywords : Marketing strategy, Knowledge, Decision to Become a Customer Abstrak Keberadaan bank syariah tentang produk dan jasa yang ditawarkan masih kurang diketahui dan dipahami oleh masyarakat Kelurahan Aek Tampang Kecamatan Padangsidimpuan Selatan dan sebagian lagi menganggap bahwa bank syariah sama saja dengan bank konvensional. Penelitian ini bertujuan untuk menganalisis pengaruh strategi pemasaran dan pengetahuan terhadap keputusan menjadi nasabah. Populasi penelitian ini adalah masyarakat Aek Tampang Kecamatan Padangsidimpuan Selatan. Dengan menggunakan rumus Slovin, sehingga jumlah sampel sebanyak 99 orang. Pengumpulan data dilakukan dengan wawancara dan penyebaran kuesioner. Analisis data dilakukan dengan bantuan aplikasi SPSS versi 23. Hasil penelitian ini menemukan bahwa strategi pemasaran tidak berpengaruh terhadap keputusan menjadi nasabah bank syariah dan pengetahuan mempunyai pengaruh terhadap keputusan menjadi nasabah. Kata Kunci: Strategi Pemasaran, Pengetahuan, Keputusan Menjadi Nasabah Kata Kunci: Strategi Pemasaran, Pengetahuan, Keputusan Menjadi Nasabah PENDAHULUAN Komunikasi antara pihak internal perusahaan dengan pihak eksternal perusahaan adalah laporan keuangan yang dihasilkan oleh perusahaan. Dalam hal ini perusahaan harus lebih profesional dan akurat dalam menyusun laporan keunganan sehingga dapat menggambarkan perusahaan secara utuh. Pada hakikatnya perusahaan bertujuan untuk menghasilkan laba yang maksimal sesuai dengan target yang ditetapkan demi meningkatkan kesejahteraan baik bagi pemilik maupun karyawan. Untuk itu pihak manajemen harus berusaha mencapai target tersebut. Target keuntungan yang dicapai oleh perusahaan tersebut harus berkualitas, dan pengukuran tingkat laba perusahaan dapat digunakan dengan perhitungan rasio rentabilitas dan profitabilitas (Kasmir, 2012:196) Pertumbuhan laba merupakan rasio perhitungan yang menggambarkan kemampuan perusahaan dalam mencapai laba bersih dengan membandingkannya pada tahun sebelumnya. Semakin meningkat rasio perhitungannya maka kondisi ini menunjukkan bahwa semakin bagus kinerja keuangan dari perusahaan tersebut (AWS & dkk, 2018:166). Laba yang dihasilkan perusahaan akan menggambarkan kemampuannya dalam membayar bunga utang, dividen, pajak pemerintahan (Hery, 2015:163). 17 JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Tabel 1 Pertumbuhan Laba Perusahaan Sub Sektor Pertanian (BEI) Periode 2013-2020. Sumber: Data diolah untuk penelitian ini KODE PERTUMBUHAN LABA(%). 2013 2014 2015 2016 2017 2018 2019 2020 AALI -0,244 0,377 -0,734 2,039 -0,000 -0,280 -0,839 2,668 JAWA -0,544 -0,261 -1,226 18,216 -0,111 -0,501 -0,058 0,088 SIMP -0,580 0,746 -0,671 0,671 0,140 -1,256 2,606 -1,529 SGRO -0,642 1,908 -0,999 0,795 -0,340 -0,790 -0,371 -1,004 LSIP -0,310 0,192 -0,317 -0,052 0,287 -0,485 -0,233 1,752 BWPT -0,306 -0,998 -1,931 1,157 -0,563 1,463 1,523 -0,050 Tabel 1 Pertumbuhan Laba Perusahaan Sub Sektor Pertanian (BEI) Periode 2013-2020. Sumber: Data diolah untuk penelitian ini KODE PERTUMBUHAN LABA(%). 2013 2014 2015 2016 2017 2018 2019 2020 AALI -0,244 0,377 -0,734 2,039 -0,000 -0,280 -0,839 2,668 JAWA -0,544 -0,261 -1,226 18,216 -0,111 -0,501 -0,058 0,088 SIMP -0,580 0,746 -0,671 0,671 0,140 -1,256 2,606 -1,529 SGRO -0,642 1,908 -0,999 0,795 -0,340 -0,790 -0,371 -1,004 LSIP -0,310 0,192 -0,317 -0,052 0,287 -0,485 -0,233 1,752 BWPT -0,306 -0,998 -1,931 1,157 -0,563 1,463 1,523 -0,050 Tabel 1 Pertumbuhan Laba Perusahaan Sub Sektor Pertanian (BEI) Periode 2013-2020. Tabel 1 memperlihatkan bahwa terdapat penurunan laba dari beberapa perusahaan yang tergabung dalam subsektor pertanian. Namun sekalipun mengalami penurunan terhadap pertumbuhan laba yang diperoleh perusahaan, harusnya tetap dilakukan analisis guna melihat apakah penurunan tersebut akibat kinerja perusahaan yang mengalami penurunan atau sebaliknya. Dalam mengukur kemampuan perusahaan melunasi kewajibannya untuk jangka pendek dapat digunakan current ratio. Pertumbuhan Laba Pertumbuhan laba adalah terjadinya perubahan laba pada laporan keuangan perusahaan dalam setiap periodenya. Dalam penelitiannya Widiyati (2019) dikatakan bahwa pertumbah laba adalah persentasi perubahan laba yang dihasilkan perusahaan dalam setahun. Besarnya perubahan laba dapat dihitung dengan mengurangi laba tahun sekarang dengan tahun sebelumnya kemudian dibagikan dengan laba pada tahun sebelumnya. Pertumbuhan laba yang dialami oleh perusahaan dapat dipengaruhi berbagai faktor diantaranya besarnya perusahaan, tingkat leverage perusahaan, umur perusahaan, tingkat penjualan dan perubahan laba dimasa lalu (Salmah & Ermeila, 2018). Perkiraan laba yang dihasilkan oleh perusahaan akan menjadi salah satu fokus perhatian para invetor dalam mengambil keputusan investasi. Laba yang mengalami perubahan secara konsisten akan lebih diminati investor ketimbang pertumbuhan laba yang mengalami fluktuasi yang tidak konsisten. PENDAHULUAN Besar kecilnya hasil yang diperoleh dengan perhitungan current rasio akan menggambarkan kemmpuan dari perusahaan tersebut dalam melunasi utangnya untuk jangka waktu pendek dengan menggunakan harta lancar. Baik buruknya tingkat likuiditas perusahaan dapat dlihat dari besar kecilnya current ratio (Krisnandi & dkk, 2019:115). Penelitian yang menggunakan current rasio sebagai varibel yang mempengaruhi pertumbuhan laba sudah banyak dilakukan sebelumnya, namun hasil temuan masing-masing belum konsisten diantaranya (Krisnandi & dkk, 2019), (Hasinah & dkk, 2019). Debt to Equity Ratio (DER) adalah salah satu rasio yang digunakan untuk menilai penggunaan utang dengan ekuitas yang dimiliki oleh perusahaan (Siregar, 2020). Debt t equity ratio yang merupakan variabel keuangan akan menunjukkan rasio suatu perusahaan yang akan berakibat pada ketidakpastian harga saham. Besar kecilnya debt to equity rasio akan menggambarkan pertumbuhan laba yang dihasilkan oleh perusahaan (Nurhayati & dkk, 2020:176). Beberapa penelitian terdahulu yang meneliti debt to equity ratio, diantaranya (Nurhayati & dkk, 2020), (Krisnandi & dkk, 2019). Return on Asset juga digunakan untuk melihat tingkat return yang dihasilkan oleh perusahaan atas penggunaan aktiva dalam operasionalnya (Sundari & Satria, 2021). Perusahaan yang mampu mengelola aktiva dengan baik akan dapat menghasilkan laba yang maksimal, demikian sebaliknya. Dalam penelitiannya Widiyati, (2019:547) ROA dapat dijadikan sebagai dasar menganalisis laba yang dihasilkan oleh perusahaan. ROA juga dapat dijadikan sebagai dasar dalam memprediksi laba perusahaan. Bukti empiris menunjukkan hasil yang masih berbeda-beda. Penelitian ini dilakukan pada perusahaan subsektor pertanian yang terdaftar di Bursa Efek Indonesia Periode 2013-2020 yang bertujuan untuk menganalisis determinasi pertumbuhan laba. 18 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Rasio Lancar (Current Ratio) Dalam mengukur kemampuan dari perusahaan untuk melunasi kewajiban- kewajibannya pada jangka waktu yang pendek, biasanya digunakan analisis rasio lancar. Dengan analisis ini akan dapat diketahui seberapa kuat posisi aktiva lancar perusahaan dalam menopang utang-utang jangka pendek secara keseluruhan. Perusahaan harus mampu melihat posisi aktiva lancarnya dengan utang lancar, karena pisisi aktiva lancar yang terlalu besar ketimbang utang lancar atau aktiva lancar yang terlalu kecil dibandingkan dengan utang lancar akan berakibat kurang baik dalam pandangan investor maupun kreditur. Untuk menentukan rasio lancar biasanya dilakukan dengan membandingkan jumlah aktiva lancar perusahan dengan utang lancar yang dicantumkan dalam laporan posisi keuangannya. Besar kecilnya rasio lancar dapat dipengaruhi berbagai hal diantaranya terjadinya distribusi dari pos aktiva lancar, tren data dari aktiva lancar dan utang angka pendek, ketentuan kredit yang dipersyaratkan perusahaan kepada para pelanggan, ketetapan waktu dalam pembayaran piutang perusahaan sesuai dengan syarat pembayaran yang disepakati, perubahan persediaan, jumlah kas dan setara kas, dll (Jumingan, 2011:124) Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… Return on Assets (ROA) Return on Assets (ROA) diguanakan dalam mengukur sejauhmana kemampuan perusahaan secara keseluruhan dalam menghasilkan keuntungan yang dibandingkan dengan jumlah aktiva yang dimiliki. Tinggi rendahnya Return on Assets (ROA) akan dapat menentukan baik atau tidaknya kondisi suatu perusahaan. Return on Assets (ROA) dapat diketahui dengan membagikan antara laba bersih yang dihasilkan perusahaan dengan total asset. Kasmir, (2012:197) tujuan analisis Return on Assets (ROA), diantaranya: a. Mengukur laba yang dihasilkan perusahaan dalam periode tertentu b. Menilai posisi laba yang dihasilkan perusahaan dari tahun sekarang dengan tahun sebelumnya c. Menganalisis perkembangan laba yang dihasilkan perusahaan dari periode ke periode berkutnya d. Menilai besarnya laba bersih sesudah pajak yang dibandingkan dengan modal sendiri perusahaan e. Mengukur produktivitas dana perusahaan secara keseluruhan baik yang bersumber dari pinjaman maupun modal sendiri f. Menilai produktivitas dana perusahaan secara keseluruhan dengan penggunaan modal sendiri Debt to Equity Ratio (DER) Debt to Equity Ratio merupakan alat yang digunakan untuk menganalisis rasio utang dengan ekuitas perusahaan. Dengan melakukan analisis ini, maka akan dapat diketahui jumlah dana yang dialokasikan peminjam dengan pemilik perusahaan, artinya berapa rupiahkah modal sendiri yang dimiliki oleh perusahaan dan menjamin utangnya. Surtina & Silistia, (2017:110) tujuan perusahaan dalam menggunakan perhitungan Debt to Equity Ratio, diantaranya: 1. Menentukan posisi kewajiban perusahaan pada pihak kreditor 2. Menilai seberapa besar kemampuan perusahaan dalam memenuhi kewajiban yang sifatnya tetap 3. Mengetahui keseimbangan antara nilai aktiva tetap dengan modal 19 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 4. Berapa besarnya aktiva yang dimiliki perusahaan dengan dibiayai oleh utang 5. Menentukan besarnya pengaruh utang perusahaan dalam mengelola aktiva 5. Menentukan besarnya pengaruh utang perusahaan dalam mengelola aktiva 6. Mengukur berapa besarnya modal sendiri dalam menjamin utang jangka panjang perusahaan 6. Mengukur berapa besarnya modal sendiri dalam menjamin utang jangka panjang perusahaan 7. Mengetahui besanya dana pinjaman yang akan segera jatuh tempo Pengaruh Current Ratio dengan Pertumbuhan Laba Current Ratio merupakan alat perhitungan dalam menganalisis kemampuan aktiva lancar yang dimiliki perusahaan dalam menjamin utang-utang lancarnya. Krisnandi & dkk, (2019:120) menemukan bahwa current ratio berpengaruh positif dengan pertumbuhan laba, artinya setiap kenaikan yang terjadi pada current ratio maka akan mengakibat menaiknya pertumbuhan laba perusahaan. Besar kecilnya perhitungan current ratio akan mempengaruhi tingkat kemampuan perusahaan dalam melunasi kewajibannya jangka pendek, artinya perusahaan yang mempunyai current ratio yang tinggi menunjukkan kemampuan aktiva lancar perusahaan dalam menjamin keberadaan utang lancar perusahaan. Dari penjelasan diatas dapat disimpulkan bahwa Current Ratio mempunyai pengaruh terhadap pertumbuhan laba perusahaan Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… Pengaruh Return on Assets (ROA) dengan Pertumbuhan Laba Analisis perhitungan Return on Assets (ROA) digunakan untuk melihat kemampuan perusahaan dalam menghasilkan keuntungan atas penggunaan aktivanya. Perusahaan yang mampu mengelola aktiva dengan produktif tentu akan mampu menghasilkan keuntungan yang lebih baik demikian sebaliknya. Dalam penelitiannya Hidayat menemukan bahwa Pertumbuhan laba dipengaruhi oleh Return on Assets (ROA) secara signifikan. Kemampuan perusahaan dalam mengelola aktiva yang diinvestasikan pemegang saham sangat berpengaruh terhadap tingkat keuntunngan yang akan diperoleh. Hal ini ditunjukkan dengan besarnya Return on Assets (ROA) yang dihasilkan. Tingginya Return on Assets (ROA) mengindikasikan bahwa tinglat laba yang dihasilkan perusahaan juga mengalami peningkatan. Dari teori dan penelitian terdahulu tersebut dapat disimpulkan bahwa Return on Assets (ROA) berpengaruh terhadap pertumbuhan laba perusahaan. Hubungan antar variabel dalam penelitian dalam digambarkan seperti berikut ini: Gambar 1. Model Penelitian Diolah untuk Penelitian ini Debt to Quick Ratio (X2) Pertumbuhan Laba Y Return on Asset (X3) Current Ratio (X1) Pertumbuhan Laba Return on Asset (X3) Gambar 1. Model Penelitian Diolah untuk Penelitian ini Pengaruh Debt to Equity Ratio (DER) dengan Pertumbuhan Laba Investor sebelum mengambil keputusan investasi, penting untuk menilai perbandingan jumlah dana yang disediakan kreditor denan pemilik perusahaan. Dalam prakteknya biasanya dilakukan dengan analisis Debt to Equity Ratio (DER). Penelitiannya (Widiyati, 2019) ditemukan bahwa tingkat pencapaian laba yang dihasilkan perusahaan dipengaruhi oleh Debt 20 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 to Equity Ratio (DER). Jika perhitungan Debt to Equity Ratio (DER) mengalami peningkatan, hal ini mengindikasikan bahwa laba yang dihasilkan oleh perusahaan akan banyak digunakan untuk menanggung utang dan bunganya kepada kreditor. Kondisi akan mengakibatkan pandangan calon investor terhadap perusahaan akan menurun karena investor beranggapan kinerja perusahaan mengalami penurunan. Sementara penelitian Tri Raharju menemukan bahwa solvabilitas mempunyai pengaruh terhadap pertumbuhan laba, namun pengaruhnya bersifat negatif. Artinya tingkat solvabilitas berbanding terbalik dengan pertumbuhan laba. Dari penjelasan tersebut dapat disimpulkan bahwa Debt to Equity Ratio (DER) berpengaruh terhadap pertumbuhan laba perusahaan HASIL DAN PEMBAHASAN Analisis Deskriptif Sumber : Hasil Pengolahan data dalam Penelitian ini Hasil diatas menunjukkan bahwa pertumbuhan laba memiliki nilai minimum -8.805000 terdapat pada perusahaan subsektor pertanian dan nilai maksumum 18.21600 pada perusahaan seubsektor pertanian, dan nilai rata-rata diperoleh 0.461702 dengan standar deviasi 545.3938. Current rasio memiliki nilai minimum 0.134000 dan nilai maksimum sebesar 5.209000 dan diperoleh rata-rata sebesar 1.297542 dengan standar deviasi variabel sebesar 71.12028. Nilai minimum 0.170000 dihasilkan oleh variabel Debt to equity rasio pada perusahaan subsektor pertanian dan nilai maksimum sebesar 13.31700 nilai rata-rata diperoleh sebesar 1.486688 dan standar deviasi variabel sebesar 222.1598. Rerurn on asset memiliki nilai minimum -8.805000 pada perusahaan subsektor pertanian dan nilai maksimum sebesar 9.638000 pada perusahaan subsektor pertanian secara keseluruhan diperoleh rata-rata sebesar 1.463313 dan standar deviasi variabel sebesar 991.8172. Metode Penelitian Populasi dalam penelitian ini adalah sebuah laporan laba rugi di Perusahaan Subsektor Pertanian yang terdapat di BEI, populasi dalam penelitian ini sebanyak 6 Perusahaan, tiap 21 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 perusahaan terdiri dari 8 Tahun laporan keuangan, sehingga jumlah populasi dalam penelitian ini sebanyak 48 data laporan keuangan. Sampel yang digunakan dalam penelitian ini adalah laporan keuangan Perusahaan Subsektor Pertanian. Data yang diambil adalah selama delapan tahun, yaitu dari periode 2013 sampai dengan tahun 2020. Pengolahan data pada penelitian ini adalah dengan menggunakan eviews. Studi dokumentasi dilakukan dalam pengumpulan data dalam penelitian ini. Dokumen yang diperlukan yaitu laporan keuangan yang berupa laporan posisi keuangan, laporan laba rugi, laporan perubahan equitas. Pengolahan data-data penelitian dilakukan dengan bantuan aplikasi eviews. Analisis yang dilakukan adalah analisis deskriptif, uji normalitas, uji asumsi klasik dan uji hipotesis. HASIL DAN PEMBAHASAN Analisis Deskriptif Hasil uji statistic deskriptif penelitian dengan bantuan Eviews: Tabel 2. Hasil Statistik Deskriptif Y X1 X2 X3 Mean 0.461702 1.297542 1.486688 1.463313 Median -0.270500 0.856000 0.904500 1.780500 Maximum 18.21600 5.209000 13.31700 9.638000 Minimum -8.805000 0.134000 0.170000 -8.805000 Std. Dev. 3.406483 1.230121 2.174122 4.593745 Skewness 2.888202 1.713272 4.026806 -0.438049 Kurtosis 17.81877 5.257580 20.83642 2.829499 Jarque-Bera 505.9255 33.67575 765.9974 1.593237 Probability 0.000000 0.000000 0.000000 0.450851 Sum 22.16168 62.28200 71.36100 70.23900 Sum Sq. Dev. 545.3938 71.12028 222.1598 991.8172 Observations 48 48 48 48 Sumber : Hasil Pengolahan data dalam Penelitian ini Estimasi Data Panel Hasil uji fixed effect 23 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Uji random effect dihasilkan seperti berikut ini: Tabel 5. Hasil uji random effect Dependent Variable: PL Method: Panel EGLS (Cross-section random effects) Date: 10/09/21 Time: 13:02 Sample: 2013 2020 Periods included: 8 Cross-sections included: 7 Total panel (unbalanced) observations: 48 Swamy and Arora estimator of component variances Variable Coefficient Std. Error t-Statistic Prob. C 2.127023 0.965787 2.202372 0.0329 X1 -0.017145 0.425683 -0.040275 0.9681 X2 -0.800026 0.255206 -3.134826 0.0031 X3 -0.261171 0.122054 -2.139795 0.0380 Effects Specification S.D. Rho Cross-section random 0.935717 0.0885 Idiosyncratic random 3.003835 0.9115 Weighted Statistics R-squared 0.178607 Mean dependent var 0.377905 Adjusted R-squared 0.122602 S.D. dependent var 3.319824 S.E. of regression 3.105595 Sum squared resid 424.3678 F-statistic 3.189169 Durbin-Watson stat 2.288639 Prob(F-statistic) 0.032762 Unweighted Statistics R-squared 0.164265 Mean dependent var 0.461702 Sum squared resid 455.8046 Durbin-Watson stat 2.130791 Sumber : Hasil Pengolahan data dalam Penelitian ini Sumber : Hasil Pengolahan data dalam Penelitian ini Dari hasil uji estimasi data panel di atas dengan pendekatan model common effect, fixed effect, random effect, maka dinyatakan model yang paling sesuai yaitu model common effect karena lebih besar dari 0,05. Pemilihan Model Estimasi Data Panel Dalam melakukan uji model estimasi dilakukan dengan uji chow, haustman, test dan uji LM (lagrange multiplier). Berdasarkan uji chow diperoleh nilai prob. Cross-section F yaitu 0.0115dan nilai Cross-section Chi-square yaitu 0.0030. berdasarkan hasil tersebut maka dapat ditentukan bahwa model yang terpilih yaitu common effect model. Estimasi Data Panel Berdasarkan uji estimasi data panel dengan pendekatan model common effect, fi 22 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 effect, dan random effect. Hasil uji menyatakan bahwa model yang paling sesuai yaitu model common effect. Hasil uji common effect disajikan sebagai berikut. effect, dan random effect. Hasil uji menyatakan bahwa model yang paling sesuai yaitu model common effect. Hasil uji common effect disajikan sebagai berikut. Tabel 3. Hasil uji common effect Dependent Variable: PL Method: Panel Least Squares Date: 10/09/21 Time: 16:20 Sample: 2013 2020 Periods included: 8 Cross-sections included: 7 Total panel (unbalanced) observations: 48 Variable Coefficient Std. Error t-Statistic Prob. C 2.103870 0.889382 2.365541 0.0225 X1 -0.060681 0.401159 -0.151265 0.8805 X2 -0.773198 0.269024 -2.874090 0.0062 X3 -0.282870 0.127659 -2.215830 0.0319 R-squared 0.167109 Mean dependent var 0.461702 Adjusted R-squared 0.110321 S.D. dependent var 3.406483 S.E. of regression 3.213090 Akaike info criterion 5.251999 Sum squared resid 454.2537 Schwarz criterion 5.407932 Log likelihood -122.0480 Hannan-Quinn criter. 5.310926 F-statistic 2.942676 Durbin-Watson stat 2.166533 Prob(F-statistic) 0.043309 Sumber : Hasil Pengolahan data dalam Penelitian ini Tabel 3. Hasil uji common effect Hasil diatas menunjukkan bahwa koefisien β variabel independen current ratio, debt to equity, return on asset > 0,05 yang berarti berpengaruh signifikan terhadap variabel dependen pertumbuhan laba. Selanjutnya hasil uji fixed effect disajikan sebagai berikut: 23 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… j y j fixed effect disajikan sebagai berikut: Tabel 4. Hasil uji fixed effect Dependent Variable: PL Method: Panel Least Squares Date: 10/09/21 Time: 12:59 Sample: 2013 2020 Periods included: 8 Cross-sections included: 7 Total panel (unbalanced) observations: 48 Variable Coefficient Std. Error t-Statistic Prob. C 2.238279 1.057484 2.116607 0.0409 X1 -0.231559 0.593046 -0.390457 0.6984 X2 -0.850844 0.260918 -3.260969 0.0023 X3 -0.144317 0.132237 -1.091352 0.2820 Effects Specification Cross-section fixed (dummy variables) R-squared 0.371326 Mean dependent var 0.461702 Adjusted R-squared 0.222429 S.D. dependent var 3.406483 S.E. of regression 3.003835 Akaike info criterion 5.220709 Sum squared resid 342.8750 Schwarz criterion 5.610542 Log likelihood -115.2970 Hannan-Quinn criter. 5.368027 F-statistic 2.493852 Durbin-Watson stat 2.751270 Prob(F-statistic) 0.023840 Sumber : Hasil Pengolahan data dalam Penelitian ini Tabel 4. Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… Uji Normalitas Untuk melihat apakah data-data variabel yang digunakan dalam penelitian berdistribusi normal dilakukan dengan uji yang dikembangkan oleh Jurqua-Bera (JB). 24 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Gambar 2. Uji Normalitas 0 1 2 3 4 5 6 7 8 9 -1.0e-15 -5.0e-16 1.0e-21 5.0e-16 Series: Standardized Residuals Sample 2013 2020 Observations 48 Mean -6.66e-17 Median -6.32e-18 Maximum 8.94e-16 Minimum -1.26e-15 Std. Dev. 3.57e-16 Skewness -0.514434 Kurtosis 4.816033 Jarque-Bera 8.713098 Probability 0.012823 Hasil dari analisis yang ditunjukkan pada gambar diatas menunjukkan bahwa nilai probabilitas JB sebesar 0,012823, sehingga dapat disimpulkan bahwa data berdistribusi normal. Gambar 2. Uji Normalitas Hasil dari analisis yang ditunjukkan pada gambar diatas menunjukkan bahwa nilai probabilitas JB sebesar 0,012823, sehingga dapat disimpulkan bahwa data berdistribusi normal. Uji Multikolinearitas Dalam menguji multikolinearitas digunakan nilai tolerance dan VIF (Variance Inflation Faktor), dimana jika nilai VIF lebih kecil dari 10 atau VIF < 10 maka artinya tidak terjadi multikolineritas terhadap data yang di uji. Sedangkan jika nilai VIF lebih besar dari 10 atau VIF > 10 maka artinya terjadi multikolineritas Tabel 6. Uji Multikolinearitas X1 X2 X3 X1 1 0.005500777855951593 -0.2633408477771594 X2 0.005500777855951593 1 -0.2743842957168812 X3 -0.2633408477771594 -0.2743842957168812 1 Sumber : Hasil Pengolahan data dalam Penelitian ini Nilai VIF dihasilkan sebesar 0.005500777855951593, -0.2633408477771594, - 0.2743842957168812. Hasil tersebut menunjukkan bahwa tidak terjadi multikolinearitas pada regresi diatas, karena nilai VIF < 10. Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… Uji Heterokedastisitas Berikut hasil uji heterokedastisitas: Berikut hasil uji heterokedastisitas: 25 JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Tabel 7. Uji Heteroskedastisitas Dependent Variable: PL Method: Panel Least Squares Date: 10/09/21 Time: 14:03 Sample: 2013 2020 Periods included: 8 Cross-sections included: 7 Total panel (unbalanced) observations: 48 Variable Coefficient Std. Error t-Statistic Prob. X1 -0.060681 0.401159 -0.151265 0.0805 X2 -0.773198 0.269024 -2.874090 0.0062 X3 -0.282870 0.127659 -2.215830 0.0319 C 2.103870 0.889382 2.365541 0.0225 R-squared 0.167109 Mean dependent var 0.461702 Adjusted R-squared 0.110321 S.D. dependent var 3.406483 S.E. of regression 3.213090 Akaike info criterion 5.251999 Sum squared resid 454.2537 Schwarz criterion 5.407932 Log likelihood -122.0480 Hannan-Quinn criter. 5.310926 F-statistic 2.942676 Durbin-Watson stat 2.166533 Prob(F-statistic) 0.043309 Sumber : Hasil Pengolahan data dalam Penelitian ini Tabel 7. Uji Heteroskedastisitas Nilai probabilitas ketiga variabel dari tabel diatas lebih kecil dari α (alpha) 0,10, sehingga disimpulkan bahwa tidak terdapat heteroskedastisitas pada penelitian ini. Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… Analisis Regresi Berganda Pengolahan data untuk regresi berganda dapat terlihat: Pengolahan data untuk regresi berganda dapat terlihat: Pengolahan data untuk regresi berganda dapat terlihat: Tabel 8. Uji Analisis Regresi Berganda Dependent Variable: PL Method: Panel Least Squares Date: 10/09/21 Time: 12:59 Sample: 2013 2020 Periods included: 8 Cross-sections included: 7 Total panel (unbalanced) observations: 48 Variable Coefficient Std. Error t-Statistic Prob. C 2.238279 1.057484 2.116607 0.0409 X1 -0.231559 0.593046 -0.390457 0.6984 X2 -0.850844 0.260918 -3.260969 0.0023 X3 -0.144317 0.132237 -1.091352 0.2820 Effects Specification Cross-section fixed (dummy variables) R-squared 0.371326 Mean dependent var 0.461702 Adjusted R-squared 0.222429 S.D. dependent var 3.406483 S.E. of regression 3.003835 Akaike info criterion 5.220709 Sum squared resid 342.8750 Schwarz criterion 5.610542 Log likelihood -115.2970 Hannan-Quinn criter. 5.368027 F-statistic 2.942676 Durbin-Watson stat 2.751270 Prob(F-statistic) 0.023840 Sumber : Hasil Pengolahan data dalam Penelitian ini Regresi berganda dapat dibuat berdasarkan hasil pengolahan data pada tabel diatas: PL= 2.238279 - 0.231559 X1 - 0.850844 X2 - 0.144317 X3+ e Sumber : Hasil Pengolahan data dalam Penelitian ini Regresi berganda dapat dibuat berdasarkan hasil pengolahan data pada tabel diatas: Regresi berganda dapat dibuat berdasarkan hasil pengolahan data pada tabel diatas: 26 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Nilai Konstanta diperoleh 2.2383 yang artinya apabila variabel independen dalam penelitian ini nilainya 0, maka pertumbuhan laba yang dihasilkan oleh perusahaan sebesar 2.2383. Koefisien dari current ratio diperoleh sebesar-0.232 artinya jika current ratio mengalami peningkatan 1 persen, maka pertumbuhan laba akan mengalami penurunan sebesar -0.232 persen dengan asumsi bahwa variabel independen yang lain dianggap tetap. Nilai koefisien debt to equity ratio diperoleh sebesar -0.850artinya jika terjadi peningkatan debt to equity rati sebesar 1 persen, maka pertumbuhan laba pada perusahaan akan mengalami penurunan sebesar -0.850 persen dengan asumsi variabel independen yang lain dianggap nilainya tetap. Nilai koefisien dari return on assets diperoleh sebesar -0.144, ini menandakan bahwa jika terjadi kenaikan terhadap return on asset 1 persen, maka pertumbuhan laba perusahaan akan mengalami penurunan sebesar -0.144 persen. Uji t Dari tabel diatas dapat dilihat bahwa pengaruh dari variabel independen terhadap variabel dependen secara parsial. Berdasarkan hasil uji t terlihat bahwa nilai untuk variabel current ratio diperoleh nilai probabilitassebesar 0.698, dimana nilai ini lebih besar dari ketentuan nilai sig yaitu 0.05. Dari hasil tersebut diketahui bahwa current ratio tidak mempunyai pengaruh terhadap pertumbuhan laba perusahaan. Untuk pengaruh debt to equity ratio terhadap pertumbuhan laba diperoleh nilai prob. 0.0023, dimana nilai ini lebih kecil dari ketentuan nilai sig yaitu 0.05. hasil ini menandakan bahwa debt to equity ratio mempunyai pengaruh terhadap pertumbuhan laba perusahaan. Nilai probabilitas dari return on asset diperoleh 0,282, dimana nilai tersebut lebih besar dari ketentuan sig (0.05). Hasil ini menyatakan bahwa return on assets tidak dapat mempengaruhi pertumbuhan laba perusahan. Pengaruh Current Ratio Terhadap Pertumbuhan Laba Perusahaan Pengaruh Current Ratio Terhadap Pertumbuhan Laba Perusahaan Current ratio merupakan rasio untuk mengukur kemampuan perusahaan dalam memenuhi kewajiban jangka pendeknya yang segera jatuh tempo dengan menggunakan aset lancar yang tersedia. Rasio ini menunjukkan hubungan aktiva lancar dengan kewajiban lancar menurut nilai-nilai rupiahnya. Rasio yang rendah dapat berarti bahwa perusahaan tidak akan sanggup melunasi utang jangka pendeknya dalam kondisi darurat. Rasio yang tinggi dianggap menguntungkan bagi kreditor-kreditor. Dalam jurnal penelitian Hendry Krisnandi dkk mengatakan “current ratio yang rendah menunjukkan bahwa Likuiditas perusahaan buruk, sebaliknya apabila current ratio yang tinggi menunjukkan Likuiditas relatif baik. Jadi dapat disimpulkan jika current ratio meningkat maka laba juga akan meningkan, dan jika current ratio mengalami penurunan maka laba juga akan menurun. Hasil analisis menunjukkan bahwa secara parsial pertumbuhan laba tidak berpengaruh signifikan terhadap pertumbuhan laba pada PT. Astra agro lestari Tbk, dapat dilihat dari perkembangan current ratio yang terus mengalami peningkatan dari tahun 2013-2020, PT. Jaya agro wattie Tbk, hal ini dapat dilihat dari perkembangan current ratio yang terus mengalami penurunan dari tahun 2013-2020, PT. Salim ivomas pratama Tbk, hal ini dapat dilihat dari perkembangan current ratio yang terus mengalami fluktuasi dari tahun 2013-2020, PT. Samporna agro Tbk, hal ini dapat dilihat dari perkembangan current ratio yang terus mengalami penurunan dari tahun 2013-2020, PT. PP london sumatera Tbk, hal ini dapat dilihat dari perkembangan current ratio yang terus mengalami peningkatan dari tahun 2013-2020, PT. Eagle high plantations Tbk, hal ini dapat dilihat dari perkembangan current ratio yang terus mengalami peningkatan dari tahun 2013-2020. Hal ini dapat dilihat dari perkembangan current ratio yang terus mengalami penurunan sementara pertumbuhan laba perusahaan juga mengalami penurunan. Oleh karena itu hal ini disebabkan oleh ketidak mampuan perusahaan dalam memanfaatkan sejumlah asset lancar yang dimiliki perusahaan dan memaksimalkan kegiatan operasional sehingga dapat mengakibatkan penumpukan persediaan atau piutang misalnya yang menyebabkan perusahaan menjadi tidak efisien karena akan memerlukan waktu yang lebih lama untuk mengubah aset tersebut menjadi keuntungan untuk perusahaan. Hal ini sesuai dengan hasil penelitian yang dilakukan oleh Anni Kholilah yang menyatakan bahawa current ratio tidak terdapat pengaruh yang signifikan terhadap pertumbuhan laba pada PT. Indofoot sukses makmur Tbk periode 2010-2017. karena aktiva lancar dari perusahaan tersebut memiliki nilai yang kecil di bawah rata-rata. Karena current ratio memiliki pengaruh negative terhadap pertumbuhan laba. Koefisien Determinasi Hasil pengolahan data dengan bantuan aplikasi Eviews yaitu: Hasil pengolahan data dengan bantuan aplikasi Eviews yaitu: Hasil pengolahan data dengan bantuan aplikasi Eviews yaitu: Tabel 9. Koefisien Determinasi (R2) R-squared 0.167109 Mean dependent var 0.461702 Adjusted R- squared 0.110321 S.D. dependent var 3.406483 S.E. of regression 3.213090 Akaike info criterion 5.251999 Sum squared resid 454.2537 Schwarz criterion 5.407932 Log likelihood -122.0480 Hannan-Quinn criter. 5.310926 F-statistic 2.942676 Durbin-Watson stat 2.166533 Prob(F- statistic) 0.043309 Sumber : Hasil Pengolahan data dalam Penelitian ini Hasil diatas menunjukkan bahwa nilai R-Squared sebesar 0.167, artinya bahwa Current Ratio, Debt to Equity Ratio dan Return on Assets dapat menjelaskan kondisi pertumbuhan laba perusahaan sebesar 16,7 % sedangan sisanya sebesar 83,29 % dijelaskan oleh variabel- variabel bebas lainnya yang tidak termuat dalam model penelitian ini. 27 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Pengaruh Return On Assets terhadap Pertumbuhan Laba Return on Asset merupakan rasio yang menunjukkan seberapa kontribusi aset dalam menciptakan laba bersih, dengan kata lain rasio ini digunakan untuk mengukur berapa besar jumlah laba bersih yang akan dihasilkan dari setiap rupiah dana yang tertanam dalam total aset. Semakin tinggi hasil pengembalian atas aset berarti semakin tinggi juga jumlah laba bersih yang dihasilkan dari setiap rupiah dana yang tertanam dalam total aset, sebaliknya semakin rendah hasil pengembalian aset berarti semakin rendah pula jumlah laba bersih yang dihasilkan dari setiap rupiah yang tertanam dalam total aset. Hasil analisis menunjukkan bahwa secara parsial return on asset tidak berpengaruh terhadap pertumbuhan laba pada PT. Astra agro lestari Tbk, PT. Jaya agro wattie Tbk, PT. Salim ivomas pratama Tbk, PT. Samporna agro Tbk, PT. PP london sumatera Tbk, PT. Eagle high plantations Tbk. Hal ini dilihat dari perkembangan return on asset yang selalu mengalami penurunan dari tahun 2013-2020, sementara pertumbuhan laba perusahaan mengalami penurunan. Hal ini dikarenakan terdapat aset yang tidak digunakan untuk proses produksi, sehingga walaupun jumlah aset yang besar tetapi tidak dapat digunakan untuk menambah laba perusahaan. Hal ini sesuai dengan hasil penelitian yang dilakukan oleh nanda revin anggani yang menyatakan bahawa return on asset tidak berpengaruh terhadap pertumbuhan laba pada perusahaan subsektor makanan dan minuman yang terdaftar di Bursa Efek Indonesia periode 2012-2016. Pengaruh Debt to Equity Ratio terhadap Pertumbuhan Laba. Debt to equity rasio merupakan rasio yang membandingkan utang perusahaan dengan total ekuitas. debt to equity ratio merupakan finansial leverage yang dipertimbangkan sebagai variabel keuangan karena secara teoritas menunjukkan rasio suatu perusahaan sehingga berdampak pada ketidak pastian harga saham. Dalam jurnal penelitian Nurhayati dkk“jika debt to equity ratio semakin besar beban utang yang harus dibayar oleh perusahaan akan berdampak pada menurunnya pertumbuhan laba, sehingga pertumbuhan laba perusahaan akan mengalami 28 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 penurunan” (Nurhayati & dkk, Analisis Faktor Yang Mempengaruhi Pertumbuhan Laba Perusahaan Pertambangan, 2020). Jadi dapat di simpulkan bahwa jika debt to equity ratio naik makaalaba akan turun, dan jika debt to equity ratio turun maka laba akan naik. Hasil analisis menunjukkan bahwa secara parsial debt to equity ratio tidak berpengaruh terhadap pertumbuhan laba pada PT. Astra agro lestari Tbk, PT. Jaya agro wattie Tbk, PT. Salim ivomas pratama Tbk, PT. Samporna agro Tbk, PT. PP london sumatera Tbk, PT. Eagle high plantations Tbk. Hal ini dilihat dari perkembangan debt to equity ratio yang selalu mengalami peningkatan dari tahun 2013-2020, sementara pertumbuhan laba perusahaan mengalami penurunan. Karena perusahaan tersebut memutuskan untuk mendahulukan membayar hutang yang jatuh tempo dan mengganti dengan laba ditahan, sehingga kegiatan operasional perusahaan akan tetap berjalan dengan maksimal dan laba tidak akan mengalami masalah. Hal ini sesuai dengan hasil penelitian yang dilakukan oleh Nanda Revin Anggani yang menyatakan bahawa debt to equity ratio tidak terdapat pengaruh yang signifikan terhadap pertumbuhan laba pada perusahaan manufaktur yang terdaftar di bursa efek Indonesia periode 2013-2016. KESIMPULAN, KETERBATASAN DAN SARAN Penelitian ini dapat disimpulkan bahwa current ratio tidak berpengaruh terhadap pertumbuhan laba, Debt to Equity Ratio berpengaruh terhadap pertumbuhan laba dan Return on Assets tidak berpengaruh terhadap Pertumbuhan laba pada perusahaan subsektor pertanian periode 2013-2020. 29 Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… JURNAL MAHASISWA AKUNTANSI SAMUDRA (JMAS) VOLUME 3 NOMOR 1, FEBRUARI 2022 Keterbatasan penelitian ini terletak pada penggunaan variabel independen yaitu current ratio, debt to equity ratio dan return on assets. Penelitian selanjutnya dapat melanjutkan penelitian ini dengan objek penelitian pada perusahaan lain atau dengan menambahkan periode pengamatan. Ajijah Harahap, Budi Gautama Siregar & Ali Hardana : Determinan Pertumbhan Laba pada Perusahaan… DAFTAR PUSTAKA AWS, R. M., & dkk. (2018). Determinan Pertumbuhan Laba Perusahaan Manufaktur Di BEI. Jurnal Riset Akuntansi dan Perpajakan, 5(2). Hasinah, N., & dkk. (2019, Februari). Kinerja Keuangan dan Pertumbuhan Laba Perusahaan Property. Edunomika, 3(1). Hery. (2015). Analisis Laporan Keuangan. Yogyakarta: Center For Academic Publishing Service. Jumingan. (2011). Analisis Laporan Keuangan. Jakarta: PT. Bumi Aksara. Kasmir. (2012). Analisis Laporan Keuangan. Jakarta: PT. Raja Grafindo Persada. Krisnandi, H., & dkk. (2019, Oktober). Pengaruh CR, IT, DER dan Ukuran Perusahaan Terhadap Kinerja Keuangan. Jurnal Rekayasa Informasi, 8(2). Nurhayati, & dkk. (2020). Analisis Faktor Yang Mempengaruhi Pertumbuhan Laba Perusahaan. Jurnal Bisnis dan Managemen, 14(3). Nurhayati, & dkk. (2020). Analisis Faktor Yang Mempengaruhi Pertumbuhan Laba Perusahaan Pertambangan. Jurnal Bisnis dan Manajemen, 14(3). Salmah, N. A., & Ermeila, S. (2018). Determinan Pertumbuhan Laba Perusahaan Perdagangan Eceran Berdasarkan Rasio Keuangan. Jurnal Manajamen dan Bisnis, 16(2). Siregar, B. G. (2020). Pengaruh Debt to Equity Ratio (DER) Terhadap Harga Saham dengan Inflasi Sebagai Variabel Moderating. Jurnal Penelitian Ekonomi Akuntansi (JENSI), 4(2), 114-124. Sundari, R., & Satria, M. (n.d.). Pengaruh ROA dan ROE Terhadap Pertumbuhan Laba Sundari, R., & Satria, M. R. (2021). Pengaruh ROA dan ROE Terhadap Pertumbuhan Laba pada Perusahaan Subsektor Wholesale. Land Jurnal, 2(1). Surtina, S., & Silistia, H. (2017). Analisis Laporan Keuangan. Jakarta: Mitra Wacana Penerbit. Surtina, S., & Silistia, H. (2017). Analisis Laporan Keuangan. Jakarta: Mitra Wacana Penerbit. Widiyati, M. (2019). Pengaruh NPM, ROA dan DER Terhadap Pertumbuhan Laba pada Perusahaan LQ-45. Jurnal riset Akuntansi Keuangan, 7(3). Widiyati, M. (2019). Pengaruh NPM, ROA dan DER Terhadap Pertumbuhan Laba pada Perusahaan LQ-45. Jurnal riset Akuntansi Keuangan, 7(3). 30
https://openalex.org/W2089918434	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0119020&type=printable	English	null	Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress	PloS one	2,015	cc-by	11,881	RESEARCH ARTICLE OPEN ACCESS ☯These authors contributed equally to this work. ☯These authors contributed equally to this work. * giselle.zenker@unifesp.br ☯These authors contributed equally to this work. * giselle.zenker@unifesp.br Citation: Silva RA, Palladino MV, Cavalheiro RP, Machado D, Cruz BLG, Paredes-Gamero EJ, et al. (2015) Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress. PLoS ONE 10(3): e0119020. doi:10.1371/journal.pone.0119020 Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress Rodrigo A. Silva1☯, Marcelly V. Palladino2☯, Renan P. Cavalheiro2, Daisy Machado1, Bread L. G. Cruz1, Edgar J. Paredes-Gamero2, Maria C. C. Gomes-Marcondes1, Willian F. Zambuzzi3, Luciana Vasques4, Helena B. Nader2, Ana Carolina S. Souza5, Giselle Z. Justo1,6* 1 Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil, 2 Departamento de Bioquímica (Campus São Paulo), Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil, 3 Departamento de Química e Bioquímica, IBB, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil, 4 Departamento de Genética e Biologia Evolutiva, Universidade de São Paulo, São Paulo, São Paulo, Brazil, 5 Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, São Paulo, Brazil, 6 Departamento de Bioquímica (Campus São Paulo) and Departamento de Ciências Biológicas (Campus Diadema), Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil Abstract Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol- induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phos- phorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant en- zymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These re- sults describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. Academic Editor: Ted S Acott, Casey Eye Institute, UNITED STATES Copyright: © 2015 Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (www.fapesp.br), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)(www.cnpq.br) and Rede Nanocosméticos/CNPq. The authors also acknowledge support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)(www.capes.gov.br). R. A. S. is supported by a PhD scholarship from FAPESP (Grant nr. 2006/ 07315-3). W. F. Z. is supported by a fellowship from CNPq (PQ-2). The funders had no role in study mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These re- sults describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. ☯These authors contributed equally to this work. * giselle.zenker@unifesp.br Introduction design, data collection and analysis, decision to publish, or preparation of the manuscript. design, data collection and analysis, decision to publish, or preparation of the manuscript. Human epidermis is a multilayered epithelium that forms the interface between the environ- ment and the organism. Due to its unique mechanical and biochemical properties, the epider- mis provides the barrier of body’s defense against environmental and physiological stressful conditions, such as UV exposition, dehydration, heat or other damaging insults such as in- flammation [1]. Keratinocytes, the major constituent of this tissue, exhibit a controlled pro- gram of differentiation that allows the dynamic and efficient recovery of skin layers [2]. However, changes in epidermal homeostasis may occur even in response to metabolically inert compounds, such as sorbitol, which is known to cause hyperosmotic stress effects in the skin [3,4]. In this condition, epidermal keratinocytes can trigger diverse intracellular signaling cascades involved in the control of proliferation and differentiation [5,6]. Moreover, depend- ing on the intensity and duration of the stimulus, keratinocytes may undergo programmed cell death [7]. Competing Interests: The authors have declared that no competing interests exist. Hyperosmotic stress initiates several adaptive responses, among them, the remodeling of the cytoskeleton is of particular importance due to its role in gene transcription and signal transduction [8,9]. In this context, keratinocytes are able to respond to hyperosmolarity through cytoskeleton remodeling [10], even though the underlying mechanisms and signaling network associated with this event have not been sufficiently defined. Recently, it was demon- strated that hyperosmotic stress induces a rapid, sustained and reversible phosphorylation of the actin-regulatory protein cofilin in kidney epithelial cells, and that this effect was mediated by the small GTPase Rho [11]. Also, mitochondrial translocation and oxidation of cofilin were shown to play a role in oxidant-induced apoptosis through loss of mitochondrial integrity [12,13]. Clearly, different biochemical mechanisms mediate cytoskeleton alterations during ap- optosis induction, such as oxidation and phosphorylation of cytoskeletal proteins. Reversible phosphorylation of tyrosine residues in proteins plays a key regulatory function in cell physiology. Protein tyrosine phosphatases (PTP) act together with protein tyrosine ki- nases (PTK) to regulate protein phosphotyrosine levels in signaling molecules, thereby mediat- ing specific changes in cellular responses, such as cell growth and differentiation, cell cycle, metabolism, and cytoskeletal function [14]. Activation of LMWPTP by Hyperosmotic Stress Abstract 1 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Introduction In particular, the 18 kDa enzymes of the low mo- lecular weight PTP (LMWPTP) family, which is encoded by the acid phosphatase locus 1 gene (ACP1), have been considered to play key regulatory functions in signaling pathways involved in cytoskeleton rearrangement [15,16]. Modulation of its activity is based on phosphorylation/ dephosphorylation of tyrosine residues and reversible oxidation of catalytic cysteine residues. Whereas phosphorylation of the conserved adjacent tyrosines, Tyr131 and Tyr132, modulates LMWPTP activity and substrates selectivity to different extents, the generation of intracellular reactive oxygen species (ROS) induces the inhibition of the enzyme through direct and indirect effects. Indeed, ROS promotes the reversible oxidation of key cysteine residues of the catalytic site leading to the inactivation of the enzyme. This event is also reinforced by the actions of ROS upon PTK since it is known that oxidation of amino acid residues in PTK promotes the activation of such enzymes [15,16,17,18]. In this respect, it has been previously demonstrated that LMWPTP oxidation leads to its inactivation and potentiates the action of PTK such as Src that can phosphorylate the phosphatase in Tyr132, contributing to its inactivation [19,20,21,22]. Herein, our aim was to investigate some events involved in keratinocytes response to hyper- osmotic stress induced by an osmotically nonionic compound, sorbitol, using a human kerati- nocyte cell line, HaCaT. In particular, given the activities displayed by LMWPTP in regulating several cellular events, including cytoskeleton functions, its involvement in such responses was studied. 2 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress Materials and Methods Materials Sorbitol, bovine serum albumin (BSA), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), trypan blue, neutral red and Protein Tyrosine Phosphatase Assay Kit—PTP- 101 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against β-actin, catalase, glutathione peroxidase (GPX), glutathione reductase (GR), phospho-PP2A (Ser8), PARP-1 [poly(ADP-ribose) polymerase], phalloidin conjugated with Alexa Fluor 488 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-cleaved caspase- 3 (Asp175) antibody conjugated with Alexa Fluor 488, anti-glyceraldehyde 3-phoshate dehydrogenase (GAPDH), anti-cofilin, anti-STAT5, anti-phospho-STAT5 (Tyr694), anti- phospho-cofilin (Ser3), anti-total Src, anti-phospho-Src (Tyr416), anti-phospho-Src (Tyr527), anti-Rac-1, anti-RhoA, anti-PKAα, and horseradish peroxidase (HRP)-linked secondary anti- rabbit and anti-mouse antibodies were bought from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 594-conjugated wheat germ agglutinin (WGA594), Protein A-Sepharose, 40,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 594 goat anti-rabbit IgG antibody were purchased from Life Technologies/Molecular Probes, Inc. (Eugene, OR, USA). Anti- LMWPTP and HRP-linked secondary anti-goat antibodies were obtained from Abcam (San Francisco, CA, USA). Paraformaldehyde and Fluoromount G were from Electron Microscopy Sciences (Hatfield, PA, USA), and annexin V conjugated to allophycocyanin (APC) was from BD Biosciences (San Jose, CA, USA). Fugene HD Transfection Reagent was obtained from Pro- mega (Madison, WI, USA). All the other chemicals and reagents used in this study were of ana- lytical grade, purchased from commercial sources. Activation of LMWPTP by Hyperosmotic Stress was used as a control (Scramble). Each target sequence does not show significant homology to other human gene sequences. HaCaT cells were seeded at 80% confluence and transfected using Fugene HD Transfection Reagent, with 40 nM of each siRNA duplex, separately, accord- ing to the manufacturer (Promega). Cells were incubated for 5 h before hyperosmotic stress was carried out. To verify the effectiveness of RNA interference, total protein was extracted and western blotting was performed. For immunofluorescence, cells grown on glass coverslips were fixed with 2% paraformalde- hyde for 30 min, washed with 0.1 mol/l glycine, and permeabilized with 0.01% saponin. After 30 min, cells were washed with PBS, pH 7.4, and incubated with Alexa Fluor 488-conjugated phalloidin in PBS/1% BSA for 2 h. Cells were then stained with primary anti-Rac-1 antibody for 2 h, washed and incubated with Alexa Fluor 594 goat anti-rabbit IgG antibody for 2 h. After washing, the nuclei were stained with DAPI for 30 min. Cells were washed and coverslips were mounted on glass slides using Fluoromount-G and examined in a laser scanning confocal mi- croscope (Leica SP8, Germany). Alexa Fluor 488 was excited at 493 nm and emission was cap- tured at 520 nm. Alexa Fluor 594 was excited at 590 nm and the emission was detected at 619 nm. For DAPI, the excitation wavelength was 405 nm and the emission was captured at 461 nm. Images presented in the figures are representative of at least three experiments. Glutathione assay. Keratinocytes (5 x 106 cells) were treated with 1 M sorbitol for 2 h, fol- lowed by cell washing with physiological solution and lysis with 2 ml of water. A volume of 3 ml of precipitant solution (1.67 g of glacial metaphosphoric acid, 0.2 g of EDTA and 30 g of NaCl in 100 ml of deionized water) was added to the lysate. After 5 min, the mixture was centrifuged and the GSH concentration was determined according to Torsoni et al. [29] with some modifi- cations. Briefly, the reaction of 1.1 ml of the supernatant with 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB) was done in the presence of 0.2 M phosphate buffered saline (PBS), pH 8.0. After 5 min of reaction, absorbance was measured at 412 nm and the GSH content calculated in rela- tion to the control (ε = 13.6 mol-1 cm-1). Control cells were treated with medium only. Methods Cell culture and sorbitol-induced hyperosmotic stress. The human keratinocyte cell line HaCaT [23,24] was gently provided by Dr. Liudmila Kodach (Amsterdam Medical Center, Amsterdam, The Netherlands). These cells were cultured in Dulbecco’s Modified Eagle’s medi- um (DMEM; Sigma Chemical Co.) supplemented with 10% fetal calf serum (FCS; Gibco, Grand Island, NY, USA), 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Viability and cell density were determined by the trypan blue dye exclusion test. Hyperosmotic stress was induced in keratinocytes (3.5 x 104 cells/ml) seeded in sextuplicates into 96-well plates in DMEM supplemented with 10% FCS. After 48 h of incubation at 37°C in a humidified atmosphere containing 5% CO2, the medium was replaced with DMEM contain- ing BSA (0.5 mg/ml) and cells were incubated for another period of 24 h. Cells were then treat- ed with different concentrations of sorbitol (0.2–2 M), diluted in serum-free culture medium, while control samples were treated with the corresponding volume of serum-free culture medi- um. All samples were incubated for 2 h at 37°C in a humidified atmosphere containing 5% CO2. After treatment, cells were harvested for analysis. Effects of sorbitol on cell viability were evaluated by the MTT reduction assay (MTT), the neutral red uptake (NRU) and the total nucleic acid content (NAC), as previously described [25,26,27]. The results were expressed as percent of the cell viability of untreated control cells (100%). Knocking down of ACP1 gene and immunofluorescence. To knockdown ACP1 gene, two different siRNAs (siAPC1-CIII and siACP1-CV) were designed based on target sequences described previously in [28] and synthesized forming siRNA duplexes (Sigma-Aldrich Co., St. Louis, MO, USA). MISSION siRNA Universal Negative Control #1 (Sigma-Aldrich Co.) 3 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress Cells undergoing phosphatidylserine exposure were then microscopically examined using an inverted laser scanning confocal microscope LSM 510 (Zeiss, Germany). The WGA594 was ex- cited with a HeNe laser (excitation = 546 nm) and light emission was detected at 560–610 nm. The annexin V-APC was excited with a HeNe laser (excitation = 633 nm) and light emission was detected at 635–700 nm. Temporal images were captured with intervals of 10 min. The in- tensity of annexin V-APC was quantified using Examiner 4.2 (Zeiss) and was normalized using WGA594 intensity. Images were prepared using the programs Examiner 4.2 (Zeiss) and Adobe Photoshop 9.2. Analysis of caspase-3 activity by flow cytometry. The endogenous levels of the large frag- ment (17/19 kDa) of activated caspase-3 were evaluated by flow cytometry. After treatment with 1 M sorbitol for 2 h, HaCaT cells were harvested by centrifugation, washed with ice-cold PBS and fixed in paraformaldehyde 2% in PBS (v/v) for 30 min. Cells were then permeabilized in PBS containing 0.01% saponin for 15 min and blocked in PBS containing 1% BSA for 30 min at room temperature. Afterwards, 10 μl of cleaved caspase-3 (Asp175) Alexa Fluor 488- conjugated antibody were added and cells were incubated in the dark at room temperature for 1 h. The antibodies were diluted according the manufacturer’s instructions (Cell Signaling Technology). Cells were then washed in PBS containing 1% BSA, resuspended in 300 μl PBS and analyzed (10.000 events were collected per sample) in a FACSCalibur flow cytometer (Bec- ton Dickinson, CA, USA) using the CellQuest software (BD). Immunoblotting. After hyperosmotic stress, HaCaT cells were washed in ice-cold PBS and protein extracts were obtained using a lysis buffer [50 mM Tris-HCl, pH 7.4, 1% Tween 20, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mmol/l Na3VO4, 1 mM NaF and protease inhibitors (1 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM 4-(2-aminoethyl) benze- nesulfonyl fluoride)] for 2 h on ice. Protein extracts were cleared by centrifugation and protein concentrations were determined using the Lowry protein assay [30]. An equal volume of 2X SDS gel loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.1% bro- mophenol blue and 20% glycerol) was added to the samples and boiled for 5 min. Equal amounts of protein (50 μg) were loaded onto SDS-PAGE and blotted onto PVDF membranes (Millipore, Bedford, MA, USA). Viability was determined by the trypan blue exclusion test. Assessment of redox status. HaCaT cells were treated with 1 M sorbitol in DMEM medi- um for 2h. After treatment, the cells were rinsed three times with cold PBS, ressuspended in 400 μl of ice cold lysis buffer [10 mmol/l phosphate buffer (pH 7.0)] and sonicated in an ice bath for 15 s. Thereafter, the total amount of protein extracts were cleared by centrifugation and the supernatant was collected and submitted to protein quantification [30] followed by as- sessment of the redox status using different assays. Catalase activity was measured by monitor- ing H2O2 decomposition determined by changes in A230 nm during 30 min incubation in reaction medium containing 50 mmol/l sodium phosphate buffer (pH 7.0), 10 mmol/l H2O2 and enzyme sample [31]. Glutathione S-transferase (GST) activity was assayed based on the conjugation of CDNB with glutathione and changes were monitored at A340 nm [32]. Oxida- tive stress was assessed based on the quantification of malondialdehyde (MDA), a secondary product of lipid peroxidation. The lipid peroxidation product MDA was determined by incuba- tion with MPO (n-methyl-2-phenylindole; Sigma), followed by measurement of the absor- bance at 590 nm [33]. The results were expressed as nmol. μg prot.-1.min-1 for catalase and GST and nM. μg prot.-1 for MDA. All the antioxidant enzyme activities were expressed as ΔOD/min/μg of protein and the absorbances were measured with an enzyme linked immuno- sorbent assay (ELISA) plate reader. Evaluation of phosphatidylserine exposure by confocal microscopy. HaCaT cells were seeded onto glass coverslips and incubated for 72 h at 37°C in a humidified atmosphere con- taining 5% CO2. Afterwards, the plasma membranes were stained with 10 μg/ml Alexa Fluor 594-conjugated WGA (WGA594) for 30 min. After washing in culture medium, HaCaT cells were treated with 1 M sorbitol in DMEM medium containing 5 μl annexin V-APC during 2 h. 4 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Hyperosmotic stress induces keratinocyte death It has been previously demonstrated the pro-apoptotic effects of hyperosmotic stress on human immortalized HaCaT keratinocytes, a well studied model of proliferating non- tumorigenic epidermal cells [34]. Herein, HaCaT cells were exposed for 2 h to a nonionic osmolyte, sorbitol, to induce hyperosmotic stress. Multiple, mechanistically different parame- ters were used to assess HaCaT cell viability: the total nucleic acid content (NAC), neutral red uptake (NRU) and reduction of the MTT [25,26,27]. As shown in Fig. 1A, the viability of HaCaT cells decreased in a dose-dependent manner in response to increasing concentrations of sorbitol (0.2–2 M). According to MTT and NRU assays, the 50% reduction in cell viability (IC50 value) was obtained with a concentration of sorbitol around 1 M. Importantly, a similar cytotoxicity profile was observed by predicting the cell number, using the NAC assay, as seen for the MTT and NRU approaches, indicating that sorbitol induces cell death. Next, we evalu- ated the characteristics of sorbitol-induced HaCaT cell death by examining typical biochemical and morphological alterations associated with apoptosis. Our results demonstrated that hyper- osmotic stress promoted the cleavage and activation of caspase-3, as demonstrated by the in- creased fluorescence intensity of its large fragment (17/19 kDa) detected directly by flow cytometry (Fig. 1B). Moreover, this is in agreement with the increased cleavage of the caspase- 3 substrate, PARP-1, as indicated by the higher levels of its small fragment in immunoblot analysis (Fig. 1C). Since translocation of phosphatidylserine from the inside to the outside surface of the plas- ma membrane is an important indicator of apoptosis induction, we followed phosphatidylser- ine exposition on the extracellular face of the membrane by annexin V staining. Confocal microscopy revealed that hyperosmolarity caused a marked increase in peripheral labeling that is consistent with an enhanced annexin V staining in a narrow line along the cell membrane (Fig. 1D). Furthermore, a time-dependent increase in annexin V/WGA ratio was found, indi- cating a 2-fold increase in annexin V fluorescence intensity relative to control after 1 h of stress exposure as long as the maximum increase (3-fold increase) was observed at 2 h (Fig. 1E). To- gether, these results corroborate with those previously reported by Diker-Cohen et al. [2], indi- cating that hyperosmolarity-provoked HaCaT cell death is mediated by apoptosis. Activation of LMWPTP by Hyperosmotic Stress or analysis of variance (ANOVA) followed by the post hoc Tukey test when more than two groups were compared, using Instat software (SigmaStat for Windows version 3.1, Systat Soft- ware Inc., USA). Differences were considered significant at P < 0.05, representing two-sided test of statistical significance. Densitometric analysis of blots was performed using the Scion Image software. Membranes were blocked in 1% fat-free dried milk or 2% BSA in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST) and incubated overnight at 4°C with appropriate primary antibody at 1:1000 dilution. After washing in TBST, membranes were in- cubated with appropriate HRP-linked secondary antibodies, at 1:5000 dilutions, in blocking buffer for 1 h. Immunoreactive bands were detected with enhanced chemiluminescence kit. LMWPTP immunoprecipitation. After hyperosmotic stress, cells were lysed in ice-cold lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% gli- cerol, 1% Triton X-100, 1 mM PMSF, 1 μg/ml leupeptin and 1 μg/ml aprotinin] for 2 h in ice. Protein extracts were cleared by centrifugation and protein concentrations were determined using the Lowry protein assay [30]. Lysates were then incubated (rotatory mixing) for 1 h with specific antibody against LMWPTP and Protein A-Sepharose at 4°C. After centrifugation, im- munoprecipitates were washed three times with HNTG sample [20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glicerol and 0.1% Triton X-100] and used for enzymatic assay, SDS-PAGE and immunoblotting as described above. LMWPTP activity assay. For determination of the tyrosine phosphatase activity, the im- munoprecipitates were washed and resuspended in 0.1 mol/l sodium acetate buffer, pH 5.5. The LMWPTP activity was analyzed using the Protein Tyrosine Phosphatase Assay Kit—PTP- 101, according to the manufacturer’s instructions (Sigma Chemical Co.). Enzyme activities were calculated in pmol PO4 2- mg-1 min-1, and expressed relative to control. Statistical analysis. All experiments were performed at least three times. Results were ex- pressed as mean ± standard deviation. Statistical analysis was performed by the Student’s t test, 5 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Modulation of expression/activation of signaling proteins associated with cytoskeletal modeling Cofilin has been implicated as an important regulator of cell fate in response to stress, due to its role in modulating actin-stressed filaments. The effects of osmotic stress on cofilin status were investigated in keratinocytes exposed to the IC50 concentration of sorbitol. As can be seen in Fig. 2A, the hyperosmotic challenge caused a substantial elevation of cofilin phosphorylation at Ser3 that was accompanied by a slight decrease in its expression, leading to a 3-fold increase in the phospho-cofilin/cofilin ratio relative to control (Fig. 2B). Concomitantly, a significant rise in the phosphorylation level of the inhibitory site of PP2A (Tyr307) was observed in this condition (Fig. 2A). These results are consistent with the view that the inhibition of PP2A may 6 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress Fig 1. Sorbitol cytotoxicity in HaCaT cells involves induction of apoptosis. (A) Confluent layers of HaCaT cells were challenged with different concentrations of sorbitol (0.2–2 M in serum-free culture medium) for 2 h, and cellular viability was assessed by three different endpoints assays [MTT reduction (MTT), neutral red uptake (NRU) and nucleic acid content (NAC)]. The results were expressed as percentage of control cell viability (100%) and represented as mean ± SD of three independent experiments run in quadruplicate. HaCaT cells exposed to 1 M sorbitol for 2 h exhibited typical signs of apoptosis. (B) Increased fluorescence intensity of the large fragment (17/19 kDa) of activated caspase-3 detected by flow cytometry in stressed (open histogram) relative to control (black histogram) cells. One representative histogram of three independent experiments for each sample is presented. (C) Increased levels of the small fragment of cleaved PARP-1 by immunoblotting. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. (D) Increased phosphatidylserine exposition on the extracellular face of the membranes of stressed compared to control cells, as shown by confocal microscopy analysis of membrane labe with WGA594 (red) and phosphatidylserine exposure labeled with annexin V-APC (blue). Images are representative of three independent experiments at t end of treatment (2 h). Bars = 20 μm. (E) Time-dependent increase in the relative fluorescence intensity of annexin V/WGA was quantified from temporal images captured with intervals of 10 min in experiment depicted in (D), using the Examiner 4.2 software. doi:10.1371/journal.pone.0119020.g001 (D) Hyperosmotic stress is associated with STAT5 activation. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSTAT5/STAT5 ratios normalized to the protein ratio of controls (1). (F) Sorbitol promotes inactivation of Src kinase. Despite the increase in protein levels of total Src kinase, a significant decrease in the phosphorylation of Y416 located on the activation loop of the kinase, and an increase of Y527, which corresponds to its inhibitory site, occur. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSrc/total Src ratios normalized to the protein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.05 and P < 0.001 compared with control. Fig 2. Effects of hyperosmotic stress on regulatory enzymes associated with cytoskeletal remodeling. HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed as described in Materials and Methods. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. β-actin or GAPDH was used as loading control. (A) Hyperosmotic stress induces cofilin phosphorylation and PP2A inhibition. To check for total cofilin expression and equality of protein loading, antibodies against cofilin and β-actin were used. (B) Densitometric analysis of the hyperosmolarity-induced cofilin phosphorylation. Data are expressed as phospho-cofilin/cofilin ratio normalized to the protein ratio of controls (1). (C) Hyperosmotic stress causes significant increases in the expression of RhoA and Rac-1, while levels of PKAα remained unchanged. (D) Hyperosmotic stress is associated with STAT5 activation. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSTAT5/STAT5 ratios normalized to the protein ratio of controls (1). (F) Sorbitol promotes inactivation of Src kinase. Despite the increase in protein levels of total Src kinase, a significant decrease in the phosphorylation of Y416 located on the activation loop of the kinase, and an increase of Y527, which corresponds to its inhibitory site, occur. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSrc/total Src ratios normalized to the protein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.05 and *P < 0.001 compared with control. doi:10.1371/journal.pone.0119020.g002 doi:10.1371/journal.pone.0119020.g002 guarantee high levels of phosphorylated cofilin in treated cells since this protein is a recognized substrate of PP2A [35]. Activation of LMWPTP by Hyperosmotic Stress Fig 2. Effects of hyperosmotic stress on regulatory enzymes associated with cytoskeletal remodeling. HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed as described in Materials and Methods. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. β-actin or GAPDH was used as loading control. (A) Hyperosmotic stress induces cofilin phosphorylation and PP2A inhibition. To check for total cofilin expression and equality of protein loading, antibodies against cofilin and β-actin were used. (B) Densitometric analysis of the hyperosmolarity-induced cofilin phosphorylation. Data are expressed as phospho-cofilin/cofilin ratio normalized to the protein ratio of controls (1). (C) Hyperosmotic stress causes significant increases in the expression of RhoA and Rac-1, while levels of PKAα remained unchanged. (D) Hyperosmotic stress is associated with STAT5 activation. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSTAT5/STAT5 ratios normalized to the protein ratio of controls (1). (F) Sorbitol promotes inactivation of Src kinase. Despite the increase in protein levels of total Src kinase, a significant decrease in the phosphorylation of Y416 located on the activation loop of the kinase, and an increase of Y527, which corresponds to its inhibitory site, occur. (E) Densitometric analysis of immunoblots was expressed as the relative intensity of phospho-YSrc/total Src ratios normalized to the protein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.05 and *P < 0.001 compared with control. Fig 2. Effects of hyperosmotic stress on regulatory enzymes associated with cytoskeletal remodeling. HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed as described in Materials and Methods. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. β-actin or GAPDH was used as loading control. (A) Hyperosmotic stress induces cofilin phosphorylation and PP2A inhibition. To check for total cofilin expression and equality of protein loading, antibodies against cofilin and β-actin were used. (B) Densitometric analysis of the hyperosmolarity-induced cofilin phosphorylation. Data are expressed as phospho-cofilin/cofilin ratio normalized to the protein ratio of controls (1). (C) Hyperosmotic stress causes significant increases in the expression of RhoA and Rac-1, while levels of PKAα remained unchanged. Modulation of expression/activation of signaling proteins associated with cytoskeletal modeling The results were expressed as mean ± standard deviation of the relative fluorescence intensities of each cell per field of view. At least three different fields were analyzed in three independent experiments doi:10.1371/journal.pone.0119020.g001 Fig 1. Sorbitol cytotoxicity in HaCaT cells involves induction of apoptosis. (A) Confluent layers of HaCaT cells were challenged with different concentrations of sorbitol (0.2–2 M in serum-free culture medium) for 2 h, and cellular viability was assessed by three different endpoints assays [MTT reduction (MTT), neutral red uptake (NRU) and nucleic acid content (NAC)]. The results were expressed as percentage of control cell viability (100%) and represented as mean ± SD of three independent experiments run in quadruplicate. HaCaT cells exposed to 1 M sorbitol for 2 h exhibited typical signs of apoptosis. (B) Increased fluorescence intensity of the large fragment (17/19 kDa) of activated caspase-3 detected by flow cytometry in stressed (open histogram) relative to control (black histogram) cells. One representative histogram of three independent experiments for each sample is presented. (C) Increased levels of the small fragment of cleaved PARP-1 by immunoblotting. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. (D) Increased phosphatidylserine exposition on the extracellular face of the membranes of stressed compared to control cells, as shown by confocal microscopy analysis of membrane labeled with WGA594 (red) and phosphatidylserine exposure labeled with annexin V-APC (blue). Images are representative of three independent experiments at the end of treatment (2 h). Bars = 20 μm. (E) Time-dependent increase in the relative fluorescence intensity of annexin V/WGA was quantified from temporal images captured with intervals of 10 min in experiment depicted in (D), using the Examiner 4.2 software. The results were expressed as mean ± standard deviation of the relative fluorescence intensities of each cell per field of view. At least three different fields were analyzed in three independent experiments. doi:10.1371/journal.pone.0119020.g001 7 / 19 7 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Hyperosmotic stress is associated with STAT5 activation It is well described that when mammalian cells are subjected to osmotic or oxidative stress they respond to such stresses through activation of JAK/STAT signaling pathways and maintenance of its activity through inhibition of protein tyrosine phosphatases such as PTP3. As recently demonstrated, in Dictyostelium cells the Ser448 and Ser747 phosphorylation of the protein- tyrosine phosphatase-3 (PTP3) is induced by a variety of stresses resulting in inhibition of its phosphatase activity and, consequently, maintenance of its substrate, STATc, activity [40]. In agreement with the data from literature, the results showed that after hyperosmotic stress both expression and activity (phosphorylation status at Tyr694) of STAT5 were increased (Fig. 2D and E). It has been shown that hyperosmotic stress induces cofilin phosphorylation through activa- tion of the Rho/ROCK/LIMK pathway. This event has been considered as a key contributor for the increase in F-actin during the stress periods. Indeed, data from the literature have shown that hyperosmotic stress induces LIMK phosphorylation leading to a rapid and sustained Rho activation. In agreement, Rho or ROCK inhibition can avoid the shrinkage-induced cofilin phosphorylation and LIMK activation [36,11]. In an opposite way, activation of Rac signaling pathways inhibits the activity of the small GTPase-RhoA through activation of Rho-GAP activ- ity [37,38]. In this way, the balance of Rac and Rho activities is fundamental and decisive for cytoskeleton rearrangement, alterations in cellular morphology and migratory behavior mainly in the determination of cofilin phosphorylation status induced by hyperosmotic stress periods [11,39]. Thus, in addition to cofilin, we analyzed by immunoblotting the expression/activation status of the small GTPases RhoA, Rac-1 and, also, PKAα. Our data showed that RhoA expres- sion was significantly increased, while levels of Rac-1 and PKAα remained unchanged (Fig. 2C) in our experimental model. The activity of Rho-A is influenced by the activity and functional status of Rho-GAP. In its phosphorylated form Rho-GAP is active, while its 8 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress dephosphorylation inactivates the protein that no more can hydrolyze de GTP in Rho, thus maintaining its active state. As demonstrated later in our work, the tyrosine phosphatase LMWPTP is activated by the hyperosmotic stress induced by sorbitol in HaCaT cells and this enzyme is able to dephosphorylate Rho-GAP and activate RhoA, suggesting that besides the in- crease in its expression, Rho-A possibly presents an increase in its activity associated with the activity of LMWPTP. Finally, our results reinforce the role of RhoA-induced intracellular sig- naling in the induction of cofilin phosphorylation and associate these events with the cytoskele- tal rearrangements induced by the hyperosmotic stress. Hyperosmotic stress induces inhibition of Src activity It is well known that Src is a pivotal mediator of cell adhesion and cytoskeleton rearrange- ments, affecting downstream protein activation, such as Rho and ROCK family members [41,42]. Thus, we next checked the expression and phosphorylation status of Src. Although sor- bitol treatment promoted an increase in protein levels, a significant reduction (>50%) in the phosphorylation of Tyr416 on Src was observed. In addition, an increase at Tyr527 phosphory- lation, which corresponds to the inhibitory site of the kinase, also occurred (Fig. 2F and G). The balance of these phosphorylations alters Src conformation leading to its inactivation, which may contribute to cell death. Therefore, a specific regulatory mechanism of Src kinase activation, acting on Tyr527 and Tyr416 located in the activation loop of the enzyme may exist in the response of keratinocytes to hyperosmotic stress conditions. PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress Fig 3. LMWPTP is markedly activated in stressed keratinocytes. HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed. (A) Hyperosmotic stress increases the activity of immunoprecipitated LMWPTP (IP-LMWPTP). P < 0.001 compared with control. (B) LMWPTP was immunoprecipitated and an anti-phosphotyrosine immunoblotting was performed. The blot was then stripped and reprobed with anti-LMWPTP antibody for normalization by densitometric analysis. (C) Hyperosmolarity increases the relative phosphorylated/non-phosphorylated LMWPTP ratios normalized to the protein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.001 compared with control. (D) Sorbitol does not affect LMWPTP expression. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with anti- LMWPTP antibody. β-actin was used as loading control. One representative immunoblot of three independent experiments is presented. doi:10.1371/journal.pone.0119020.g003 Fig 3. LMWPTP is markedly activated in stressed keratinocytes. HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed. (A) Hyperosmotic stress increases the activity of immunoprecipitated LMWPTP (IP-LMWPTP). P < 0.001 compared with control. (B) LMWPTP was mmunoprecipitated and an anti-phosphotyrosine immunoblotting was performed. The blot was then stripped and reprobed with anti-LMWPTP antibody for ormalization by densitometric analysis. (C) Hyperosmolarity increases the relative phosphorylated/non-phosphorylated LMWPTP ratios normalized to the rotein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.001 compared with control. D) Sorbitol does not affect LMWPTP expression. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with anti- MWPTP antibody β-actin was used as loading control One representative immunoblot of three independent experiments is presented Fig 3. LMWPTP is markedly activated in stressed keratinocytes. HaCaT cells were exposed to 1 M sorbitol for 2 h, harvested and lysed. (A) Hyperosmotic stress increases the activity of immunoprecipitated LMWPTP (IP-LMWPTP). P < 0.001 compared with control. (B) LMWPTP was immunoprecipitated and an anti-phosphotyrosine immunoblotting was performed. The blot was then stripped and reprobed with anti-LMWPTP antibody for normalization by densitometric analysis. (C) Hyperosmolarity increases the relative phosphorylated/non-phosphorylated LMWPTP ratios normalized to the protein ratio of controls (1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.001 compared with control. (D) Sorbitol does not affect LMWPTP expression. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with anti- LMWPTP antibody. β-actin was used as loading control. One representative immunoblot of three independent experiments is presented. doi:10.1371/journal.pone.0119020.g003 Hyperosmotic stress induces LMWPTP activation in keratinocytes Given the general importance of PTP activity in the regulation of many crucial events associat- ed with cellular physiology and, in particular, the ability of the LMWPTP to interact with Src kinase [18,20,43], the possible involvement of this phosphatase in the control of keratinocyte response to hyperosmotic stress was evaluated. To address this issue, we first compared LMWPTP expression and activity between control and stressed keratinocytes. HaCaT cells were treated for 2 h with 1 M sorbitol or medium and the activity of immunoprecipitated- LMWPTP (IP-LMWPTP) was assessed. The results showed that hyperosmotic stress caused a large increase (3.5-fold) in LMWPTP specific activity (Fig. 3A) compared with the control group. Furthermore, immunoblot analysis of Tyr phosphorylation status on IP-LMWPTP also corroborates this result, as the level of phospho-Tyr in IP-LMWPTP was increased in stressed keratinocytes (Fig. 3B-C). In addition, the expression levels of LMWPTP were evaluated in total protein extracts by immunoblotting and no changes in the amounts of LMWPTP were in- duced by sorbitol exposition (Fig. 3D). Altogether, these results indicated a positive action of hyperosmotic stress in the modulation of LMWPTP activity. 9 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress Fig 4. Changes in the cellular redox status induced by hyperosmotic stress. (A) Hyperosmotic stress dramatically increases GSH concentration in HaCaT cells, especially considering the reduced number of viable cells after stress. Viability was determined by the trypan blue exclusion method. All the esults were expressed as percentage of control (100%) and represented as mean ± SD of three independent experiments run in triplicate. P < 0.05 compared with control. (B) Hyperosmotic stress causes significant increases in the expression of glutathione peroxidase (GPX) and glutathione reductase GR), while levels of catalase remained unchanged. After hyperosmotic stress, cells were harvested and lysed. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. β-act was used as loading control. (C) The influence of hyperomostic stress on the malondialdehyde content (nM.ug prot.-1) and (D) glutathione-S-transferase an catalase activity (nmol.ug prot.-1.min-1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.05 compared with control. Fig 4. Changes in the cellular redox status induced by hyperosmotic stress. (A) Hyperosmotic stress dramatically increases GSH concentration in HaCaT cells, especially considering the reduced number of viable cells after stress. Viability was determined by the trypan blue exclusion method. All the results were expressed as percentage of control (100%) and represented as mean ± SD of three independent experiments run in triplicate. P < 0.05 compared with control. (B) Hyperosmotic stress causes significant increases in the expression of glutathione peroxidase (GPX) and glutathione reductase (GR), while levels of catalase remained unchanged. After hyperosmotic stress, cells were harvested and lysed. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. β-actin was used as loading control. (C) The influence of hyperomostic stress on the malondialdehyde content (nM.ug prot.-1) and (D) glutathione-S-transferase and catalase activity (nmol.ug prot.-1.min-1). Results were represented as mean ± standard deviation of three independent experiments. P < 0.05 compared with control. doi:10.1371/journal.pone.0119020.g004 of antioxidant enzymes in sorbitol-treated HaCaT cells were performed by western blotting and activity assays. As indicated in Fig. 4 (Fig. Hyperosmotic stress modulates antioxidant defenses in keratinocytes A role for oxidative signaling in hyperosmotic stress has been suggested in different cell models [44]. Importantly, beside its phosphorylation status, the redox environment also modulates LMWPTP activity by oxidizing a cysteine residue in the active site that is crucial for its catalytic activity. Thus, in view of the importance of the cellular redox status in modulating the activity of PTP in general [14,15], including the LMWPTP [27], analyses of the expression and activity PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 10 / 19 doi:10.1371/journal.pone.0119020.g004 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Role of LMWPTP in hyperosmotic stress As demonstrated by the results obtained in this work, it is clear the effects of hyperosmotic stress in keratinocytes LMWPTP activity. However, while the data shows the stimulatory ac- tion of hyperosmotic stress in the activity of LMWPTP, nothing is known about the effects of this activation in the molecular events associated with HaCaT response to such stress. To ad- dress this point, the expression of this phosphatase was knocking down in HaCaT cells using specific small interfering RNA against ACP1 gene, and its effects were analyzed before and after cell treatment with 1 M sorbitol. Efficiency of knockdowns was confirmed by the reduced levels of LMWPTP protein expression, obtaining better results with transfection of cells with 40 nM of siACP1-CV (Fig. 5A and data not shown). Thus, all experiments involving siRNA knockdown were conducted using 40 nM siACP1-CV. g The analysis of data obtained in non-stressed HaCaT cells showed that this phosphatase is, indeed, an active modulator of Src kinase, FAK and STAT5 expression/activity. Comparing the phosphorylation status of residues Tyr416 and Tyr527 before and after LMWPTP knockdown, it is clear that this phosphatase contributes for inactivation of Src kinase in normal, non- stressed cells. In fact, after LMWPTP knockdown, the cells presented a significant increase in Tyr416 phosphorylation, while a reduction in the phosphorylation levels at Tyr527 occurred (Fig. 5A and D). In agreement, LMWPTP knockdown resulted in an increase in Tyr577 phosphorylation of FAK protein (Fig. 5B), a known site of Src kinase activity. These results demonstrated that LMWPTP is an important regulator of Src activity in HaCaT cells, thus cor- roborating with other works showing Src kinase as a substrate of this phosphatase [43]. Inter- estingly, LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells (Fig. 5A), mainly in the status of Src kinase phosphorylation and activity. As demonstrated pre- viously, treatment of HaCaT cells with sorbitol induces a reduction in Src kinase phosphorylat- ed at Tyr416, which was accompanied by an increase in Tyr527 phosphorylation levels. When we compare these results with those obtained in cells exposed to sorbitol but transfected with siACP1-CV, we observed an increase in Tyr416 phosphorylated Src kinase, while no significant alteration in Tyr527 phosphorylation status occurred (Fig. 5D). These results demonstrated the important role played by LMWPTP during hyperosmotic stress in the regulation of Tyr416 phosphorylation status and Src kinase activation. As demonstrated by Nimnual et al. Activation of LMWPTP by Hyperosmotic Stress detoxification of cytotoxic and genotoxic compounds and in the prevention of tissue oxidative damages. In addition to their role in catalyzing the conjugation of electrophilic substrates to GSH, these enzymes also carry out a range of other functions in cells, such as the removal of re- active oxygen species and regeneration of S-thiolated proteins (both of which are consequences of oxidative stress), catalysis of conjugations with endogenous ligands, and catalysis of reac- tions in metabolic pathways not associated with detoxification [45,46]. Finally, the effectiveness of glutathione-based antioxidant defense mechanism is reinforced by the diminished levels of lipid peroxidation in treated cells, as indicated by the reduced concentration of MDA, a sec- ondary product of lipid peroxidation (Fig. 4C). Previous work has pointed out the importance of the glutathione/glutathione reductase/ NADPH system in the reversible oxidation of LMWPTP, underlying GSH as the major non- protein thiol in mammalian cells with protective action against the irreversible oxidation of this phosphatase [47]. Based on the results shown here, it is possible to conclude that antioxi- dant defense mechanisms guarantee the availability of GSH to maintain LMWPTP cysteine residue in its reduced form, thus contributing for the activity of this enzyme. PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 4A, 4B) treatment of cells with sorbitol had no difference in cata- lase expression but increased the levels of reduced glutathione (GSH) and upregulated the ex- pression of glutathione reductase (GR) and glutathione peroxidase (GPX). Also, although no significant differences in catalase activity were observed after exposition of cells to sorbitol, glu- tathione S-transferase (GST) has its activity increased in such condition (Fig. 4D). GST is a member of the superfamily of multifunctional dimeric proteins involved in the cellular PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 11 / 19 Activation of LMWPTP by Hyperosmotic Stress Fig 5. Effect of LMWPTP knockdown with siACP1-CV on expression/activity of Rac-1, Src, FAK and STAT5. HaCaT cells were transfected with siACP1-CV 40 nM, exposed to 1 M sorbitol for 2 h, harvested and lysed as described in Materials and Methods. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. GAPDH was used as loading control. The effects of ACP1 knockdown in Rac-1, Src, FAK and STAT5 proteins were evaluated in HaCaT cells before and after exposition to sorbitol-induced stress (A). (B-E) Densitometric analysis of the results normalized to the protein content of LMWPTP-expressing cells (value equal to 1) comparing stressed and non-stressed cells. Results were represented as mean ± standard deviation of three independent experiments. P < 0.001 compared with control. Fig 5. Effect of LMWPTP knockdown with siACP1-CV on expression/activity of Rac-1, Src, FAK and STAT5. Fig 5. Effect of LMWPTP knockdown with siACP1-CV on expression/activity of Rac-1, Src, FAK and STAT5. HaCaT cells were transfected with siACP1-CV 40 nM, exposed to 1 M sorbitol for 2 h, harvested and lysed as described in Materials and Methods. Equal amounts of total protein (50 μg) from cell lysates were loaded per lane and blotted with specific antibodies. One representative immunoblot of three independent experiments is presented. GAPDH was used as loading control. The effects of ACP1 knockdown in Rac-1, Src, FAK and STAT5 proteins were evaluated in HaCaT cells before and after exposition to sorbitol-induced stress (A). (B-E) Densitometric analysis of the results normalized to the protein content of LMWPTP-expressing cells (value equal to 1) comparing stressed and non-stressed cells. Results were represented as mean ± standard deviation of three independent experiments. *P < 0.001 compared with control. doi:10.1371/journal.pone.0119020.g005 and inhibition of LMWPTP. As mentioned before, our model of sorbitol-induced hyperosmo- tic stress did not cause alteration in Rac-1 expression, which is in agreement with our results showing increased activity of LMWPTP. In addition, since LMWPTP is able to dephosphory- late Rho-GAP, contributing to the maintenance of active Rho-A, it is possible to suggest that beside the demonstrated increase in Rho expression, the activity of this protein is also in- creased. Interestingly, our results showed that LMWPTP silencing in HaCaT cells (without ex- position to sorbitol) increased Rac-1 expression, suggesting that LMWPTP also influences the expression of this small GTPase (Fig. 5A and B). The same pattern of results was obtained when expression of Rac-1 was analyzed by confocal microscopy (Fig. 6). In addition, the results also showed that when LMWPTP-siRNA transfected cells were treated with sorbitol, a lack of inhibition of Rac-1 expression was observed compared with its expression in Scramble-siRNA transfected cells (Fig. 5A and C). The same pattern of results was obtained when expression of Rac was analysed through confocal microscopy (Fig. 6). These results demonstrated that LMWPTP is involved in the mechanisms associated with Rac-1 expression and that the expres- sion of this phosphatase is involved in the effects of sorbitol treatment in Rac-1 during hyper- osmotic stress. How LMWPTP activity inhibits the expression of Rac-1 is still an unknown issue. Moreover, its effect on cytoskeleton was also suggested as these results were followed by changes in F-actin organization, with a different distribution at the leading edge (Fig. 6). and inhibition of LMWPTP. As mentioned before, our model of sorbitol-induced hyperosmo- tic stress did not cause alteration in Rac-1 expression, which is in agreement with our results showing increased activity of LMWPTP. In addition, since LMWPTP is able to dephosphory- late Rho-GAP, contributing to the maintenance of active Rho-A, it is possible to suggest that beside the demonstrated increase in Rho expression, the activity of this protein is also in- creased. Interestingly, our results showed that LMWPTP silencing in HaCaT cells (without ex- position to sorbitol) increased Rac-1 expression, suggesting that LMWPTP also influences the expression of this small GTPase (Fig. 5A and B). The same pattern of results was obtained when expression of Rac-1 was analyzed by confocal microscopy (Fig. 6). In addition, the results also showed that when LMWPTP-siRNA transfected cells were treated with sorbitol, a lack of inhibition of Rac-1 expression was observed compared with its expression in Scramble-siRNA transfected cells (Fig. 5A and C). The same pattern of results was obtained when expression of Rac was analysed through confocal microscopy (Fig. 6). These results demonstrated that LMWPTP is involved in the mechanisms associated with Rac-1 expression and that the expres- sion of this phosphatase is involved in the effects of sorbitol treatment in Rac-1 during hyper- osmotic stress. Role of LMWPTP in hyperosmotic stress [48], cytoskeletal rearrangements based on Rac-1 activa- tion involve the production of reactive oxygen species (ROS) and the consequent oxidation 12 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 doi:10.1371/journal.pone.0119020.g006 Discussion Hyperosmotic stress removes water out of cell causing cell shrinkage and increasing ionic strength, which ultimately can lead to alterations in cellular architecture compartments, dena- turation of proteins, and perturbation of cell functions [2,52,53]. Additionally, osmotic shock activates cytoskeleton reorganization, although less is known about the biochemical mecha- nisms involved in this response. In this work, exposure of HaCaT cells to sorbitol demonstrates that hyperosmotic stress induces apoptosis in keratinocytes, as previously reported by Diker- Cohen et al. [2], since a marked increase in annexin V staining, caspase-3 activation and PARP-1 cleavage were demonstrated. In addition, cytoskeleton rearrangements were also trig- gered by sorbitol during cell death as indicated by Src kinase inhibition, LMWPTP activation, alterations in Rho and Rac-1 expression, and changes in F-actin organization. Several aspects of the physiological action of LMWPTP in the regulation of cellular metabo- lism have been described in different biological conditions. Of interest, the ability of LMWPTP to interact with Src kinase in the control of cellular differentiation and cytoskeleton rearrange- ment has been studied [18,20,43]. The activity of the Src kinase mediates cell adhesion and cy- toskeleton rearrangements, thus affecting downstream protein activation [41,42]. Our data demonstrated that exposure of HaCaT keratinocytes to sorbitol-induced osmotic stress inhib- ited the Src kinase activity, as indicated by the downregulation of phospho-Tyr416 located in the activation loop of the enzyme and the increase in phospho-Tyr527, a known inhibitory site of this kinase. As mentioned before, the Src kinase pathway acts in osmotically stressed kerati- nocytes by inhibiting cell death [1,7], supporting the idea that its inhibition might contribute to the observed cell death. Concomitantly to Src inhibition, we reported the activation of LMWPTP, which was also demonstrated by the evaluation of phospho-Tyr levels in immuno- precipitated LMWPTP. Interestingly, LMWPTP is able to dephosphorylate Src kinase mainly at Tyr416 [18], thus explaining the reduction in Tyr416 phosphorylated form of Src kinase ob- served in our study. It is very important to mention that the regulation of LMWPTP is based on phosphorylation/dephosphorylation and a change in ROS concentration modulates revers- ible oxidation of cysteine residues. The activity of protein kinases is finely regulated in cells since these proteins are key players in the regulation of signaling pathways. How LMWPTP activity inhibits the expression of Rac-1 is still an unknown issue. Moreover, its effect on cytoskeleton was also suggested as these results were followed by changes in F-actin organization, with a different distribution at the leading edge (Fig. 6). Since STAT5 is a known substrate of LMWPTP [49,50] and HaCaT cells exposed to sorbitol showed an increase in STAT5 expression and Tyr694 phosphorylation levels, a possible role of 13 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress Fig 6. Rac-1 and F-actin staining in LMWPTP silenced HaCaT cells. HaCaT cells were transfected with siACP1-CV 40 nM and exposed to 1 M sorbi 2 h. Rac-1 distribution and the organization of the actin filaments (F-actin) were evaluated by laser confocal microscopy after incubation of the cells with specific antibody for Rac-1, followed by staining with Alexa Fluor 594 goat anti-rabbit IgG antibody (red), and Alexa Fluor 488-conjugated phalloidin (gre The nuclei were stained with DAPI (blue). Bar = 20 μm. Representative results of 3 independent experiments. doi:10.1371/journal.pone.0119020.g006 Fig 6. Rac-1 and F-actin staining in LMWPTP silenced HaCaT cells. HaCaT cells were transfected with siACP1-CV 40 nM and exposed to 1 M sorbitol for 2 h. Rac-1 distribution and the organization of the actin filaments (F-actin) were evaluated by laser confocal microscopy after incubation of the cells with specific antibody for Rac-1, followed by staining with Alexa Fluor 594 goat anti-rabbit IgG antibody (red), and Alexa Fluor 488-conjugated phalloidin (green). The nuclei were stained with DAPI (blue). Bar = 20 μm. Representative results of 3 independent experiments. doi:10.1371/journal.pone.0119020.g006 14 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress LMWPTP in STAT5 phosphorylation during sorbitol stress was evaluated in HaCaT cells. First, the results showed that in non-stressed cells LMWPTP silencing caused a marked de- crease in STAT5 Tyr694 phosphorylation, indicating a positive correlation between LMWPTP activity and STAT5 activation (Fig. 5A and E). Curiously, when these cells were treated with sorbitol, the levels of STAT5 Tyr694 phosphorylation did not change significantly (Fig. 5A and E). Although phosphorylated STAT5 is known as a LMWPTP substrate, our results indi- cate that in the hyperosmotic stress context, LMWPTP indirectly contributes to the mainte- nance of STAT5 phosphorylation. It is known that STAT5 phosphorylation at Tyr694 is associated, mainly, to the activation of JAK2, although other kinases could exert the same activ- ity, as the case of the leukemia-related BCR-ABL tyrosine kinase. Also, the reversible tyrosine phosphorylation of the STAT proteins is regulated by SH2-domain-containing protein tyrosine phosphatase-2 (SHP-2), PTP1-B, as well as serine protein phosphatase 2A (PP2A) [51]. Studies are necessary to uncover the effects of LMWPTP on the activity of such phosphatases and ki- nases in order to improve our knowledge on the functions of this phosphatase in hyperosmotic stress conditions. PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Discussion It is thought that the redox status of the intracellular milieu exerts high influence in the regulation of both protein kinases and phosphatases, leading to the activation of the former and inactivation of the later group. In this respect, our results PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 15 / 19 Activation of LMWPTP by Hyperosmotic Stress demonstrating that Src activity was inhibited by sorbitol-induced stress, in contrast to the acti- vation of LMWPTP, reinforce the modulation of ROS action by antioxidant mechanisms [22]. Previous work showed the importance of the glutathione/glutathione reductase/NADPH sys- tem in the reversible oxidation of LMWPTP, which is dependent on the availability of GSH [47]. In this way, our results indicate that GSH maintains the intracellular redox balance and protects the sulfenic derivative of LMWPTP from further oxidation, driving the formation of a more stable product, and creating ideal conditions for LMWPTP action in response to hyper- osmotic stress. In addition to GSH, upregulation of GR and GPX expression, and raise in GST activity were also detected after treatment of HaCaT with sorbitol. The effectiveness of this an- tioxidant response could be observed through the reduced lipid peroxidation, inhibition of Src activity and, importantly, LMWPTP activity. A recent report has demonstrated that LMWPTP also plays a role in modulating Src activity in osteoblast differentiation [18]. According to these authors, modest activation of LMWPTP leads to dephosphorylation of the kinase at Tyr527 and Tyr416, the inhibitory and stimulatory residues of the kinase activity, respectively. Although our results demonstrate an increase in Tyr527 phosphorylation after exposure of keratinocytes to sorbitol, a significant reduction in Tyr416 was also observed, suggesting an action of LMWPTP in such event. Why the phos- phatase could not dephosphorylate Tyr527 is a theme for a further study but we can hypothe- size that this event is associated with the selectivity of the enzyme towards its substrate depending on its phosphorylation status. Indeed, phosphorylation of LMWPTP in Tyr132 resi- due is able to regulate the selectivity of the phosphatase towards diverse substrates. Once LMWPTP is phosphorylated at Tyr132 the enzyme becomes able to interact with SH2 domain- containing molecules such as Grb2, a known adaptor protein in signaling pathways. Discussion Further- more, as Tyr132 is located close to the active site, the interaction of Grb2-like proteins with Tyr132-phosphorylated LMWPTP could limit the access of the ligand-binding surface and ac- tive site entrance, leading to enzyme inactivation or selection of substrates by size [54]. Possi- bly, in our model, LMWPTP becomes able to dephosphorylate Src at Tyr416, but unable to dephosphorylate this enzyme at Tyr527. In addition to Src, other cytoskeletal proteins were altered in keratinocytes by hyperosmotic shock. Alteration in F-actin organization and increased protein expression was found for the small GTPases RhoA and Rac-1, whereas cofilin expression was decreased and its phosphoryla- tion at Ser3 was increased. Cofilin is an actin-binding protein that is required for actin filament disassembly and rearrangement. In physiological conditions, PP2A, a Ser/Thr phosphatase, is able to control cofilin activation by dephosphorylating its Ser3 residue [55]. Thus, our results demonstrating strong inhibition of PP2A during hyperosmotic shock is consistent with the in- activation of cofilin in keratinocytes. Finally, the direct action of LMWPTP in the molecular events associated with keratinocyte hyperosmotic stress response was demonstrated by the effects of knocking down the ACP1 gene. Inhibition of LMWPTP expression was able to attenuate the effects of sorbitol in HaCaT cells mainly at the control of Src and STAT5 activities and Rac expression. Indeed, even in non-stressed cells LMWPTP showed to be a key player in the regulation of expression and ac- tivity of Src, FAK and Rac proteins. References 1. Burg MB, Ferraris JD, Dmitrieva NI. Cellular response to hyperosmotic stresses. Physiol Rev. 2007; 87: 1441–1474. PMID: 17928589 2. Diker-Cohen T, Koren R, Ravid A. Programmed cell death of stressed keratinocytes and its inhibition by vitamin D: the role of death and survival signaling pathways. Apoptosis. 2006; 11: 519–534. PMID: 16532377 3. Ravid A, Rubinstein E, Gamady A, Rotem C, Liberman UA, Koren R. Vitamin D inhibits the activation of stress-activated protein kinases by physiological and environmental stresses in keratinocytes. J Endo- crinol. 2002; 173: 525–532. PMID: 12065242 4. Pan X, Kane LA, Van Eyk JE, Coulombe PA. Type I keratin 17 protein is phosphorylated on serine 44 by p90 ribosomal protein S6 kinase 1 (RSK1) in a growth- and stress-dependent fashion. 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Conclusions In summary, our work provides new contribution to the mechanisms involved in the response of keratinocytes to hyperosmotic shock, in which LMWPTP may play an important role in cy- toskeleton rearrangement. To the best of our knowledge, up until now, there was no study de- scribing a role for LMWPTP in keratinocytes upon exposure to stressful conditions. 16 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0119020 March 17, 2015 Activation of LMWPTP by Hyperosmotic Stress Acknowledgments The authors also acknowledge support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Rede Nanocosméticos/CNPq. R.A.S. is supported by a PhD schol- arship from FAPESP (Grant nr. 2006/07315–3). The authors would like to thank Prof. Carmen V. Ferreira (Departamento de Bioquímica, Universidade Estadual de Campinas) for the helpful discussions and comments on the manuscript. W.F.Z. is supported by a fellowship from CNPq (PQ-2). 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https://openalex.org/W3099405462	http://doc.rero.ch/record/326171/files/454_2014_Article_9614.pdf	English	null	Coloring Intersection Graphs of Arc-Connected Sets in the Plane	Discrete and computational geometry/Discrete & computational geometry	2,014	cc-by	11,099	Discrete Comput Geom (2014) 52:399–415 DOI 10.1007/s00454-014-9614-5 Discrete Comput Geom (2014) 52:399–415 DOI 10.1007/s00454-014-9614-5 Coloring Intersection Graphs of Arc-Connected Sets in the Plane Michał Laso´n · Piotr Micek · Arkadiusz Pawlik · Bartosz Walczak Received: 7 February 2014 / Revised: 21 May 2014 / Accepted: 5 July 2014 / Published online: 13 August 2014 Received: 7 February 2014 / Revised: 21 May 2014 / Accepted: 5 July 2014 / Published online: 13 August 2014 © The Author(s) 2014. This article is published with open access at Springerlink.com Abstract A family of sets in the plane is simple if the intersection of any subfamily is arc-connected, and it is pierced by a line L if the intersection of any member with L is a nonempty segment. It is proved that the intersection graphs of simple families of compact arc-connected sets in the plane pierced by a common line have chromatic number bounded by a function of their clique number. Keywords Geometric intersection graphs · Chromatic number · χ-boundedness 1 Introduction A proper coloring of a graph is an assignment of colors to the vertices of the graph such that no two adjacent ones are assigned the same color. The minimum number of colors sufﬁcient to color a graph G properly is called the chromatic number of G and M. Laso´n · B. Walczak École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland M. Laso´n · B. Walczak École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 123 123 123 123 400 Discrete Comput Geom (2014) 52:399–415 denoted by χ(G). The maximum size of a clique (a set of pairwise adjacent vertices) in a graph G is called the clique number of G and denoted by ω(G). It is clear that χ(G) ⩾ω(G). The chromatic and clique numbers of a graph can be arbitrarily far apart. There are various constructions of graphs that are triangle-free (have clique number 2) and still have arbitrarily large chromatic number. The ﬁrst one was given in 1949 by Zykov [16], and the one perhaps best known is due to Mycielski [11]. However, these classical constructions require a lot of freedom in connecting vertices by edges, and many important classes of graphs derived from speciﬁc (e.g. geometric) representations have chromatic number bounded in terms of the clique number. A class of graphs is called χ-bounded if there is a function f : N →N such that χ(G) ⩽f (ω(G)) holds for any graph G from the class. In this paper, we focus on the relation between the chromatic number and the clique number for geometric intersection graphs. The intersection graph of a family of sets F is the graph with vertex set F and edge set consisting of pairs of intersecting elements of F. We consider ﬁnite families F of arc-connected compact sets in the plane which are simple in the sense that the intersection of any subfamily of F is also arc-connected. We usually identify the family F with its intersection graph and use such terms as chromatic number, clique number or χ-boundedness referring directly to F. In the one-dimensional case of subsets of R, the only arc-connected compact sets are closed intervals. They deﬁne the class of interval graphs, which have chromatic number equal to their clique number. 123 1 Introduction The study of the chromatic number of intersection graphs of geometric objects in higher dimensions was initiated in the seminal paper of Asplund and Grünbaum [1], where they proved that the families of axis-aligned rectanglesinR2 areχ-bounded.Ontheotherhand,Burling[2]showedthatintersection graphs of axis-aligned boxes in R3 with clique number 2 can have arbitrarily large chromatic number. Since then, a lot of research focused on proving χ-boundedness of the families of geometric objects in the plane with various restrictions on the kind of objects considered, their positions, or the way they can intersect. Gyárfás [5,6] proved that the families of chords of a circle are χ-bounded. This was generalized by Kostochka and Kratochvíl [8] to the families of convex polygons inscribed in a circle. Kim et al. [7] showed that the families of homothetic (uniformly scaled) copies of a ﬁxed convex compact set in the plane are χ-bounded. Fox and Pach [3] showed that the intersection graphs of any arc-connected compact sets in the plane that do not contain a ﬁxed bipartite subgraph H have chromatic number bounded by a function of H. This easily implies that the families of pseudodiscs, that is, closed disc homeomorphs in the plane the boundaries of any two of which cross at most twice, are χ-bounded. Note that families of pseudodiscs are simple. The above-mentioned results of [7] and [3] are actually stronger—they state that the number of edges of the intersection graph of a respective family F is bounded by f (ω(F))\|F\| for some function f . A family of sets F is pierced by a line L if the intersection of any member of F with L is a nonempty segment. McGuinness [9] proved that the families of L-shapes (shapes consisting of a horizontal and a vertical segments of arbitrary lengths, forming the letter ‘L’) pierced by a ﬁxed vertical line are χ-bounded. Later [10], he showed that the triangle-free simple families of compact arc-connected sets in the plane pierced 123 Discrete Comput Geom (2014) 52:399–415 401 by a common line have bounded chromatic number. Suk [15] proved χ-boundedness of the simple families of x-monotone curves intersecting a ﬁxed vertical line. In this paper, we generalize the results of McGuinness, allowing any bound on the clique number, and of Suk, removing the x-monotonicity condition. by a common line have bounded chromatic number. 1 Introduction Suk [15] proved χ-boundedness of the simple families of x-monotone curves intersecting a ﬁxed vertical line. In this paper, we generalize the results of McGuinness, allowing any bound on the clique number, and of Suk, removing the x-monotonicity condition. Theorem 1 The class of simple families of compact arc-connected sets in the plane pierced by a common line is χ-bounded. By contrast, Pawlik et al. [12,13] proved that there are intersection graphs of straight-line segments (or geometric sets of many other kinds) with clique number 2 and arbitrarily large chromatic number. This justiﬁes the assumption of Theorem 1 that the sets are pierced by a common line. The best known upper bound on the chromatic number of simple families of curves in the plane with clique number ω is log n log ω O(log ω) due to Fox and Pach [4]. The bound on the chromatic number following from our proof of Theorem 1 is double exponential in terms of the clique number. The ultimate goal of this quest is to understand the border line between the classes of graphs (and classes of geometric objects) that are χ-bounded and those that are not. In a preliminary version of this paper, we proposed the following two problems. Problem 1 Are the families (not necessarily simple) of x-monotone curves in the plane pierced by a common vertical line χ-bounded? Problem 2 Are the families of curves in the plane pierced by a common line χ-bounded? Rok and Walczak [14] proved recently that the answers to both these questions are positive. However, the bound on the chromatic number in terms of the clique number resulting from their proof is enormous (greater than an exponential tower), which is much worse than the double exponential bound of Theorem 1. 2 Topological Preliminaries To obtain Ai, replace the part of Ai−1 between y1 and y2 by any arc between y1 and y2 in X ∩Yi (which exists because X ∩Yi is arc-connected). Clearly, Ai is simple with respect to Yi. Since X ∩Yi is disjoint from each of Y1, . . . , Yi−1, Ai remains simple with respect to Y1, . . . , Yi−1. ⊓⊔ ⊓⊔ A Jordan curve is the image of a continuous map φ : [0, 1] →R2 such that φ(0) = φ(1) and φ is injective on [0, 1). The famous Jordan curve theorem states that if C ⊂R2 is a Jordan curve, then R2∖C has exactly two arc-connected components, one bounded and one unbounded. An extension of this, called Jordan-Schönﬂies theorem, adds that there is a homeomorphism of R2 that maps C to a unit circle, the bounded arc-connected component of R2 ∖C to the interior of this circle, and the unbounded arc-connected component of R2 ∖C to the exterior of the circle. We will use a special case of the Jordan curve theorem for arcs in the closed upper halfplane R × [0, +∞). Namely, if x and y are two points on the horizontal axis R × {0} and A is an arc between x and y such that A ∖{x, y} ⊂R × (0, +∞), then the set (R × [0, +∞)) ∖A has exactly two arc-connected components, one bounded and one unbounded. This in particular implies that for any four points x1, x2, y1 and y2 in this order on the horizontal axis, every arc in R × [0, +∞) between x1 and y1 intersects every arc in R × [0, +∞) between x2 and y2. 2 Topological Preliminaries All the geometric sets that considered in this paper are assumed to be subsets of the Euclidean plane R2 or, further in the paper, subsets of the closed upper halfplane R × [0, +∞). An arc between points x, y ∈R2 is the image of a continuous injective map φ : [0, 1] →R2 such that φ(0) = x and φ(1) = y. A set X ⊂R2 is arc-connected if any two points of X are connected by an arc in X. The union of two arc-connected sets that have non-empty intersection is itself arc-connected. More generally, if X is a family of arc-connected sets whose intersection graph is connected, then X is arc-connected. For a set X ⊂R2, the relation {(x, y) ∈X2 : X contains an arc between x and y} is an equivalence, whose equivalence classes are the arc-connected components of X. Every arc-connected component of an open set is itself an open set. 2 All families of sets that we consider are ﬁnite. A family F of sets in R2 is simple if the intersection of any subfamily of F is arc-connected (possibly empty). A set X is simple with respect to a family Y if {X} ∪Y is simple. 12 3 402 Discrete Comput Geom (2014) 52:399–415 Lemma 2 Let X be a compact arc-connected set and Y be a family of compact arc- connected sets such that X is simple with respect to Y and the intersections of the members of Y with X are pairwise disjoint. Between any points x1, x2 ∈X, there is an arc A ⊂X that is simple with respect to Y. Proof Let Y = {Y1, . . . , Yn}. For i ∈{0, . . . , n}, we construct an arc Ai ⊂X between x1 and x2 that is simple with respect to {Y1, . . . , Yi}. As X is arc-connected, we pick A0 to be any arc between x1 and x2 within X. We construct Ai from Ai−1 as follows. If Ai−1∩Yi = ∅, then we take Ai = Ai−1. Otherwise, let y1 and y2 be respectively the ﬁrst and the last points on Ai−1 that belong to Yi (which exist as Ai ∩Yi is non-empty and compact). 3 Grounded Families In Theorem 1, a family F compact arc-connected sets in the plane is assumed to be pierced by a common line. We assume without loss of generality that this piercing line is the horizontal axis R × {0} and call it the baseline. The base of a set X, denoted by base(X), is the intersection of X with the baseline. We ﬁx a positive integer k and assume ω(F) ⩽k. The intersection graph of the bases of the members of F is an interval graph, so it can be properly colored with k colors. To ﬁnd a proper coloring of F with a number of colors bounded in terms of k, we can restrict our attention to one color class in the coloring of this interval graph. Therefore, without loss of generality, we assume that no two members of F intersect on the baseline and show that F can be colored properly with a bounded number of colors. Moreover, it is clear that the families F+ = {X ∩(R×[0, +∞)): X ∈F} and F−= {X ∩(R×(−∞, 0]): X ∈F} are simple. It sufﬁces to obtain proper colorings φ+ and φ−of F+ and F−, respectively, with bounded numbers of colors, since then F may be colored by pairs (φ+, φ−). We only focus on coloring F+, as F−can be 12 123 403 Discrete Comput Geom (2014) 52:399–415 Fig. 1 A grounded family of sets Fig. 1 A grounded family of sets handled by symmetry. To simplify notation, we rename F+ to F. Therefore, each se X ∈F is assumed to satisfy the following: handled by symmetry. To simplify notation, we rename F+ to F. Therefore, each set X ∈F is assumed to satisfy the following: • X ⊂R × [0, +∞), • X ∩R × {0} is a non-empty interval, • X is compact and arc-connected, and F is assumed to be simple. Any set that satisﬁes the conditions above is called grounded, and any simple family of grounded sets with pairwise disjoint bases is also called grounded (see Fig. 1). All the geometric sets that we consider from now on are contained in R×[0, +∞). To prove Theorem 1, it sufﬁces to show the following. Proposition 3 For k ⩾1, there is ξk such that χ(F) ⩽ξk holds for any grounded family F with ω(F) ⩽k. 3 Grounded Families The case k = 1 is trivial, and the case k = 2 with some additional assumptions meant to avoid topological pathologies was settled by McGuinness [10]. We write X ≺Y if base(X) is entirely to the left of base(Y). The relation ≺is a total order on a grounded family and naturally extends to its subfamilies (or any other families of grounded sets with pairwise disjoint bases): for example, X ≺Y denotes that X ≺Y for any Y ∈Y. For grounded sets X1 and X2 such that X1 ≺X2, we deﬁne F(X1, X2) = {Y ∈F : X1 ≺Y ≺X2}. For a grounded set X, we deﬁne F(−∞, X) = {Y ∈F : Y ≺X} and F(X, +∞) = {Y ∈F : X ≺Y}. The proof of Proposition 3 heavily depends on two decomposition lemmas, which given a grounded family with large chromatic number ﬁnd its subfamily with large chromatic number and some special properties. The ﬁrst one is a reformulation of Lemma 2.1 in [9]. Lemma 4 Let F be a grounded family with χ(F) > 2a(b + 1), where a, b ⩾0. There is a subfamily H of F that satisﬁes χ(H) > a and χ(F(H1, H2)) > b for any intersecting H1, H2 ∈H. Lemma 4 Let F be a grounded family with χ(F) > 2a(b + 1), where a, b ⩾0. There is a subfamily H of F that satisﬁes χ(H) > a and χ(F(H1, H2)) > b for any intersecting H1, H2 ∈H. Lemma 4 Let F be a grounded family with χ(F) > 2a(b + 1), where a, b ⩾0. There is a subfamily H of F that satisﬁes χ(H) > a and χ(F(H1, H2)) > b for any intersecting H1, H2 ∈H. Proof We partition F into subfamilies F0 ≺· · · ≺Fn so that χ(Fi) = b + 1 for 0 ⩽i < n. This can be done by adding sets to F0 in the increasing ≺-order until we get χ(F0) = b+1, then following the same procedure with the remaining sets to form F1, and so on. Let F0 = i F2i and F1 = i F2i+1. Since χ(F0∪F1) > 2a(b+1), Proof We partition F into subfamilies F0 ≺· · · ≺Fn so that χ(Fi) = b + 1 for 0 ⩽i < n. 3 Grounded Families This can be done by adding sets to F0 in the increasing ≺-order until we get χ(F0) = b+1, then following the same procedure with the remaining sets to form F1, and so on. Let F0 = i F2i and F1 = i F2i+1. Since χ(F0∪F1) > 2a(b+1), 12 12 3 3 404 Discrete Comput Geom (2014) 52:399–415 Fig. 2 An externally supported family of sets we have χ(Fk) > a(b + 1) for k = 0 or k = 1. We now color each F2i+k properly using the same set of b + 1 colors. This coloring induces a partitioning of the entire Fk into subfamilies H0, . . . , Hb such that for 0 ⩽i ⩽n, 0 ⩽j ⩽b the family Fi ∩H j is independent. We set H = H j, where H j has the maximum chromatic number among H0, . . . , Hb. Since χ(Fk) > a(b +1), we have χ(H) > a. It remains to show that χ(F(H1, H2)) > b for H1, H2 ∈H with H1 ∩H2 ̸= ∅. Indeed, such sets H1 and H2 must lie in different families F2i1+k and F2i2+k, respectively, so χ(F(H1, H2)) ⩾χ(F2i1+k+1) = b + 1 > b, as required. ⊓⊔ Fig. 2 An externally supported family of sets Fig. 2 An externally supported family of sets we have χ(Fk) > a(b + 1) for k = 0 or k = 1. We now color each F2i+k properly using the same set of b + 1 colors. This coloring induces a partitioning of the entire Fk into subfamilies H0, . . . , Hb such that for 0 ⩽i ⩽n, 0 ⩽j ⩽b the family Fi ∩H j is independent. We set H = H j, where H j has the maximum chromatic number among H0, . . . , Hb. Since χ(Fk) > a(b +1), we have χ(H) > a. It remains to show that χ(F(H1, H2)) > b for H1, H2 ∈H with H1 ∩H2 ̸= ∅. Indeed, such sets H1 and H2 must lie in different families F2i1+k and F2i2+k, respectively, so χ(F(H1, H2)) ⩾χ(F2i1+k+1) = b + 1 > b, as required. ⊓⊔ ⊓⊔ For a set X, we deﬁne ext(X) to be the only unbounded arc-connected component of (R × [0, +∞)) ∖X. For a grounded family F, we deﬁne ext(F) = ext( F). 3 Grounded Families A subfamily G of a grounded family F is externally supported in F if for any X ∈G there exists Y ∈F such that Y ∩X ̸= ∅and Y ∩ext(G) ̸= ∅(see Fig. 2). The idea behind the following lemma is due to Gyárfás [5] and was subsequently used in [9,10,15]. Lemma 5 Let F be a grounded family with χ(F) > 2a, where a ⩾1. There is a subfamily G of F that is externally supported in F and satisﬁes χ(G) > a. Proof For convenience, we restrict F to its connected component with maximum chromatic number. Let X0 be the ≺-least member of F. For i ⩾0, let Fi be the family of members of F that are at distance i from X0 in the intersection graph of F. It follows that F0 = {X0} and, for \|i −j\| > 1, each member of Fi is disjoint from each member of F j. Clearly, χ( i F2i) > a or χ( i F2i+1) > a, and therefore there is d ⩾1 with χ(Fd) > a. We claim that Fd is externally supported in F. Fix Xd ∈Fd, and let X0, . . . , Xd be a shortest path from X0 to Xd in the intersection graph of F. Since X0 ∩ext(Fd) ̸= ∅and X0, . . . , Xd−2 are disjoint from Fd, we have X0, . . . , Xd−2 ⊂ext(Fd). Thus Xd−1 ∩ext(Fd) ̸= ∅and Xd−1 ∩Xd ̸= ∅. ⊓⊔ 4 Cliques and Brackets Let F be a grounded family with ω(F) ⩽k. A k-clique in F is a family of k pairwise intersecting members of F. For a k-clique K, we denote by int(K) the only arc- connected component of (R × [0, +∞)) ∖ K containing the part of the baseline 123 Discrete Comput Geom (2014) 52:399–415 405 S int( ) int( ) K B K Fig. 3 A k-bracket B with k-clique K and support S betweenthetwo≺-least members of K. Ak-bracket inF is asubfamilyofF consisting of a k-clique K and a set S called the support such that S ≺K or K ≺S and S ∩int(K) ̸= ∅. For such a k-bracket B, we denote by int(B) the only arc-connected component of (R × [0, +∞)) ∖ B containing the part of the baseline between S and K (see Fig. 3). S int( ) int( ) K B K Fig. 3 A k-bracket B with k-clique K and support S Fig. 3 A k-bracket B with k-clique K and support S betweenthetwo≺-least members of K. Ak-bracket inF is asubfamilyofF consisting of a k-clique K and a set S called the support such that S ≺K or K ≺S and S ∩int(K) ̸= ∅. For such a k-bracket B, we denote by int(B) the only arc-connected component of (R × [0, +∞)) ∖ B containing the part of the baseline between S and K (see Fig. 3). betweenthetwo≺-least members of K. Ak-bracket inF is asubfamilyofF consisting of a k-clique K and a set S called the support such that S ≺K or K ≺S and S ∩int(K) ̸= ∅. For such a k-bracket B, we denote by int(B) the only arc-connected component of (R × [0, +∞)) ∖ B containing the part of the baseline between S and K (see Fig. 3). Lemma 6 Let F be a grounded family, X, Y, Z ∈F, and X ∩Y ̸= ∅. Let C1 and C2 be any two distinct arc-connected components of (R × [0, +∞)) ∖(X ∪Y). Let z, z′ ∈Z ∩C1. If every arc between z and z′ within Z intersects C2, then every such arc intersects both X and Y. Lemma 6 Let F be a grounded family, X, Y, Z ∈F, and X ∩Y ̸= ∅. Let C1 and C2 be any two distinct arc-connected components of (R × [0, +∞)) ∖(X ∪Y). Let z, z′ ∈Z ∩C1. 4 Cliques and Brackets If every arc between z and z′ within Z intersects C2, then every such arc intersects both X and Y. Proof Suppose there is an arc A ⊂Z between z and z′ such that A ∩X = ∅or A ∩Y = ∅. If A ∩X = ∅and A ∩Y = ∅, then A ⊂C1, so A ∩C2 = ∅. Now, suppose A ∩X = ∅and A ∩Y ̸= ∅. Let y and y′ be respectively the ﬁrst and last points of A in Y. Since Z ∩Y is arc-connected, there is an arc A′ in Z that is simple with respect to Y and goes along A from z to y, then to y′ inside Y, and ﬁnally along A to z′. It follows that A′ ⊂C1 ∪Y, so A′ ∩C2 = ∅. The case that A ∩X ̸= ∅and A ∩Y = ∅ is symmetric. ⊓⊔ Corollary 7 Let F be a grounded family, K be a k-clique in F, and X ∈F. If x, y ∈X ∩int(K) (or x, y ∈X ∩ext(K)) and every arc between x and y within X intersects ext(K) (int(K), respectively), then X intersects every member of K. Proof Let K = {K1, . . . , Kk} and K1 ≺int(K) ≺K2 ≺· · · ≺Kk. The statement follows directly from Lemma 6 and the fact that int(K) and ext(K) belong to distinct arc-connected components of (R × [0, +∞)) ∖(K1 ∪Ki) for 2 ⩽i ⩽k. ⊓⊔ Corollary 8 Let F be a grounded family and B be a bracket in F with clique K and support S. Let X ∈F. If X ∩int(B) ̸= ∅and X ∩ext(B) ̸= ∅, then X intersects S or every member of K. Proof Let x ∈X ∩int(B) and x′ ∈X ∩ext(B), and suppose X ∩S = ∅. By the Jordan curve theorem, every arc between x and x′ within X must intersect S ∪int(K) and thus int(K). Since x, x′ ∈X ∩ext(K), it follows from Corollary 7 that X intersects every member of K. ⊓⊔ 12 3 Discrete Comput Geom (2014) 52:399–415 406 5 Proof of Proposition 3 The proof goes by induction on k. Proposition 3 holds trivially for k = 1 with ξ1 = 1. Therefore, we assume that k ⩾2 and that the statement of the proposition holds for k −1. This context of the induction step is maintained throughout the entire remaining part of the paper. A typical application of the induction hypothesis looks as follows: if F is a grounded family with ω(F) ⩽k, G ⊂F, and there is X ∈F ∖G intersecting all members of G, then ω(G) ⩽k −1 and thus χ(G) ⩽ξk−1. , ( ) ⩽ χ( ) ⩽ξk 1 Deﬁne βk = 8kξ2 k−1, δk,k = 0, δk, j = βk + 2δk, j+1 + 2ξk−1(kξk−1 + k + 2) + for k −1 ⩾j ⩾0, and ﬁnally ξk = 2k+2(δk,0 + 2ξk−1 + 1). ( ) χ( ) ξk 1 Deﬁne βk = 8kξ2 k−1, δk,k = 0, δk, j = βk + 2δk, j+1 + 2ξk−1(kξk−1 + k + 2) + 2 for k −1 ⩾j ⩾0, and ﬁnally ξk = 2k+2(δk,0 + 2ξk−1 + 1). , We say that a grounded set X (a grounded family X) is surrounded by a set S if X (every member of X, respectively) is disjoint from S ∪ext(S). For a set S and a grounded set R such that base(R) is surrounded by S, let cut(R, S) denote the closure of the unique arc-connected component of R ∖S containing base(R). For a set S and a grounded family R of sets whose bases are surrounded by S, let cut(R, S) = {cut(R, S): R ∈R}. First, we present a technical lemma, which generalizes similar statements from [10] (Lemma 3.2) and [15] (Lemma 4.1), and which we will prove in Sect. 6. Loosely speaking, it says that one can color properly, with the number of colors bounded in terms of k, all the members of F surrounded by a set S which intersect cut(R, S) for any set R ∈F intersecting S. Lemma 9 Let S be a compact set and R∪D be a grounded family with the following properties: • the base of every member of R is surrounded by S, • every member of R intersects S, • D is surrounded by S, • every member of D intersects cut(R, S), • ω(R ∪D) ⩽k. (iv) every member of F intersecting ext(S) and some member of G also intersects each of S1, . . . , Sj. (iii) the sets S1, . . . , Sj pairwise intersect, It follows that χ(D) ⩽βk. Since the chromatic number of a graph is the maximum chromatic number of its connected component, there is G′′ ⊂G′ ∖D such that the intersection graph of G′′ is connected and χ(G′′) > 2δk, j +2ξk−1(kξk−1+k +2)+2. Partition G′′ into three subfamilies X, Y, Z so that X ≺Y ≺Z and χ(X) = χ(Z) = δk, j +(k+1)ξk−1+1. It follows that χ(Y) > 2ξk−1(kξk−1 + 1). Apply Lemma 4 to ﬁnd H ⊂Y such that χ(H) > ξk−1 and for any intersecting H1, H2 ∈H we have χ(Y(H1, H2)) > kξk−1. Since χ(H) > ξk−1, there is a k-clique K ⊂H. The members of Y intersecting K have chromatic number at most kξk−1, so there is P ∈Y that is contained in int(K). Since F j−1 is externally supported in F j, there is Sj ∈F j such that Sj ∩P ̸= ∅ and Sj ∩ext(S′) ⊃Sj ∩ext(F j−1) ̸= ∅. Therefore, since S′ and G′ satisfy (iv), Sj intersects each of S1, . . . , Sj−1 and thus (iii) holds for S1, . . . , Sj. We show that Sj ≺G′′ or G′′ ≺Sj. Suppose that neither of these holds. It follows that base(Sj) is surrounded by S′, which yields Sj ∈R. Moreover, base(Sj) is surrounded by G′′, as the intersection graph of G′′ is connected. Therefore, we have cut(Sj, S′) ∩ G′′ ̸= ∅, so there is X ∈G′′ such that X ∩cut(Sj, S′) ̸= ∅. This means that X ∈D, which contradicts the deﬁnition of G′′. Now, we have Sj ∩int(K) ⊃Sj ∩P ̸= ∅and Sj ≺K or K ≺Sj, so the k-clique K and the support Sj form a k-bracket. Let S = S′ ∪K ∪{Sj}. If Sj ≺G′′, then Sj ≺X ≺K. In this case, let G be the family of those members of X that are disjoint from K ∪Sj. It is clear that (i) holds. Since χ(X) > δk, j + (k + 1)ξk−1 and the members of X intersecting K ∪Sj have chromatic number at most (k + 1)ξk−1, we have χ(G) > δk, j, so (ii) holds. Since ext(S) ⊂ext(K ∪{Sj}), it follows from Corollary 8 that every member of F intersecting ext(S) and some member of G intersects Sj. Hence (iv) holds. It follows that χ(D) ⩽βk. Suppose for the sake of contradiction that there is a grounded family F with ω(F) ⩽k and χ(F) > ξk = 2k+2(δk,0 + 2ξk−1 + 1). A repeated application of Lemma 5 yields a sequence of families F = Fk+1 ⊃Fk ⊃· · · ⊃F0 such that Fi is externally supported in Fi+1 and χ(Fi) > 2i+1(δk,0 + 2ξk−1 + 1), for 0 ⩽i ⩽k. The following claim is the core of the proof. Claim 10 For 0 ⩽j ⩽k, there are families S, G ⊂F j and sets S1, . . . , Sj ∈S with the following properties: Claim 10 For 0 ⩽j ⩽k, there are families S, G ⊂F j and sets S1, . . . , Sj ∈S with the following properties: (i) G is surrounded by S, (iii) the sets S1, . . . , Sj pairwise intersect, (iv) every member of F intersecting ext(S) and some member of G also intersects each of S1, . . . , Sj. 123 407 Discrete Comput Geom (2014) 52:399–415 Proof The proof goes by induction on j. First, let j = 0. Apply Lemma 4 to ﬁnd H ⊂F0 such that χ(H) > 1 and for any intersecting H1, H2 ∈H we have χ(F0(H1, H2)) > δk,0 + 2ξk−1. Since χ(H) > 1, such two intersecting H1, H2 ∈H exist. Let S = {H1, H2} and G be the family of those members of F0(H1, H2) that are disjoint from H1 ∪H2. It is clear that (i) holds. Since the members of F0(H1, H2) intersecting H1 ∪H2 have chromatic number at most 2ξk−1, we have χ(G) > δk,0, so (ii) holds. The conditions (iii) and (iv) are satisﬁed vacuously. Now, assume that j ⩾1 and the claim holds for j −1, that is, there are families S′, G′ ⊂F j−1 and sets S1, . . . , Sj−1 ∈S′ satisfying (i)–(iv). Let R = R ∈F : base(R) is surrounded by S′ and R ∩ S′ ̸= ∅ , D = D ∈G′ : D ∩ cut(R, S′) ̸= ∅ . It follows from Lemma 9 that χ(D) ⩽βk and thus χ(G′∖D) > 2δk, j +2ξk−1(kξk−1+ k +2)+2. Claim 11 χ(cut(R, S′)) ⩽k. Claim 11 χ(cut(R, S′)) ⩽k. Proof Consider a relation < on cut(R, S′) deﬁned as follows: R′ 1 < R′ 2 if and only if R′ 1 ≺R′ 2 and R′ 1 ∩R′ 2 = ∅. It is clear that < is irreﬂexive and antisymmetric. It is also transitive, which follows from the fact that if R′ 1, R′ 2, R′ 3 ∈R, R′ 1 ≺R′ 2 ≺R′ 3, and R′ 1 ∩R′ 3 ̸= ∅, then R′ 2 ∩(R′ 1 ∪R′ 3) ̸= ∅. Therefore, < is a strict partial order. The intersection graph of cut(R, S′) is the incomparability graph of <, so it is perfect, which implies χ(cut(R, S′)) = ω(cut(R, S′)) ⩽k. ⊓⊔ Proof Consider a relation < on cut(R, S′) deﬁned as follows: R′ 1 < R′ 2 if and only if R′ 1 ≺R′ 2 and R′ 1 ∩R′ 2 = ∅. It is clear that < is irreﬂexive and antisymmetric. It is also transitive, which follows from the fact that if R′ 1, R′ 2, R′ 3 ∈R, R′ 1 ≺R′ 2 ≺R′ 3, and R′ 1 ∩R′ 3 ̸= ∅, then R′ 2 ∩(R′ 1 ∪R′ 3) ̸= ∅. Therefore, < is a strict partial order. The intersection graph of cut(R, S′) is the incomparability graph of <, so it is perfect, which implies χ(cut(R, S′)) = ω(cut(R, S′)) ⩽k. ⊓⊔ ⊓⊔ By Claim 11, there is a coloring φ of R with k colors such that for any R1, R2 ∈R with φ(R1) = φ(R2), we have cut(R1, S′) ∩cut(R2, S′) = ∅. For a color c, let Rc = {R ∈R: φ(R) = c} and Dc = {D ∈D: D ∩ cut(Rc, S′) ̸= ∅}. We are going to show that χ(Dc) ⩽8ξ2 k−1. Once this is obtained, we will have χ(D) ⩽ c χ(Dc) ⩽8kξ2 k−1 = βk. Since the sets cut(R, S′) for R ∈Rc are pairwise disjoint, the curve S′ and the families cut(Rc, S′) and Dc satisfy the assumptions of Lemma 9. To simplify the notation, we assume for the remainder of the proof that S = S′, R = cut(R, S′) for every R ∈Rc, R = cut(Rc, S′), and D = Dc. By Jordan-Schönﬂies theorem, the segment pq and the arc S form a Jordan curve which is the boundary of a set J homeomorphic to a closed disc. It follows that χ(D) ⩽βk. If G′′ ≺Sj, then let G be the family of those members of Z that are disjoint from K ∪Sj. An analogous argument shows that (i), (ii), and (iv) are satisﬁed. ⊓⊔ ⊓⊔ Let S, G, and S1, . . . , Sk be as guaranteed by Claim 10 for j = k. By (ii), we have χ(G) > 0, so there is P ∈G. Since Fk is externally supported in F, there is Sk+1 ∈F such that Sk+1 ∩P ̸= ∅and Sk+1 ∩ext(S) ̸= ∅. By (iii) and (iv), we conclude that S1, . . . , Sk+1 pairwise intersect. This contradicts the assumption that ω(F) ⩽k, thus completing the proof of Proposition 3. Let S, G, and S1, . . . , Sk be as guaranteed by Claim 10 for j = k. By (ii), we have χ(G) > 0, so there is P ∈G. Since Fk is externally supported in F, there is Sk+1 ∈F such that Sk+1 ∩P ̸= ∅and Sk+1 ∩ext(S) ̸= ∅. By (iii) and (iv), we conclude that S1, . . . , Sk+1 pairwise intersect. This contradicts the assumption that ω(F) ⩽k, thus completing the proof of Proposition 3. 12 3 408 Discrete Comput Geom (2014) 52:399–415 p q Fig. 4 The setting of the proof of Lemma 9: the set S with a dashed arc S′ and three sets from R Fig. 4 The setting of the proof of Lemma 9: the set S with a dashed arc S′ and three sets from R 6 Proof of Lemma 9 The proof of Lemma 9 goes along similar lines to the proof of Lemma 4.1 in [15]. Since D is surrounded by S, there is an arc S′ ⊂S such that D is surrounded by S′. We can assume without loss of generality that base(S′) = {p, q} for some points p and q on the baseline such that p ≺D ≺q. For every R ∈R we have cut(R, S) ⊂cut(R, S′). Hence every member of D intersects cut(R, S′) (see Fig. 4). • ω(R ∪D) ⩽k. Claim 11 χ(cut(R, S′)) ⩽k. In this new setting, S is an arc and R∪D is a grounded family with the following properties: • the base of every member of R is surrounded by S, • the base of every member of R is surrounded by S, • every member of R is contained in J and intersects S, • the members of R are pairwise disjoint, • D is surrounded by S, • every member of D intersects R, 123 409 Discrete Comput Geom (2014) 52:399–415 We enumerate the members of R as R1, . . . , Rm in the ≺-order, that is, so that R1 ≺ · · · ≺Rm. We are going to show that χ(D) ⩽8ξ2 k−1. Claim 12 For 1 ⩽i < j ⩽m, any arc in J between Ri and R j intersects all Ri+1, . . . , R j−1. Proof Let A be an arc in J between points xi ∈Ri and x j ∈R j. For any R ∈ {Ri+1, . . . , R j−1}, base(R) is surrounded by Ri ∪A ∪R j and we have R ∩(Ri ∪A ∪ R ) ̸ ∅ R ∩S ̸ ∅Si R i di j i t f R d R h R ∩A ̸ ∅ ⊓⊔ 1, . . . , R j−1}, base(R) is surrounded by Ri ∪A ∪R j and we have R ∩(Ri ∪A ∪ = ∅, as R ∩S ̸= ∅. Since R is disjoint from Ri and R j, we have R ∩A ̸= ∅. ⊓⊔ j j R j) ̸= ∅, as R ∩S ̸= ∅. Since R is disjoint from Ri and R j, we have R ∩A ̸= ∅. A point x ∈J is a neighbor of Ri if there is an arc in J between x and Ri disjoint from all R1, . . . , Rm except Ri. It follows from Claim 12 that each point in J is a neighbor of at most two consecutive sets of R1, . . . , Rm. For 1 ⩽i < m, let Ii denote the set of points in J that are neighbors of Ri and Ri+1. Claim 13 Any arc-connected subset of J intersects an interval of sets in the sequence R1, I1, R2, . . . , Im−1, Rm. Proof Let X be an arc-connected subset of J. Claim 11 χ(cut(R, S′)) ⩽k. First, we show that if X intersects Ri and Ri+1, then it also intersects Ii. This is guaranteed by the compactness of Ri and Ri+1. Indeed, take a ⊂-minimal arc in J between Ri and Ri+1. By Claim 12, the interior of this arc is disjoint from all R1, . . . , Rm and therefore must lie in Ii. Now, let i be the least index such that X ∩(Ri ∪Ii) ̸= ∅and j be the greatest index such that X ∩(I j−1 ∪R j) ̸= ∅. Let A be an arc in X between points xi ∈X ∩(Ri ∪Ii) and x j ∈X ∩(I j−1 ∪R j). Since xi is a neighbor of Ri, there is an arc Ai ⊂J between xi and Ri disjoint from all R1, . . . , Rm except Ri. Similarly, since x j is a neighbor of R j, there is an arc A j ⊂J between x j and R j disjoint from all R1, . . . , Rm except R j. There is an arc ¯A ⊂Ai ∪A ∪A j between Ri and R j. By Claim 12, ¯A intersects all Ri+1, . . . , R j−1. But Ri+1, . . . , R j−1 are disjoint from Ai and A j, hence they intersect A. ⊓⊔ For convenience, deﬁne I0 = Im = ∅. For D ∈D, deﬁne leftclip(D) = D ∖Ii and rightclip(D)= D∖I j, wherei and j are chosen so that Ri+1 is the ﬁrst and R j is the last member of R intersecting D (see Fig. 5). This deﬁnition extends to families M ⊂D: leftclip(M)={leftclip(M): M ∈M} and rightclip(M)={rightclip(M): M ∈M}. For convenience, deﬁne I0 = Im = ∅. For D ∈D, deﬁne leftclip(D) = D ∖Ii and rightclip(D)= D∖I j, wherei and j are chosen so that Ri+1 is the ﬁrst and R j is the last member of R intersecting D (see Fig. 5). This deﬁnition extends to families M ⊂D: leftclip(M)={leftclip(M): M ∈M} and rightclip(M)={rightclip(M): M ∈M}. Claim 14 leftclip(D) and rightclip(D) are simple families of compact arc-connected sets. Proof We present the proof only for leftclip(D), as for rightclip(D) it is analogous. The sets I1, . . . , Im−1 are open in J, as they are arc-connected components of the set J ∖ R, which is open in J. Claim 11 χ(cut(R, S′)) ⩽k. Each member of leftclip(D) is a difference of a compact set in D and one of I1, . . . , Im−1 and thus is compact as well. Proof We present the proof only for leftclip(D), as for rightclip(D) it is analogous. The sets I1, . . . , Im−1 are open in J, as they are arc-connected components of the set J ∖ R, which is open in J. Each member of leftclip(D) is a difference of a compact set in D and one of I1, . . . , Im−1 and thus is compact as well. To prove that leftclip(D) is simple and consists of arc-connected sets, we need to show that leftclip(M) is arc-connected for any M ⊂ D. Let x, y ∈ leftclip(M) ⊂ M. Since M is arc-connected, Lemma 2 provides us with an arc A ⊂ M between x and y that is simple with respect to R. It suf- ﬁces to show A ⊂leftclip(M) for each M ∈M. To this end, ﬁx M ∈M 12 3 410 Discrete Comput Geom (2014) 52:399–415 q p R I R I R I R I R Fig. 5 A dashed arc S, sets from R spanned between the baseline and S, and sets from D with D ∖ leftclip(D) marked for each D ∈D Fig. 5 A dashed arc S, sets from R spanned between the baseline and S, and sets from D with D leftclip(D) marked for each D ∈D and let Ri be the ≺-least member of R intersecting M. Suppose there is a point z ∈A ∩(M ∖leftclip(M)) ⊂Ii−1. Since x, y ∈leftclip(M) ⊂m j=i(R j ∪I j), it follows from Claim 13 that the parts of A from x to z and from z to y intersect Ri. This and z /∈Ri contradict the simplicity of A with respect to Ri. ⊓⊔ Claim 15 leftclip(D) and rightclip(D) have clique number at most k −1. Proof Again,wepresenttheproofonlyforleftclip(D).LetK beacliqueinleftclip(D). By Claim 14, each member of K is arc-connected. Therefore, by Claim 13, each mem- ber of K intersects an interval of sets in the sequence R1, I1, . . . , Rm−1, Im−1, Rm, the ﬁrst set in this interval being of the form R j. Since the members of K pairwise intersect, these intervals also pairwise intersect, which implies that they all contain a common R j. Claim 11 χ(cut(R, S′)) ⩽k. , Jm, each homeomorphic to a closed disc, so that Ji−1 ∩Ji = Qi for 1 ⩽i ⩽m. It is clear that each arc-connected subset of J intersects an interval of sets in the sequence J0, Q1, J1, . . . , Qm, Jm. Since Q1, . . . , Qm are simple with respect to M, so are J0, . . . , Jm. S as moving from p to q. Moreover, the arcs Q1, . . . , Qm partition J into m + 1 sets J0, . . . , Jm, each homeomorphic to a closed disc, so that Ji−1 ∩Ji = Qi for 1 ⩽i ⩽m. It is clear that each arc-connected subset of J intersects an interval of sets in the sequence J0, Q1, J1, . . . , Qm, Jm. Since Q1, . . . , Qm are simple with respect to M, so are J0, . . . , Jm. Since the sets J0, . . . , Jm are homeomorphic to a closed disc and so are rectangles with bottom sides base(J0), . . . , base(Jm), there are homeomorphisms μ0, . . . , μm such that • μi is constant on base(Ji) and maps Ji onto a rectangle with bottom side base(Ji) for 0 ⩽i ⩽m, • μi is constant on base(Ji) and maps Ji onto a rectangle with bottom side base(Ji) for 0 ⩽i ⩽m, • μi−1 and μi agree on Qi for 1 ⩽i ⩽m. Thus μ0 ∪· · · ∪μm is a homeomorphism between J and a rectangle with bottom side base(J), and it extends to a homeomorphism μ of R × [0, +∞) whose restriction to each Ji is μi. Let τ0, . . . , τm be horizontal translations such that τ0(u) ≺· · · ≺ τm(u) for a point u on the baseline. Let x1, . . . , xm be the x-coordinates of the points u1, . . . , um, respectively, so that ui = (xi, 0) for 1 ⩽i ⩽m. Deﬁne • ˆJ0 = μ−1((−∞, x1] × [0, +∞)), • ˆJi = μ−1([xi, xi+1] × [0, +∞)) for 1 ⩽i < m, • ˆJm = μ−1([xm, +∞) × [0, +∞)), • ˆQi = μ−1({xi} × [0, +∞)) for 1 ⩽i ⩽m, • σi = τi ◦μ for 0 ⩽i ⩽m. Note that Ji ⊂ˆJi and Qi ⊂ˆQi. Claim 11 χ(cut(R, S′)) ⩽k. Thus K ∪{R j} is a clique, and ω(R ∪D) ⩽k yields \|K\| ⩽k −1. ⊓⊔ Recall that we need to show χ(D) ⩽8ξ2 k−1. Deﬁne Recall that we need to show χ(D) ⩽8ξ2 k−1. Deﬁne • DL = {X ∈D: base(leftclip(X)) ̸= ∅}, DR {X ∈D: base(rightclip(X)) ̸ ∅} • DR = {X ∈D: base(rightclip(X)) ̸= ∅}. Since each member of D intersects at least one of R1, . . . , Rm, we have DL ∪DR = D. Therefore, it is enough to show that χ(DL) ⩽4ξ2 k−1 and χ(DR) ⩽4ξ2 k−1. We only present the proof of χ(DR) ⩽4ξ2 k−1, as the proof of the other inequality is analogous. Since each member of D intersects at least one of R1, . . . , Rm, we have DL ∪DR = D. Therefore, it is enough to show that χ(DL) ⩽4ξ2 k−1 and χ(DR) ⩽4ξ2 k−1. We only present the proof of χ(DR) ⩽4ξ2 k−1, as the proof of the other inequality is analogous. By Claims 14 and 15, rightclip(DR) is a grounded family with clique number at most k−1. This and the induction hypothesis yield χ(rightclip(DR)) ⩽ξk−1. We ﬁx a coloring φR of DR with ξk−1 colors so that φR(X) ̸= φR(Y) for any X, Y ∈DR with rightclip(X) ∩rightclip(Y) ̸= ∅. Let M ⊂DR be a family of sets having the same color in φR. In particular, we have rightclip(X)∩rightclip(Y) = ∅for any X, Y ∈M. It remains to prove that χ(M) ⩽4ξk−1. We show how to construct a coloring φL of M with ξk−1 colors such that leftclip(X)∩leftclip(Y) = ∅for any X, Y ∈M with φL(X) = φL(Y). We exploit the fact that members of M have pairwise disjoint intersections with each Ri to simplify the topology of M and R1, . . . , Rm. Recall that S is an arc with base(S) = {p, q}. For 1 ⩽i ⩽m, by Lemma 2, there is an arc Qi ⊂Ri between base(Ri) and Ri ∩S that is simple with respect to M. We assume without loss of generality that base(Qi) = {ui} and Qi ∩S = {vi}. The points v1, . . . , vm occur in this order on 123 12 411 Discrete Comput Geom (2014) 52:399–415 S as moving from p to q. Moreover, the arcs Q1, . . . , Qm partition J into m + 1 sets J0, . . . • Qi ⊂Ri for 1 ⩽i ⩽m, Claim 11 χ(cut(R, S′)) ⩽k. For a set X, deﬁne X⋆= σ0(X ∩ˆJ0) ∪[σ0(X ∩ˆQ1), σ1(X ∩ˆQ1)] ∪σ1(X ∩ˆJ1) ∪· · · ∪[σm−1(X ∩ˆQm), σm(X ∩ˆQm)] ∪σm(X ∩ˆJm) where [Y, Z] denotes the rectangle with left side Y and right side Z. It is clear that the map X →X⋆preserves compactness and arc-connectedness and is compatible with unions and intersections. In particular, M⋆= {X⋆: X ∈M} is a grounded family with intersection graph isomorphic to that of M (see Fig. 6). where [Y, Z] denotes the rectangle with left side Y and right side Z. It is clear that the map X →X⋆preserves compactness and arc-connectedness and is compatible with unions and intersections. In particular, M⋆= {X⋆: X ∈M} is a grounded family with intersection graph isomorphic to that of M (see Fig. 6). In the remainder of the proof, we will deal with M⋆and R⋆ 1, . . . , R⋆ m, but for sim- plicity we relabel them to M and R1, . . . , Rm, respectively. We also relabel I ⋆ 0, . . . , I ⋆ m to I0, . . . , Im, Q⋆ 1, . . . , Q⋆ m to Q1, . . . , Qm, S⋆to S, J ⋆to J, and σ0(J0), . . . , σm(Jm) to J0, . . . , Jm. The following properties clearly follow: • J0, . . . , Jm are pairwise disjoint rectangles, • Qi is a rectangle whose left side is the right side of Ji−1 and whose right side is the left side of Ji, for 1 ⩽i ⩽m, • every arc-connected subset of J intersects an interval of sets in the sequence J0, Q1, J1, . . . , Qm, Jm, • Qi ⊂Ri for 1 ⩽i ⩽m • every arc-connected subset of J intersects an interval of sets in the sequence J0, Q1, J1, . . . , Qm, Jm, • Qi ⊂Ri for 1 ⩽i ⩽m, • the intersection of any member of M with any Ji is arc-connected, • the intersection of any member of M with any Qi is a rectangle or horizontal segment spanning the entire width of Qi. Claim 11 χ(cut(R, S′)) ⩽k. 12 3 3 412 Discrete Comput Geom (2014) 52:399–415 p u u u q 3 2 2 3 1 1 σ ( p) σ (u ) σ (u ) σ (u ) σ (u ) σ (u ) σ (u ) σ (q) Fig. 6 The transformation X →X⋆; top: a family M before transformation; bottom: the families M⋆ (including the marked regions) and M+ (excluding the marked regions), and connections of the sets in M⋆to the baseline p u u u q 3 2 2 3 1 1 3 p u u u q 2 3 1 σ ( p) σ (u ) σ (u ) σ (u ) σ (u ) σ (u ) σ (u ) σ (q) Fig. 6 The transformation X →X⋆; top: a family M before transformation; bottom: the families M (including the marked regions) and M+ (excluding the marked regions) and connections of the sets i σ ( p) σ (u ) σ (u ) σ (u ) σ (u ) σ (u ) σ (u ) σ (q) Fig. 6 The transformation X →X⋆; top: a family M before transformation; bottom: the families M (including the marked regions) and M+ (excluding the marked regions) and connections of the set Fig. 6 The transformation X →X⋆; top: a family M before transformation; bottom: the families M⋆ (including the marked regions) and M+ (excluding the marked regions), and connections of the sets in M⋆to the baseline 123 Claim 16 There is a grounded family M′ with intersection graph isomorphic to that of leftclip(M). Doing so for all 413 Discrete Comput Geom (2014) 52:399–415 i with 1 ⩽i < m, we transform M+ into a grounded family M′ with intersection graph isomorphic to the intersection graph of M+ and hence of leftclip(M). ⊓⊔ i with 1 ⩽i < m, we transform M+ into a grounded family M′ with intersection graph isomorphic to the intersection graph of M+ and hence of leftclip(M). ⊓⊔ Claim 16 allows us to use the induction hypothesis on leftclip(M) to obtain a coloring φL of M with ξk−1 colors such that leftclip(X) ∩leftclip(Y) = ∅for any X, Y ∈M with φL(X) = φL(Y). Let N ⊂M be a family of sets having the same color in φL. In particular, we have leftclip(X) ∩leftclip(Y) = ∅and rightclip(X) ∩ rightclip(Y) = ∅for any X, Y ∈N. The following claim completes the proof of Lemma 9. The planarity argument used in the proof applies the ideas of McGuinness [10]. Claim 16 There is a grounded family M′ with intersection graph isomorphic to that of leftclip(M). Claim 16 There is a grounded family M′ with intersection graph isomorphic to that of leftclip(M). Proof Let Mi = {X ∈M: X ∩Ri ̸= ∅} for 1 ⩽i ⩽m. It follows that leftclip(X) = X ∖Ii for every X ∈Mi+1 ∖Mi and 1 ⩽i < m. We can assume without loss of generality that each member of Mi+1 ∖Mi intersects Qi+1 for 1 ⩽i < m, as those that do not are isolated vertices in the intersection graph of leftclip(M) and thus do not inﬂuence the existence of M′. For X ∈Mi+1∖Mi and1 ⩽i < m,let X+ denotethepartof X totherightof Qi+1 including the right side of Qi+1, that is, X+ = X∩(Ji+1∪Qi+2∪Ji+2∪· · ·∪Qm∪Jm). It follows that X+ ⊂leftclip(X) ⊂X+ ∪Ri+1 and X+ is compact and arc-connected. For X ∈M1, let X+ = X = leftclip(X). Let M+ = {X+ : X ∈M}. Since the intersections of the members of leftclip(M) with each Ri+1 are pairwise disjoint, the intersection graphs of M+ and leftclip(M) are isomorphic. We show how to extend the sets X+ for which base(X+) = base(leftclip(X)) = ∅to connect them to the baseline without creating any new intersections (see Fig. 6). Let 1 ⩽i < m, X ∈Mi+1 ∖Mi, and base(X+) = ∅. Thus base(X) ⊂base(Ji). For every Y ∈Mi, it is an immediate consequnce of the Jordan curve theorem and arc-connectedness of X ∩Ji and (Y ∪Ri) ∩Ji that Y ∩Qi+1 is empty or lies above X ∩Qi+1. Therefore, we can connect X+ to base(Qi+1) by an arc inside Qi+1 that is disjoint from any other Y + ∈M+. Moreover, all these arcs for X ∈Mi+1 ∖Mi with base(X+) = ∅can be drawn so that they are pairwise disjoint. Doing so for all Let 1 ⩽i < m, X ∈Mi+1 ∖Mi, and base(X+) = ∅. Thus base(X) ⊂base(Ji). For every Y ∈Mi, it is an immediate consequnce of the Jordan curve theorem and arc-connectedness of X ∩Ji and (Y ∪Ri) ∩Ji that Y ∩Qi+1 is empty or lies above X ∩Qi+1. Therefore, we can connect X+ to base(Qi+1) by an arc inside Qi+1 that is disjoint from any other Y + ∈M+. Moreover, all these arcs for X ∈Mi+1 ∖Mi with base(X+) = ∅can be drawn so that they are pairwise disjoint. Proof Deﬁne We are to show that G is planar. For X ∈Ni and 1 ⩽i < m, the set X ∩Ji is arc-connected and thus entirely contained in one member of C. It is clear that C L X ̸= C R X for X ∈N L ∩N R. Consider the graph G with vertex set C and edges connecting C L X and C R X for all X ∈N L ∩N R. We are to show that G is planar. p We construct sets V ⊂E ⊂ N with the following properties: We construct sets V ⊂E ⊂ N with the following properties: (i) V is a ﬁnite subset of C, (ii) E is a ﬁnite union of arcs with endpoints in V , pairwise disjoint outside of V , (iii) E ∩C is arc-connected for every component C ∈C, (iii) E ∩C is arc-connected for every component C ∈C, (iv) every X ∈N L ∩N R contains an arc in E between C L X and C R X (iv) every X ∈N L ∩N R contains an arc in E between C L X and C R X. The construction proceeds by induction on the members of N. We start with V con- taining one (arbitrary) point in each member of C, and with E = V . They clearly satisfy (i)–(iii). Then, for each X ∈N L ∩N R, we enlarge V and E to satisfy the condition (iv) for X, as follows. Since C L X ∪X ∪C R X is arc-connected, there is an arc A ⊂C L X ∪X ∪C R X between E ∩C L X and E ∩C R X. We can furthermore assume that A ∩E ∩C L X = {vL} and A ∩E ∩C R X = {vR}. This implies that A ∩E = {vL, vR}, as X ∖(C L X ∪C R X) is disjoint from any member of N ∖{X}. We add vL and vR to V and A to E. After processing all X ∈N L ∩N R, the resulting sets V and E satisfy (i)–(iv). The construction proceeds by induction on the members of N. We start with V con- taining one (arbitrary) point in each member of C, and with E = V . They clearly satisfy (i)–(iii). Proof Deﬁne • N L = {X ∈N : X ∖leftclip(X) ̸= ∅}, R • N L = {X ∈N : X ∖leftclip(X) ̸= ∅}, R • N R = {X ∈N : X ∖rightclip(X) ̸= ∅}, L L • N R = {X ∈N : X ∖rightclip(X) ̸= ∅}, L L • N L i = {X ∈N L : X ∖leftclip(X) ⊂Ji} for 1 ⩽i < m, • N L i = {X ∈N L : X ∖leftclip(X) ⊂Ji} for 1 ⩽i < m, • N R i = {X ∈N R : X ∖rightclip(X) ⊂Ji} for 1 ⩽i < m, • Ni = N L i ∪N R i , • N R i = {X ∈N R : X ∖rightclip(X) ⊂Ji} for 1 ⩽i < m, • Ni = N L i ∪N R i , • C L X to be the arc-connected component of ( Ni) ∩Ji containing X ∖leftclip(X), for X ∈N L i and 1 ⩽i < m, • C L X to be the arc-connected component of ( Ni) ∩Ji containing X ∖leftclip(X), for X ∈N L i and 1 ⩽i < m, for X ∈N L i and 1 ⩽i < m, • C R X to be the arc-connected component of ( Ni)∩Ji containing X ∖rightclip(X), for X ∈N R i and 1 ⩽i < m, • C R X to be the arc-connected component of ( Ni)∩Ji containing X ∖rightclip(X), for X ∈N R i and 1 ⩽i < m, N for X ∈N R i and 1 ⩽i < m, i • C to be the family of arc-connected components of ( Ni) ∩Ji for 1 ⩽i < m, that is, C = {C L X : X ∈N L} ∪{C R X : X ∈N R}. • C to be the family of arc-connected components of ( Ni) ∩Ji for 1 ⩽i < m, that is, C = {C L X : X ∈N L} ∪{C R X : X ∈N R}. For X ∈Ni and 1 ⩽i < m, the set X ∩Ji is arc-connected and thus entirely contained in one member of C. It is clear that C L X ̸= C R X for X ∈N L ∩N R. Consider the graph G with vertex set C and edges connecting C L X and C R X for all X ∈N L ∩N R. Claim 17 χ(N) ⩽4. Proof Deﬁne Proof Deﬁne X • if X ∈N R, then ψ(X) ̸= φ(C R X). Such a coloring exists, because φ(C L X) ̸= φ(C R X) for any X ∈N L ∩N R. To see that ψ is a proper coloring of N, consider some X, Y ∈N such that X ≺Y and X ∩Y ̸= ∅. Since φL(X) = φL(Y) and φR(X) = φR(Y) we have leftclip(X) ∩leftclip(Y) = ∅ and rightclip(X) ∩rightclip(Y) = ∅. Therefore, (X ∖rightclip(X)) ∩(Y ∖leftclip(Y)) = X ∩Y ̸= ∅. (X ∖rightclip(X)) ∩(Y ∖leftclip(Y)) = X ∩Y ̸= ∅. In particular, we have X ∈N R and Y ∈N L. The set X ∩Y is arc-connected, so C R X = C L Y . This yields ψ(X) ̸= φ(C R X) = φ(C L Y ) = ψ(Y). ⊓⊔ ⊓⊔ Acknowledgments M. Laso´n, P. Micek, and A. Pawlik were supported and B. Walczak was partially supported by Ministry of Science and Higher Education of Poland Grant 884/N-ESF-EuroGIGA/10/2011/0 within ESF EuroGIGA project GraDR. B. Walczak was partially supported by Swiss National Science Foundation Grant 200020-144531. 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. Proof Deﬁne Then, for each X ∈N L ∩N R, we enlarge V and E to satisfy the condition (iv) for X, as follows. Since C L X ∪X ∪C R X is arc-connected, there is an arc X X A ⊂C L X ∪X ∪C R X between E ∩C L X and E ∩C R X. We can furthermore assume that A ∩E ∩C L X = {vL} and A ∩E ∩C R X = {vR}. This implies that A ∩E = {vL, vR}, as X ∖(C L X ∪C R X) is disjoint from any member of N ∖{X}. We add vL and vR to V and A to E. After processing all X ∈N L ∩N R, the resulting sets V and E satisfy (i)–(iv). A ⊂C L X ∪X ∪C R X between E ∩C L X and E ∩C R X. We can furthermore assume that A ∩E ∩C L X = {vL} and A ∩E ∩C R X = {vR}. This implies that A ∩E = {vL, vR}, as X ∖(C L X ∪C R X) is disjoint from any member of N ∖{X}. We add vL and vR to V and A to E. After processing all X ∈N L ∩N R, the resulting sets V and E satisfy (i)–(iv). We have thus obtained a planar representation of a graph H with vertex set V and edge set consisting of maximal arcs in E internally disjoint from V . It follows from 12 3 414 Discrete Comput Geom (2014) 52:399–415 (iii) that the subgraph of H induced on V ∩C is connected for every C ∈C. Consider the graph obtained from H by contracting V ∩C for every C ∈C. Its vertices represent members of C, and by (iv), its edges connect the vertices representing C L X and C R X for all X ∈N L ∩N R. Hence this graph is isomorphic to G. This shows that G is planar, as a contraction of a planar graph is planar. 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https://openalex.org/W4210577241	https://www.frontiersin.org/articles/10.3389/fonc.2021.842169/pdf	English	null	Application of Intravoxel Incoherent Motion Diffusion-Weighted Imaging in Predicting and Monitoring Early Efficacy of Anti-Angiogenic Therapy in the C6 Glioma Rat Model	Frontiers in oncology	2,022	cc-by	7,199	1 Department of Radiology, First Afﬁliated Hospital of Anhui Medical University, Hefei, China, 2 Department of Nuclear Medicine, The First Afﬁliated Hospital of Anhui Medical University, Hefei, China, 3 Department of Basic Medical Sciences, School of Basic Medical Sciences, Anhui Medical University, Hefei, China Edited by: Guang Yang, Imperial College London, United Kingdom Edited by: Guang Yang, Imperial College London, United Kingdom Reviewed by: Zixing Huang, Sichuan University, China Zeyu Xiao, Jinan University, China Correspondence: Xiaohu Li zhaorenlxh@126.com Yongqiang Yu ayfyyuyongqiang@126.com †These authors have contributed equally to this work and share ﬁrst authorship Objective: To investigate the feasibility of intravoxel incoherent motion (IVIM) diffusion- weighted imaging (DWI) in evaluating early effects of anti-angiogenic therapy in the C6 glioma rat model. Reviewed by: Zixing Huang, Sichuan University, China Zeyu Xiao, Jinan University, China Reviewed by: Zixing Huang, Sichuan University, China Zeyu Xiao, Jinan University, China Methods: Twenty-six rats of the C6 glioma model were randomly divided into a treatment group (received bevacizumab) and a control group (physiological saline). IVIM-DWI was performed on days 0, 1, 3, 5, and 7 after anti-angiogenic therapy and tumor growth and IVIM-DWI parameters were dynamically observed. Hematoxylin and eosin, CD34 microvessel density (MVD), proliferation of cell nuclear antigen (PCNA), and Hif-a staining were performed on day 7. One-way ANOVA was used to compare intra-group differences and an independent-samples t-test was used to compare inter-group differences of MRI parameters. Correlations between IVIM-DWI parameters, tumor size, and pathological results were analyzed. Correspondence: Xiaohu Li zhaorenlxh@126.com Yongqiang Yu ayfyyuyongqiang@126.com †These authors have contributed equally to this work and share ﬁrst authorship Correspondence: Xiaohu Li zhaorenlxh@126.com Yongqiang Yu ayfyyuyongqiang@126.com †These authors have contributed equally to this work and share ﬁrst authorship Specialty section: This article was submitted to Cancer Imaging and Image-directed Interventions, a section of the journal Frontiers in Oncology Received: 23 December 2021 Accepted: 31 December 2021 Published: 28 January 2022 Specialty section: This article was submitted to Cancer Imaging and Image-directed Interventions, a section of the journal Frontiers in Oncology Results: The relative change in tumor volume (DVolume) in the two groups differed signiﬁcantly on days 5 and 7 (p = 0.038 and p < 0.001). The perfusion-related parameters D- and f-values decreased in the treatment group and demonstrated signiﬁcant differences compared with the control group on days 3, 5, and 7 (p = 0.033, p < 0.001, and p < 0.001, respectively). ORIGINAL RESEARCH published: 28 January 2022 doi: 10.3389/fonc.2021.842169 The diffusion-related parameters ADC and D-values increased in the treatment group and were found to be signiﬁcantly differently different from the control group on days 5 and 7 (both p < 0.001). The initial D-value showed a negative correlation with DVolume (g = −0.744, p < 0.001), whereas the initial D-value and relative change of D-value had a positive correlation with DVolume (g = 0.718, p < 0.001 and g = 0.800, p < 0.001, respectively). MVD was strongly positively correlated with D-value (r = 0.886, p = 0.019), PCNA was negatively correlated with ADC- and D-values (r = −0.848, p = 0.033; and r = −0.928 p = 0.008, respectively), and Hif-1a was strongly negatively correlated with D-value (r = −0.879, p = 0.010). Received: 23 December 2021 Accepted: 31 December 2021 Published: 28 January 2022 Application of Intravoxel Incoherent Motion Diffusion-Weighted Imaging in Predicting and Monitoring Early Efﬁcacy of Anti-Angiogenic Therapy in the C6 Glioma Rat Model Weishu Hou 1†, Yangyang Xue 1,2†, Yinfeng Qian 1, Hongli Pan 1, Man Xu 1, Yujun Shen 3, Xiaohu Li 1 and Yongqiang Yu 1* Cell Culture and Animal Model Establishment High-grade glioma is the most aggressive primary malignant tumor of the central nervous system in adults and has a high expression of vascular endothelial growth factor (VEGF) (1). VEGF plays an essential role in regulating angiogenesis of glioma and, thus, anti-angiogenesis therapy targeting the VEGF signaling pathway has been recognized as an effective targeted therapy method for malignant glioma (2, 3). Recently the recombinant humanized monoclonal antibody bevacizumab (Avastin®, Bev), an anti-angiogenesis medicine, has been widely used as a treatment, either alone or in combination with traditional chemotherapy, for recurrent glioblastoma and newly diagnosed high-grade glioma (4–6). C6 glioma cells were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, PR, China). The cells were kept in the incubator at 37°C in 5% carbon dioxide, and cultured in high glucose Dulbecco’s Modiﬁed Eagle’s Medium (Abcam, Cambridge, UK). After C6 cells had reached approximately 90% conﬂuency, they were digested with 0.05% trypsin and washed twice with phosphate buffered saline (Abcam, Cambridge, UK). Finally, the C6 cell suspension, at a concentration of 1 × 107 cells/ml, was prepared to establish the glioma orthotopic rat model. Male Sprague–Dawley rats (purchased from Animal Center of Anhui Province, No. SCXK-Wan-2017–001) aged 6–8 weeks were anesthetized by intraperitoneal injection with 10% chloral hydrate (0.3 ml/100 g), and the glioma orthotopic rat model was established using a stereotactic apparatus (Kopf, Cayunga, CA, USA). The heads of the rats were shaved, disinfected with 0.1% povidone-iodine, and ﬁxed on the stereotactic apparatus. An HP- 4 dental drill bit was used to drill a hole in the skull at the following three coordinates: 1 mm to the anterior arcuate suture, 3 mm to the right of the sagittalis suture, and at a depth of 5 mm. Finally, 10 ml of the C6 cell suspension was injected into the caudate nucleus with a 0.5-ml microsyringe at a rate of 1 ml/min. However, to date, how to monitor the effects of anti- angiogenesis therapy sensitively, dynamically, and non- invasively is still a major challenge of glioma therapy. Diffusion-weighted imaging (DWI) is a functional magnetic resonance imaging (MRI) sequence used to evaluate or predict treatment outcome of malignant tumors; however, the apparent diffusion coefﬁcient (ADC) has been shown to be inﬂuenced by both the diffusion of water molecules and microcirculation due to the mono-exponential model of DWI (7, 8). MRI Scanning Prior to MRI, all rats were anesthetized with 3% isoﬂurane and continuous anesthesia with 1%–2% isoﬂurane using an anesthesia machine for small animals (Suzhou Zhongzhi Medical Technologies, China), and their core temperature maintained at 37°C. MRI scans were then performed using a 3.0-T MRI system (Discovery 750w, GE Medical System, Milwaukee, WI, USA) with a custom-built eight-channel receiver coil for animals (GE Medical System). Both the treated and control groups underwent an initial MRI scan before treatment (day 0) and then subsequent MRI sequences on days 1, 3, 5 and 7 after treatment with Bev or vehicle. MRI sequences included axial fast spin-echo T1-weighted imaging (T2W1) with the following parameters: repetition time/echo time (TR/TE) = Citation: Hou W, Xue Y, Qian Y, Pan H, Xu M, Shen Y, Li X and Yu Y (2022) Application of Intravoxel Incoherent Motion Diffusion-Weighted Imaging in Predicting and Monitoring Early Efﬁcacy of Anti-Angiogenic Therapy in the C6 Glioma Rat Model. Front. Oncol. 11:842169. doi: 10.3389/fonc.2021.842169 January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org Hou et al. IVIM for Glioma Treatment Response Conclusion: IVIM-DWI was sensitive and accurate in predicting and monitoring the effects of early anti-angiogenesis therapy in a C6 glioma rat model. Conclusion: IVIM-DWI was sensitive and accurate in predicting and monitoring the effects of early anti-angiogenesis therapy in a C6 glioma rat model. Keywords: intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI), glioma, angiogenesis inhibitors, therapy, vascular normalization Therapy of the Glioma Model py Rat models that were established successfully were randomly divided into a treatment group and control group. Rats in the treatment group were administered with Bev (Roche, Shanghai, China) diluted with 0.9% physiological saline, while rats in the control group were administered with vehicle (0.9% physiological saline). The two groups received intraperitoneal injection of Bev or vehicle, respectively, at a dose of 5 mg/kg once daily for 7 days. Therefore, the purpose of this study was to observe the early dynamics of IVIM-DWI parameters during anti- angiogenic therapy to explore the accuracy of IVIM-DWI in anti-angiogenic therapy outcome via determining the angiogenesis, cell proliferation, and hypoxia of cells in the C6 glioma rat model. Cell Culture and Animal Model Establishment Recently, an intravoxel incoherent motion DWI (IVIM-DWI) has been developed that can separate microcirculation from restricted Brownian self-diffusion, and has been applied to evaluate the microcirculation of tumors after anti-angiogenic therapy several types of animal models (9–11). However, few studies have focused on the early and dynamic changes of IVIM-DWI parameters during therapy and the correlation between IVIM- DWI parameters and histological assessment. Ethics Statement All treated procedures were approved by the Ethics Committee for Animal Experimentation of Anhui Medical University of Anhui Province, China (Approval no. SCXK-Wan-2017–001) and were conducted in strict accordance with the Guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org 2 IVIM for Glioma Treatment Response Hou et al. 400 ms/9.5 ms; slice thickness = 2.0 mm; ﬁeld of view (FOV) = 60 mm × 48 mm; and number of excitations (NEX) = 4.0. For axial spin echo T2-weighted imaging, the parameters included the following: TR/TE = 2,000 ms/46 ms; slice thickness = 2.0 mm; FOV = 60 mm × 48 mm; and NEX = 3.0. For IVIM DWI scans, single-shot echo-planar imaging DWI sequence was performed with nine b-values of 0, 10, 50, 100, 200, 400, 600, 800, and 1000 s/mm2. Other parameters included TR/TE = 3000 ms/102.4 ms; ﬂip angle, 90°; matrix = 64 × 64; FOV = 90 mm × 90 mm; section thickness = 2.0 mm; and NEX= 4.0, with a total scanning time of 6 min and 15 s. identifying three “hotspots” with dense positive cells in a low-power ﬁeld (40×) and the number of CD34-positive cells was counted manually under a high-power ﬁeld (200×); MVD was then calculated as the average number of the three “hotspots”. Expression of PCNA and Hif1-a was assessed using ImageJ software (National Institutes of Health). Effect of Bev on Tumor Volume Effect of Bev on Tumor Volume Overall, 26 out of 30 rats underwent a successful glioma orthotopic procedure; two rats died due to intracranial hemorrhage, and no tumors were found in an additional two rats. (Figure 1). There was no bleeding, weight loss, or other serious side effects during the treatment course of Bev or vehicle in our study. The tumor volume of the control and treatment groups before treatment (day 0) was 23.4 ± 5.58 mm3 and 25.2 ± 4.33 mm3, respectively, indicating no signiﬁcant difference between the two groups (p = 0.76). After treatment with Bev, the average tumor volume of the two groups differed signiﬁcantly on day 7 (90.7 ± 13.4 mm3 vs. 75.3 ± 11.5 mm3, p = 0.022, Figure 2A). The relative change in tumor size was calculated as DVolume = (volumen −volume0)/volume0 × 100%. The DVolume in the control group was 29.1% ± 5.4% on day 1, 85.1% ± 13.5% on day 3, 200.5% ± 45.4% on day 5, and 287.6% ± 58.4% on day 7, while that of the treatment group was 27.7% ± 4.7% on day 1, 42.1% ± 12.3% on day 3, 138.8% ± 25.7% on day 5, and 198.8% ± 42.3% on day 7. The DVolume in the two groups differed signiﬁcantly on days 5 and day 7 (p = 0.038 and p < 0.001, respectively, (Figure 2B). Imaging Analysis All MRI data were transferred to a post-processing workstation AW4.6 (GE Healthcare, USA), and tumor volume and IVIM- DWI parameter measurements were performed independently by one radiologist with 12 years’ experience in MRI diagnosis. For the calculation of the glioma volume, the region of interest (ROI) was drawn along the edge of tumor on T2WI images to obtain the surface area of the tumor in each slice (mm2), after which the surface area of all the slices was summed and multiplied by the slice thickness (2 mm) to determine the total tumor volume (mm3). For the quantitative measurement of IVIM-DWI parameters, MRI data were analyzed using MADC software (GE Healthcare, USA) in the AW4.6 post-processing workstation. First, the IVIM-DWI parameter (apparent diffusion coefﬁcient, ADC; diffusion coefﬁcient, D; pseudodiffusion coefﬁcient, D; and perfusion fraction, f) maps were generated automatically using MADC software. Next, due to the low signal-to-noise ratio of IVIM-DWI image, the ROI was set in the section that demonstrated the largest tumor diameter according to both axial T2WI image and DWI image with a b-value of 1000 s/ mm2. The ROI (12–20 mm3) was drawn manually along the edge of the tumor in order to cover as much of the solid part as possible and exclude necrotic and hemorrhagic areas. Finally, the average values of IVIM-DWI quantitative parameters were calculated automatically. Statistical Analysis All statistical processes were performed by SPSS 19.0 (IBM, Armonk, NY, USA). The tumor volume and quantitative IVIM-DWI parameters were expressed as the mean ± standard deviation. A one-way analysis of variance (ANOVA) with post- hoc test (Bonferroni test) was used to compare IVIM-DWI parameters among the ﬁve time points in the treatment and control group. An independent-samples t-test was used to compare IVIM-DWI parameters between the treatment and control groups. Pearson’s analysis was used to analyze the correlation between IVIM-DWI parameters and the average tumor growth rate. Pearson’s or Spearman’s correlation tests were used to analyze the correlation between IVIM-DWI parameters and histological assessment in the treatment group. p < 0.05 indicated a signiﬁcant difference. Histological Assessment g After the last MRI scan on the 7th day of treatment, all rats were sacriﬁced by cervical dislocation. Then, the brain tissue was removed, ﬁxed with 4% paraformaldehyde, dehydrated with 70% alcohol, embedded with parafﬁn, and dissected into axial sections. To ensure that the tumor sections corresponded to IVIM-DWI images as accurately as possible, sections were processed sequentially at a thickness of 4 mm, in the same orientation as the axial MRI plane. Finally, hematoxylin and eosin staining was performed according to standard procedures. To evaluate neoangiogenesis and cell proliferation of hypoxic glioma cells, sections were further analyzed by immunohistochemical staining using CD34 (1:250; Abcam), PCNA (PCNA, 1:100; Santa Cruz Biotechnology, CA, USA), and Hif1-a (1:100; Abcam) monoclonal antibodies. A pathologist with 10 years of experience reviewed the microvessel density (MVD) of CD34-positive cells and PCNA and Hif1-a expression. MVD calculation was determined by After the last MRI scan on the 7th day of treatment, all rats were sacriﬁced by cervical dislocation. Then, the brain tissue was removed, ﬁxed with 4% paraformaldehyde, dehydrated with 70% alcohol, embedded with parafﬁn, and dissected into axial sections. To ensure that the tumor sections corresponded to IVIM-DWI images as accurately as possible, sections were processed sequentially at a thickness of 4 mm, in the same orientation as the axial MRI plane. Finally, hematoxylin and eosin staining was performed according to standard procedures. IVIM-DWI Parameter Images To evaluate neoangiogenesis and cell proliferation of hypoxic glioma cells, sections were further analyzed by immunohistochemical staining using CD34 (1:250; Abcam), PCNA (PCNA, 1:100; Santa Cruz Biotechnology, CA, USA), and Hif1-a (1:100; Abcam) monoclonal antibodies. A pathologist with 10 years of experience reviewed the microvessel density (MVD) of CD34-positive cells and PCNA and Hif1-a expression. MVD calculation was determined by g The gliomas were generally located in the right cerebral hemisphere with scattered hemorrhage, and tumors, as visualized on T2WI and IVIM-DWI (b = 1,000 s/mm2) images, were veriﬁed withs HE staining (Figure 3). T2WI image showed that tumors grew slowly from days 0 to day 7 in the treatment group and had indicative hyperintensities on DWI January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org 3 Hou et al. IVIM for Glioma Treatment Response FIGURE 1 \| The experiment ﬂow chart of glioma model establishment, anti-angiogenic therapy, and MRI scan. ADC, apparent diffusion coefﬁcient; D, diffusion coefﬁcient; D, pseudodiffusion coefﬁcient; f, perfusion fraction. FIGURE 1 \| The experiment ﬂow chart of glioma model establishment, anti-angiogenic therapy, and MRI scan. ADC, apparent diffusion coefﬁcient; D, diffusion coefﬁcient; D, pseudodiffusion coefﬁcient; f, perfusion fraction. good. The interclass correlation coefﬁcient of the IVIM-DWI parameters is summarized in Table 1. images and hypointensities on ADC maps, with colors, ranging from blue to red, representing values ranging from low to high. Pseudocolor maps indicated that the D-values increased gradually while D- and f-values decreased during the 7 days (Figure 4). Intraobserver and Interobserver Intraobserver and Interobserver IVIM-DWI Parameter Analyses The dynamic changes of the IVIM-DWI parameters in the control and treatment groups at each time point are displayed in Table 2. ADC-value in the treatment group increased gradually while it decreased in the control group during the therapy course, with the two groups displaying signiﬁcant differences among the ﬁve time points (treatment group: F = 18.72, p < 0.001; control group: Agreement of IVIM-DWI Parameters The reproducibility of ADC, D, and D* ranged from good to excellent and the reproducibility of f ranged from moderate to A B FIGURE 2 \| Dynamic of tumor volume size (A) and average tumor growth rate DVolume (B) of the treated and control group during anti-angiogenic course. p < 0.05, p < 0.01, *p < 0.001. Error bars denote standard errors. A B B B FIGURE 2 \| Dynamic of tumor volume size (A) and average tumor growth rate DVolume (B) of the treated and control group during anti-angiogenic course. p < 0.05, p < 0.01, p < 0.001. Error bars denote standard errors. January 2022 \| Volume 11 \| Article 842169 4 Frontiers in Oncology \| www.frontiersin.org Hou et al. IVIM for Glioma Treatment Response A C D B FIGURE 3 \| The glioma on T2WI (A) and IVIM-DWI (b = 1,000 s/mm2) (B) images. Corresponding HE staining showed atypical tumor cells (C original magniﬁcation ×4; D original magniﬁcation ×10). A B B A C D C D D FIGURE 3 \| The glioma on T2WI (A) and IVIM-DWI (b = 1,000 s/mm2) (B) images. Corresponding HE staining showed atypical tumor cells (C original magniﬁcation ×4; D original magniﬁcation ×10). I (A) and IVIM-DWI (b = 1,000 s/mm2) (B) images. Corresponding HE staining showed atypical tumor cells (C original magniﬁcation F = 7.98, p < 0.001). Multiple comparisons between the two groups are shown in Figures 5A, B. For intergroup analyses, the baseline ADC-values were 0.585 ± 0.112 ×10−3 mm2/s and 0.643 ± 0.290 × 10−3 mm2/s in the control and treatment groups, respectively, which displayed no signiﬁcant difference. However, the ADC- values in the treatment group were signiﬁcantly higher than those in the control group from day 3 onwards (day 3: p = 0.033; day 5: p < 0.001; day 7: p < 0.001, Figure 6A). By contrast, the D-value of the control group increased markedly with time (F = 4.23, p = 0.008). The results of multiple comparisons between the two groups are shown in Figures 5E, F. After treatment, D-values in the treatment group were signiﬁcantly lower than those in the control group on days 3, 5, and 7 (day 3: p = 0.005; day 5: p < 0.001; day 7: p < 0.001, Figure 6C). Agreement of IVIM-DWI Parameters The f-value of the treatment group decreased slightly during the treatment course while that of the control group increased during the 7 days; however, there was no difference among the ﬁve time points in the two groups (treatment group: F = 0.95, p = 0.45; control group: F = 2.08, p = 0.11, Figures 5G, H). Only the f-value on the 7th day showed a signiﬁcant difference between the two groups (p = 0.008, Figure 6D). The D-value in the treatment group increased daily while it signiﬁcantly decreased in the control group. There was signiﬁcant difference of D-value among the ﬁve time points in both treatment and control groups (treatment: F = 33.17, p < 0.001; control group: F = 23.64, p < 0.001). Comparison results between the two groups are shown in Figures 5C, D. For intergroup analyses, the baseline D-values were 0.274 ± 0.052 × 10−3 mm2/s and 0.288 ± 0.050 ×10−3 mm2/s in the control and treatment groups, respectively, which displayed no signiﬁcant difference. Following daily treatment of Bev, the D-values in the treatment group were signiﬁcantly higher compared with those in the control group on days 5 and day 7 (both p < 0.001, Figure 6B). Frontiers in Oncology \| www.frontiersin.org Correlation Between DVolume and IVIM Parameters The initial D-value showed a moderate negative correlation with DVolume (g = −0.744, p < 0.001), while the initial D-value and relative change of D-value (DD) demonstrated moderate positive correlations with DVolume (D: g = 0.718, p < 0.001; DD: g = 0.800, p < 0.001, Figure 7). No correlation between other initial or relative changes of IVIM parameters and tumor size were found. For the perfusion-related parameters, the D-value of the treatment group decreased gradually and displayed signiﬁcant differences among the ﬁve time points (F = 11.08, p < 0.001). January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org 5 Hou et al. IVIM for Glioma Treatment Response FIGURE 4 \| Axial T2WI, DWI (b = 1000), ADC, and pseudocolor maps of D, D, and f at different time points in the treated group. T2WI image showed the tumor grew slowly from day 0 to day 7 (arrows) and presented hyperintensity on DWI images and hypointensity on ADC maps (arrows). Color ranging from blue to red represented values ranging from low to high. D-values increased gradually (arrows), while D- and f-values decreased during the 7 days (arrows). FIGURE 4 \| Axial T2WI, DWI (b = 1000), ADC, and pseudocolor maps of D, D, and f at different time points in the treated group. T2WI image showed the tumor grew slowly from day 0 to day 7 (arrows) and presented hyperintensity on DWI images and hypointensity on ADC maps (arrows). Color ranging from blue to red represented values ranging from low to high. D-values increased gradually (arrows), while D- and f-values decreased during the 7 days (arrows). FIGURE 4 \| Axial T2WI, DWI (b = 1000), ADC, and pseudocolor maps of D, D, and f at different time points in the treated group. T2WI image showed the tumor grew slowly from day 0 to day 7 (arrows) and presented hyperintensity on DWI images and hypointensity on ADC maps (arrows). Color ranging from blue to red represented values ranging from low to high. D-values increased gradually (arrows), while D- and f-values decreased during the 7 days (arrows). TABLE 1 \| The interobserver and intraobserver reproducibility of IVIM-DWI parameters. Interobserver Intraobserver ADC 0.944 (0.702–0.956) 0.903 (0.783–0.936) D 0.778 (0.692–0.895) 0.895 (0.744–0.921) D* 0.856 (0.603–0.881) 0.842 (0.689–0.911) f 0.648 (0.497–0.785) 0.731 (0.558–0.802) The 95% conﬁdence intervals of interclass correlation coefﬁcient (ICC) were in parentheses. Correlation Between DVolume and IVIM Parameters ADC, apparent diffusion coefﬁcient; D, diffusion coefﬁcient; D, pseudodiffusion coefﬁﬁcient; f, perfusion fraction. TABLE 1 \| The interobserver and intraobserver reproducibility of IVIM-DWI parameters. The 95% conﬁdence intervals of interclass correlation coefﬁcient (ICC) were in parentheses. DISCUSSION CD34-positive tubules are a pivotal feature of microvascular growth and were used to calculate the density of neovascularization in our study. Our study showed that D- value had a signiﬁcant positive correlation with MVD, which indicated that the perfusion-related IVIM parameters could reﬂect the change of blood ﬂow accurately in glioma models following intervention of anti-angiogenic therapy. Besides, although previous research found that the f-value had more potential to predict the tumor response after antiangiogenic therapy, there was no explicit correlation between MVD and f in our present study, a result that was likely because of the obvious image noise of the f pseudo-color image (21). Based on our previous study, the orthotopic C6 glioma model exhibited greater growth rate with less necrosis or hemorrhage area from day 14 to day 21 after C6 cell transplantation (12). Therefore, we started anti-angiogenesis therapy from the 14th day after establishing the glioma model and continued the therapy for 7 days to obtain the early dynamic changes in IVIM-DWI parameters. Based on the dual-exponential model of IVIM-DWI, the perfusion-related parameters f and D* are inﬂuenced by the blood volume and velocity of the tumor (13). In our study, the f and D-values demonstrated a pronounced reduction in the treatment group, which proved the effect of Bev-mediated inhibition of VEGF-dependent vascular growth. Besides, the positive correlation between the initial D and DVolume indicated that tumors with high blood perfusion responded better to anti-angiogenic therapy. This result was essentially in agreement with previous studies that the D* and f-values of malignant tumors decreased signiﬁcantly after anti-angiogenic therapy (14–16). g g The diffusion-related parameters ADC and D reﬂect the microscopic Brownian motion of water molecules in tumor tissue (22). The decreased tumor blood perfusion following Bev treatment resulted in a decrease in cell proliferation, and resulted in a signiﬁcant increase in ADC and D-values from day 5. Together, the data indicated that the change of diffusion- related parameters occurred later than that of perfusion-related parameters. The degree of proliferation can be reﬂected by PCNA because it is strongly associated with DNA replication in tumor cells (23). The decrease of tumor cells after anti- angiogenic therapy was also conﬁrmed by the signiﬁcant differences in PCNA scores between the control and treatment groups on day 7. Histological Staining and Correlations With IVIM-DWI Parameters Comparisons of IVIM-DWI parameters between the control and treated groups at the same time point were performed by the independ ADC, apparent diffusion coefﬁcient; D, diffusion coefﬁcient; D, pseudodiffusion coefﬁﬁcient; f, perfusion fraction. strongly correlated with ADC-value and the D-value (ADC: r = −0.848, p = 0.033; D: r = −0.928, p = 0.008, Table 4). strongly correlated with ADC-value and the D-value (ADC: r = −0.848, p = 0.033; D: r = −0.928, p = 0.008, Table 4). “normalization of tumor vasculature” theory (17, 18). However, the transient increase of perfusion-related MRI parameters was not found in the current IVIM-DWI research, suggesting that there was no direct correlation between IVIM-DWI perfusion- related parameters and DCE-MRI perfusion-related parameters. In fact, previous research has indicated that the perfusion parameters of DCE-MRI and IVIM-DWI were not completely consistent and possibly even contrasting. Indeed, IVIM-DWI provides information on the total blood transit through a voxel and is focused on the intravascular blood movement, while DCE- MRI provides information on the exchange of contrast agents between the intravascular and extravascular spaces (19, 20). Regarding the Hif-1a, both the nuclei and cytoplasm of cells were stained brown in Hif-1a-stained sections. Hif-1a-positive cells were mainly distributed at the edge of the tumor necrosis area. The Hif-1a-positive area in the treatment group was markedly smaller than that of the control group, and showed a signiﬁcant difference (p < 0.001, Table 2 and Figure 8). The score of Hif-1a was strongly correlated with D-value (r = −0.879, P = 0.010, Table 4). Frontiers in Oncology \| www.frontiersin.org January 2022 \| Volume 11 \| Article 842169 Histological Staining and Correlations With IVIM-DWI Parameters CD34. Vascular endothelial cells were stained yellow and brown in CD34-stained sections. The CD34-positive vessels in the control group were dilated and distorted while they were tiny and regular in the treatment group, with a signiﬁcant difference in the MVD between the two groups on day 7 (p < 0.001, Table 3 and Figure 8). In addition, MVD was strongly correlated with D-value (r = 0.886, p = 0.019, Table 4). Regarding PCNA, the cell nuclei were stained brown in PCNA-stained sections. The score of PCNA-positive cells in the treatment group was markedly lower than that in the control group on day 7 (p < 0.001, Table 2 and Figure 8). PCNA was January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org IVIM for Glioma Treatment Response Hou et al. TABLE 2 \| Comparison of IVIM-DWI parameters between the control and treated groups at different time points before and after antiangiogenic therapy. Day 0 Day 1 Day 3 Day 5 Day 7 ADC (×10−3 mm2/s) Control 0.585 ± 0.112 0.577 ± 0.126 0.487 ± 0.130 0.450 ± 0.150 0.363 ± 0.140 Treated 0.643 ± 0.290 0.668 ± 0.271 0.733 ± 0.182 0.846 ± 0.278 0.968 ± 0.340 p 0.215 0.055 0.033 <0.001 <0.001 D (×10−3 mm2/s) Control 0.274 ± 0.052 0.268 ± 0.040 0.192 ± 0.087 0.145 ± 0.058 0.086 ± 0.039 Treated 0.288 ± 0.050 0.265 ± 0.064 0.396 ± 0.150 0.499 ± 0.016 0.630 ± 0.290 p 0.730 0.798 0.052 <0.001 <0.001 D* (×10−3 mm2/s) Control 5.797 ± 1.642 5.946 ± 1.620 7.233 ± 2.760 8.189 ± 2.830 9.061 ± 1.870 Treated 6.374 ± 1.730 5.758 ± 1.450 4.446 ± 1.709 3.475 ± 1.440 3.290 ± 1.102 p 0.505 0.810 0.005 <0.001 <0.001 f (%) Control 65.833 ± 11.502 59.837 ± 14.230 68.440 ± 22.003 69.164 ± 22.340 80.335 ± 28.037 Treated 69.525 ± 11.320 67.325 ± 12.530 59.355 ± 20.026 55.329 ± 24.703 44.858 ± 14.930 p 0.581 0.368 0.498 0.304 0.008 Comparisons of IVIM-DWI parameters between the control and treated groups at the same time point were performed by the independent-samples t-test. ADC, apparent diffusion coefﬁcient; D, diffusion coefﬁcient; D, pseudodiffusion coefﬁﬁcient; f, perfusion fraction. arameters between the control and treated groups at different time points before and after antiangiogenic therapy. DISCUSSION The negative correlation between diffusion- Our previous study of DCE-MRI data from the orthotopic C6 glioma model showed slightly increased Ktrans and Kep on day 1 after anti-angiogenic therapy, which may be the result of decreased leakage and increased circulation presented in the January 2022 \| Volume 11 \| Article 842169 January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org 7 Hou et al. IVIM for Glioma Treatment Response A B C D E F G H FIGURE 5 \| Box plots comparing the IVIM-DWI parameters in the control groups (A–D) and treated group (E–H) at different time points. The four IVIM-DWI parameters between the two groups all showed opposite increased or decreased trends. ANOVA followed by the Bonferroni test was used in multiple comparison of different time points. Error bars denote standard errors. A A B B C D D D C C D F E F E G H G FIGURE 5 \| Box plots comparing the IVIM-DWI parameters in the control groups (A–D) and treated group (E–H) at different time points. The four IVIM-DWI parameters between the two groups all showed opposite increased or decreased trends. ANOVA followed by the Bonferroni test was used in multiple comparison of different time points. Error bars denote standard errors. related parameters and PCNA in the treatment group indicated that ADC and D-values could accurately reﬂect the cell density after anti-angiogenic therapy. The correlation between ADC- value and PCNA was weaker than that between D-value and PCNA, probably because the ADC-value was inﬂuenced by both microscopic water diffusion and blood perfusion of the tumor. hypoxia is related to distorted neovascularization and proliferative cell density of tumors (25). In our study, we used Hif-1a as the pathological gold standard to evaluate the hypoxia of high-grade glioma and found that there was positive correlation between D-value and Hif-1a. The increased D- value indicated the reduced tumor cell density, which resulted in the decreased oxygen consumption of tumor cells. Our results conﬁrmed that the diffusion-related parameter D-value could reﬂect the hypoxia of glioma after anti-angiogenic therapy. Unfortunately, we did not ﬁnd any correlation between Hypoxia reduces the responsiveness of tumors to conventional chemotherapy and radiotherapy, and is an important factor of anti-angiogenic therapy failure for high- grade glioma (24). CONCLUSIONS The animal study was approved by the Ethics Committee for Animal Experimentation of Anhui Medical University of Anhui Province, China (Approval no. SCXK-Wan-2017–001) and was conducted in strict accordance with the Guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. In conclusion, IVIM-DWI showed decreased perfusion-related parameters and increased diffusion-related parameters were consistent with histological staining in the Bev-treated glioma model. The present study demonstrated that IVIM-DWI was sensitive and accurate in predicting and monitoring the effect of early anti-angiogenic therapy in the C6 glioma rat model. DISCUSSION Groups MVD PCNA Hif1-a Control 38.890 ± 6.571 10.896 ± 2.326 5.677 ± 1.213 Treated 18.328 ± 4.256 4.875 ± 1.191 2.940 ± 0.046 p <0.001 <0.001 <0.001 Comparisons of histological assessment between the control and treated groups on day 7 were performed using the independent-samples t-test. FIGURE 8 \| IHC staining of CD34, PCNA, and Hif-1a in the control and treated group on day 7 after therapy. Positive cells are indicated with arrows. The CD34- positive vessels in the control group were dilated and distorted while they were tiny and regular in the treatment group. The PCNA-positive cells in the treatment group was lower than that in the control group. The Hif-1a-positive area in the treatment group was smaller than that of the control group. TABLE 4 \| Correlation coefﬁcients between IVIM-DWI and immunohistochemistry scores of the treated group on day 7 after antiangiogenic therapy. MVD PCNA Hif-1a Pearson’s r p Spearman r p Spearman r p ADC 0.767 0.075 −0.848 0.033 −0.278 0.594 D 0.527 0.283 −0.928 0.008* −0.879 0.010* D* 0.886 0.019* 0.866 0.019* −0.626 0.052 f 0.802 0.055 −0.314 0.544 −0.463 0.355 Pearson correlation was used for the comparison between MVD and IVIM-DWI parameters; Spearman’s analysis was used for comparisons between PCNA, Hif-1a, and IVIM DWI parameters (p < 0.05). n coefﬁcients between IVIM-DWI and immunohistochemistry scores of the treated group on day 7 after antiangiogenic therapy. TABLE 4 \| Correlation coefﬁcients between IVIM-DWI and immunohistochemistry scores of the treated group on day 7 after antian DISCUSSION Previous research has conﬁrmed that January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org 8 IVIM for Glioma Treatment Response Hou et al. A B C D FIGURE 6 \| Line graphs comparing the IVIM-DWI parameters of the treated and control group on different time points. The four IVIM-DWI parameters between the two groups all showed opposite trends during therapy (A–D). The independent-samples t-test was used. p < 0.05, p < 0.01, p < 0.001. A B D C D C FIGURE 6 \| Line graphs comparing the IVIM-DWI parameters of the treated and control group on different time points. The four IVIM-DWI parameters between the two groups all showed opposite trends during therapy (A–D). The independent-samples t-test was used. p < 0.05, p < 0.01, p < 0.001. perfusion-related parameters of IVIM-DWI and Hif-1a, which may be related to the complex changes in blood vessels after anti- angiogenic therapy. temozolomide was not used in this study because we aimed to explore the correlation between IVIM-DWI parameters and immunohistochemical indicators, rather than to conﬁrm the mechanism of anti-angiogenic medicines. Thus, future studies should focus on investigating the effects of anti-angiogenic therapy combined with traditional chemotherapy in the same model using IVIM-DWI. There were some limitations in the study. First, because of the limited sample size, the correlation between IVIM-DWI parameters and histological assessment was only analyzed at the end of follow-up. Second, combined therapy with A B C FIGURE 7 \| Scatter diagrams of correlations between the relative change of tumor size (DVolume) and IVIM-DWI parameters. (A) The initial D-value showed moderate negative correlation with DVolume. (B, C) Initial D-value and relative change of D-value (DD) demonstrated moderate positive correlation with DVolume. The Pearson correlation analysis was used. C A B FIGURE 7 \| Scatter diagrams of correlations between the relative change of tumor size (DVolume) and IVIM-DWI parameters. (A) The initial D-value showed moderate negative correlation with DVolume. (B, C) Initial D*-value and relative change of D-value (DD) demonstrated moderate positive correlation with DVolume. The Pearson correlation analysis was used. January 2022 \| Volume 11 \| Article 842169 9 Frontiers in Oncology \| www.frontiersin.org IVIM for Glioma Treatment Response Hou et al. TABLE 3 \| The scores and statistical differences analysis of MVD, PCNA, and Hif1-a between the control and treated groups on day 7 after antiangiogenic therapy. REFERENCES 16. Martens RM, Koopman T, Lavini C, Ali M, Peeters CFW, Noij DP, et al. 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This work was supported by the Natural Science Foundation of Anhui Province [No. 2008085QH381]. DATA AVAILABILITY STATEMENT WH and YX contributed to conceptualization. YX, HP, and MX contributed to the cell and animal experiments. YQ, WH, and HP contributed to MRI scan and MRI data analysis. YS The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org 10 IVIM for Glioma Treatment Response Hou et al. REFERENCES Hou W, Xue Y, Tang W, Pan H, Xu M, Li X, et al. Evaluation of Tumor Hypoxia in C6 Glioma Rat Model With Dynamic Contrast-Enhanced Magnetic Resonance Imaging. 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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. 15. Song T, Yao Q, Qu J, Zhang H, Zhao Y, Qin J, et al. The Value of Intravoxel Incoherent Motion Diffusion-Weighted Imaging in Predicting the Pathologic Response to Neoadjuvant Chemotherapy in Locally Advanced Esophageal Squamous Cell Carcinoma. Eur Radiol (2021) 31(3):1391–400. doi: 10.1007/ s00330-020-07248-z January 2022 \| Volume 11 \| Article 842169 Frontiers in Oncology \| www.frontiersin.org
https://openalex.org/W3128935392	https://www.nature.com/articles/s41438-021-00473-7.pdf	English	null	LlWRKY39 is involved in thermotolerance by activating LlMBF1c and interacting with LlCaM3 in lily (Lilium longiflorum)	Horticulture research	2,021	cc-by	11,256	© The Author(s) 2021 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/. Ding et al. Horticulture Research (2021) 8:36 https://doi.org/10.1038/s41438-021-00473-7 Ding et al. Horticulture Research (2021) 8:36 https://doi.org/10.1038/s41438-021-00473-7 Horticulture Research www.nature.com/hortres A R T I C L E O p e n A c c e s s Abstract WRKY transcription factors (TFs) are of great importance in plant responses to different abiotic stresses. However, research on their roles in the regulation of thermotolerance remains limited. Here, we investigated the function of LlWRKY39 in the thermotolerance of lily (Lilium longiﬂorum ‘white heaven’). According to multiple alignment analyses, LlWRKY39 is in the WRKY IId subclass and contains a potential calmodulin (CaM)-binding domain. Further analysis has shown that LlCaM3 interacts with LlWRKY39 by binding to its CaM-binding domain, and this interaction depends on Ca2+. LlWRKY39 was induced by heat stress (HS), and the LlWRKY39-GFP fusion protein was detected in the nucleus. The thermotolerance of lily and Arabidopsis was increased with the ectopic overexpression of LlWRKY39. The expression of heat-related genes AtHSFA1, AtHSFA2, AtMBF1c, AtGolS1, AtDREB2A, AtWRKY39, and AtHSP101 was signiﬁcantly elevated in transgenic Arabidopsis lines, which might have promoted an increase in thermotolerance. Then, the promoter of LlMBF1c was isolated from lily, and LlWRKY39 was found to bind to the conserved W-box element in its promoter to activate its activity, suggesting that LlWRKY39 is an upstream regulator of LlMBF1c. In addition, a dual-luciferase reporter assay showed that via protein interaction, LlCaM3 negatively affected LlWRKY39 in the transcriptional activation of LlMBF1c, which might be an important feedback regulation pathway to balance the LlWRKY39-mediated heat stress response (HSR). Collectively, these results imply that LlWRKY39 might participate in the HSR as an important regulator through Ca2+-CaM and multiprotein bridging factor pathways. LlWRKY39 is involved in thermotolerance by activating LlMBF1c and interacting with LlCaM3 in lily (Lilium longiﬂorum) Liping Ding1,2, Ze Wu1,2,3, Renda Teng1,2, Sujuan Xu1,2, Xing Cao4,5, Guozhen Yuan1,2, Dehua Zhang1,2 and Nianjun Teng 1,2 Introduction must produce various defense mechanisms against high temperature, including the accumulation of heat shock proteins (HSPs) and complex regulatory networks as established by transcription factors (TFs)4,5. High temperature is one of the unfavorable factors affecting the growth of plants, generally impairing pho- tosynthetic activity and negatively affecting cell division and growth1. Extreme high temperatures may result in a series of morphoanatomical and physiochemical changes in plant cells and even lead to severe economic losses in crops and other economically important plants2,3. Plants Lily (Lilium spp.) is one of the most popular cut ﬂower products worldwide because of its attractive shape and color6. Lily adapts well to cool conditions but is sensitive to high temperatures (>30 °C), which not only reduces the quality of cut ﬂowers but also leads to the degeneration of the bulb7. However, high temperatures will become an unavoidable environmental stress factor in the future because of the irreversible trend in global warming8,9. Therefore, an understanding of the HSR mechanisms of lily under HS is essential to improve the thermotolerance of lily. Correspondence: Nianjun Teng (njteng@njau.edu.cn) 1Key Laboratory of Landscaping Agriculture, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China 2 2Baguazhou Science and Technology Innovation Center of Modern Horticulture Industry, Nanjing 210043, China Full list of author information is available at the end of the article These authors contributed equally: Liping Ding, Ze Wu Correspondence: Nianjun Teng (njteng@njau.edu.cn) 1Key Laboratory of Landscaping Agriculture, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China 2Baguazhou Science and Technology Innovation Center of Modern Horticulture Industry, Nanjing 210043, China Full list of author information is available at the end of the article These authors contributed equally: Liping Ding, Ze Wu © The Author(s) 2021 © The Author(s) 2021 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/. Page 2 of 14 Ding et al. Horticulture Research (2021) 8:36 TFs play major roles in increasing the stress tolerance of plants since they can regulate critical downstream genes by binding to cis-elements in gene promoters10,11. In the HSR, HS transcription factors (HSFs) can directly regulate the expression of downstream genes by binding to HS elements (HSEs; nGAAnnTTCn) in the promoters of downstream genes in response to HS12,13. Currently, most studies on thermotolerance in lily focus on HSFs. The overexpression of LlHSFA1 and LlHSFA2 from lily in Arabidopsis can enhance the thermotolerance of trans- genic lines6,7,14. Two HSFA3 homologs of lily, LlHSFA3A and LlHSFA3B, increase the thermotolerance of transgenic Arabidopsis plants, possibly through a proline-mediated pathway15. In addition, LlHSFA3A and LlHSFA3B from lily can form a regulatory mechanism involving heat- inducible alternative splicing to sustain balance in the HSR16. Furthermore, lily LlDREB2B, a member of the DREB subfamily of the ERF/AP2 TF family, can increase the basal thermotolerance (BT) and acquired thermo- tolerance (AT) of transgenic Arabidopsis17. expression of target stress genes and thus increase plant stress tolerance29,32. In recent years, increasing evidence has shown that WRKYs are related to thermotolerance33–36. However, studies on WRKY TFs are primarily focused on crops or model plants such as rice, wheat, and Arabidopsis, and little research has been carried out on lily. In A. thaliana, the group II WRKY protein AtWRKY39 may play a positive role in thermotolerance by regulating the coop- eration between the SA- and JA-activated signaling pathways34. Here, we identify a WRKY-IId factor in lily, LlWRKY39, which was induced by HS and can interact with LlCaM3 in a Ca2+-dependent manner. The ther- motolerance of transgenic plants increased with the overexpression of LlWRKY39. © The Author(s) 2021 Further analysis indicated that LlWRKY39 can bind to the promoter of LlMBF1c and activate its expression. However, the interaction between LlCaM3 and LlWRKY39 negatively affected the transactivation of LlMBF1c induced by LlWRKY39, which may imply feedback regulation of LlWRKY39 to maintain a balance during the HSR. The results of this study may help to reveal the biological function and mechanism of LlWRKY39 under HS and provide an important theore- tical foundation for further perfecting the HS signal transduction network regulated by lily TFs. Multiprotein bridging factor 1 (MBF1) is a highly con- served transcriptional coactivator with various forms involved in the regulation of diverse processes, such as oxidative stress, hormone-regulated seed germination, and translation18–20. In Arabidopsis, MBF1 cofactors are enco- ded by three genes: AtMBF1a (AT2G42680), AtMBF1b (AT3G58680), and AtMBF1c (AT3G24500)21. Among these genes, AtMBF1c is related to thermotolerance and func- tions upstream of salicylic acid (SA), ethylene, and trehalose signaling22,23. A previous study reported that AtMBF1c is regulated by AtHsfA1 cofactors because an hsfa1 quadruple mutant shows suppressed expression of AtMBF1c during HS24. Despite much information available indicating how plant MBF1c genes respond to HS, many questions about the relationship between MBF1c and other important TFs, e.g., WRKY TFs, remain. LlWRKY39 is a heat-inducible member of the WRKY group IId family of transcription factors We searched AtWRKY39 from the Arabidopsis database with the online TAIR tool (https://www.arabidopsis.org/) and selected putative WRKY39 from the pollen tran- scriptome database of ‘little kiss’37. Then, we cloned puta- tive WRKY39 from ‘white heaven’. Thus, the candidate was designated LlWRKY39. The full-length cDNA sequence of LlWRKY39 contains an 858-bp open reading frame (ORF) that encodes a 285-amino acid protein. The phylogenetic tree including all WRKY proteins in Arabidopsis showed that LlWRKY39 is closely related to AtWRKY39, AtWRKY74, and AtWRKY21 (Supplementary Fig. S1), which suggests that LlWRKY39 is a member of the WRKY group IId family. To identify the basic characteristics of LlWRKY39, amino acid sequence alignment of LlWRKY39, AtWRKY39, AtWRKY74, and AtWRKY21 was performed. The sequences exhibited a similar signature, which included a WRKYGQK sequence, one zinc-binding motif C–X5–C–X23–H–X1–H, one HARF (RTGHARFRR[A/G] P) motif, a nuclear localization site (NLS), and a potential calmodulin (CaM)-binding domain (CBD) (Fig. 1b). Then, we investigated the evolutionary relationship between LlWRKY39 and WRKY39 factors in different plants, including Arabidopsis, tomato, apple, wheat, brachypodium, date, oil palm, and pineapple. The phylogenetic results indicated that the closest relationship was established WRKY TFs can participate in multiple adverse responses during plant growth and development25–27. The typical feature of WRKY TFs is the WRKY domain, which contains an invariant WRKYGQK sequence and a zinc ﬁnger motif (CX4–5CX22–23HXH or CX7CX23HXC)28,29. The WRKY superfamily is classiﬁed into three groups based on the number of conserved WRKY signatures (two WRKY sequences in group I and one WRKY sequence in groups II and III) and the composition of the zinc ﬁnger motif in which the zinc ﬁnger motif of groups I and II is CX4–5CX22–23HXH and the zinc ﬁnger motif of group III is CX7CX23HXC22. Group II is further classiﬁed into ﬁve subgroups (IIa to IIe) based on different conserved short motifs22,28. WRKY TFs can recognize and bind to W-box elements (TTGACC/T) in the promoters of resistance- related genes. The core sequence TGAC is essential for WRKY recognition, as indicated by different binding experiments30,31. This type of binding can regulate the Page 3 of 14 Ding et al. Horticulture Research (2021) 8:36 Fig. 1 LlWRKY39 is a heat-inducible member of the WRKY group IId of transcription factors. a Structural diagram of the WRKY IId transcription factor. b Multiple alignments of LlWRKY39 with AtWRKY39 (AT3G04670), AtWRKY74 (AT5G28650), and AtWRKY21 (AT2G30590) using the Clustal W algorithm with default parameters. LlWRKY39 is a heat-inducible member of the WRKY group IId family of transcription factors The approximate 60-amino acid WRKY domain, CaM-binding domain (CBD), HARF motif, and nuclear localization site (NLS) are indicated by black boxes. c Phylogenetic relationship of LlWRKY39 with other plant WRKY proteins was determined by the neighbor-joining method with 500 bootstrap replicates. LlWRKY39 is highlighted with a purple triangle. The following abbreviations are used to indicate the scientiﬁc names of plants with WRKY: AC ananas comosus, EG elaeis guineensis, PD phoenix dactylifera, BD brachypodium distachyon, TA triticum aestivum, AT arabidopsis thaliana, MD malus domestics, Sl solanum lycopersicum. d Relative expression of LlWRKY39 in lily leaves under HS for different lengths of time. Each bar indicates the mean ± SD of three repeated experiments (P < 0.05 and P < 0.01, Student’s t test). e Analysis of the promoter activity in N. benthamiana leaves under room temperature (RT) and HS (37 °C 2 h). f The relative value of LUC/REN. The ratio of LUC/REN at RT was set to 1 for normalization. All the values represent the mean ± SD of three repeated experiments (P < 0.05, Student’s t test) Fig. 1 LlWRKY39 is a heat-inducible member of the WRKY group IId of transcription factors. a Structural diagram of the WRKY IId transcription factor. b Multiple alignments of LlWRKY39 with AtWRKY39 (AT3G04670), AtWRKY74 (AT5G28650), and AtWRKY21 (AT2G30590) using the Clustal W algorithm with default parameters. The approximate 60-amino acid WRKY domain, CaM-binding domain (CBD), HARF motif, and nuclear localization site (NLS) are indicated by black boxes. c Phylogenetic relationship of LlWRKY39 with other plant WRKY proteins was determined by the neighbor-joining method with 500 bootstrap replicates. LlWRKY39 is highlighted with a purple triangle. The following abbreviations are used to indicate the scientiﬁc names of plants with WRKY: AC ananas comosus, EG elaeis guineensis, PD phoenix dactylifera, BD brachypodium distachyon, TA triticum aestivum, AT arabidopsis thaliana, MD malus domestics, Sl solanum lycopersicum. d Relative expression of LlWRKY39 in lily leaves under HS for different lengths of time. Each bar indicates the mean ± SD of three repeated experiments (P < 0.05 and P < 0.01, Student’s t test). e Analysis of the promoter activity in N. benthamiana leaves under room temperature (RT) and HS (37 °C 2 h). f The relative value of LUC/REN. The ratio of LUC/REN at RT was set to 1 for normalization. LlWRKY39 is a heat-inducible member of the WRKY group IId family of transcription factors All the values represent the mean ± SD of three repeated experiments (P < 0.05, Student’s t test) Page 4 of 14 Ding et al. Horticulture Research (2021) 8:36 between LlWRKY39 and pineapple AcWRKY39 (Fig. 1c), which are both noncereal monocot species. between LlWRKY39 and pineapple AcWRKY39 (Fig. 1c), which are both noncereal monocot species. LlWRKY39 under HS, we conducted a transient luciferase reporter assay with Nicotiana benthamiana leaves. The LUC signal increased profoundly after subjection to HS, although under normal conditions, the LUC signal was very low, which implied that HS activated the promoter activity of LlWRKY39 (Fig. 1e, f). Thus, these results indicate that LlWRKY39 is a heat-inducible WRKY group IId factor. LlWRKY39 under HS, we conducted a transient luciferase reporter assay with Nicotiana benthamiana leaves. The LUC signal increased profoundly after subjection to HS, although under normal conditions, the LUC signal was very low, which implied that HS activated the promoter activity of LlWRKY39 (Fig. 1e, f). Thus, these results indicate that LlWRKY39 is a heat-inducible WRKY group IId factor. LlWRKY39 can be rapidly induced under HS at 37 °C for 0.5 h, and following prolonged HS for 12 h, the expression of LlWRKY39 is enhanced signiﬁcantly (Fig. 1d). The expression results suggested that LlWRKY39 is induced by high temperature over a long period. To further examine the underlying mechanism of LlWRKY39 under HS, we isolated and analyzed the promoter of LlWRKY39. Different kinds of cis-elements were found in the promoter of LlWRKY39, such as light-responsive elements (I-BOX and GATA-BOX), an element for pollen-speciﬁc expression (POLLEN1), a binding site for WRKY TFs (W-box), CCAAT-BOX, and others, indicating that LlWRKY39 may be regulated by different types of TFs (Supplementary Table S1). Among these motifs, CCAAT-BOX is related to HS, and it can act cooperatively with HSEs to increase HS promoter activity38. To identify the promoter activity of LlCaM3 interacts with LlWRKY39 by binding the CBD In the BIFC assay, LlWRKY39 with the CBD interacted with LlCaM3, but LlWRKY39 lacking the CBD did not (Fig. 2e). Notably, the interaction signal was observed throughout the cytoplasm and nucleus, which may be explained by the deletion of the NLS of LlWRKY39 and LlCaM3 in the cytoplasm and nucleus30. In a split-ubiquitin assay with yeast cells, similar results were observed (Supplementary Fig. S3), which implies that the interaction between LlCaM3 and LlWRKY39 depends on the CBD region. cytoplasmic and nuclear protein39. Here, we found that the ﬂuorescence of the LlWRKY39-GFP fusion protein appeared only in the nucleus (Fig. 2b), which might explain why the ﬂuorescence emitted by LlWRKY39 interacting with LlCaM3 appeared only in the nucleus. Similarly, ﬁreﬂy luciferase complementation imaging (FLC) assays also showed that LlWRKY39 interacted with LlCaM3 (Fig. 2c). However, compared with that of homologous genes, the CBD of LlWRKY39 is not highly conserved (Fig. 2d). Therefore, we sought to determine whether the interaction between LlWRKY39 and LlCaM3 depends on this domain. We truncated LlWRKY39 into two parts, one segment with the CBD and one without the CBD. In the BIFC assay, LlWRKY39 with the CBD interacted with LlCaM3, but LlWRKY39 lacking the CBD did not (Fig. 2e). Notably, the interaction signal was observed throughout the cytoplasm and nucleus, which may be explained by the deletion of the NLS of LlWRKY39 and LlCaM3 in the cytoplasm and nucleus30. In a split-ubiquitin assay with yeast cells, similar results were observed (Supplementary Fig. S3), which implies that the interaction between LlCaM3 and LlWRKY39 depends on the CBD region. ion chelator ethylene glycol tetraacetic acid (EGTA) treatments. The interaction signal in the leaves treated with CaCl2 was stronger than that in the leaves treated with water, but the interaction signal was signiﬁcantly repressed in the leaves treated with EGTA, which implies that the LlCaM3–LlWRKY39 interaction might depend on Ca2+ (Fig. 3). LlCaM3 interacts with LlWRKY39 by binding the CBD In the protein sequence assay, a potential CBD was found in LlWRKY39 (Fig. 1b), which suggests that LlWRKY39 is a CaM-binding protein (CBP). Therefore, we evaluated whether LlWRKY39 interacted with a heat- inducible CaM, LlCaM3, from lily (Supplementary Fig. S2). Bimolecular ﬂuorescence complementation (BIFC) showed that the ﬂuorescence generated upon LlWRKY39 interacting with LlCaM3 was emitted only in the nucleus (Fig. 2a). A previous study showed that LlCaM3 is a Fig. 2 Interaction between LlWRKY39 and LlCaM3 by binding the CaM-binding domain. a BIFC assay. Fluorescence signals were observed using a confocal microscope. Bars = 50 μm. b Transient expression of LlWRKY39 in N. benthamiana leaves. Bar = 50 μm. c FLC assay. LUC signals were observed using a CCD camera. d Alignment of the CBD among WRKY group IId members in Arabidopsis, oil palm, wheat, and lily. Identical amino acids are shaded in black and gray. e Interaction of the CBD of LlWRKY39 and LlCaM3 as determined by BIFC. Bar = 50 μm Fig. 2 Interaction between LlWRKY39 and LlCaM3 by binding using a confocal microscope. Bars = 50 μm. b Transient expression Fig. 2 Interaction between LlWRKY39 and LlCaM3 by binding the CaM-binding domain. a BIFC assay. Fluorescence signals were observed using a confocal microscope. Bars = 50 μm. b Transient expression of LlWRKY39 in N. benthamiana leaves. Bar = 50 μm. c FLC assay. LUC signals were observed using a CCD camera. d Alignment of the CBD among WRKY group IId members in Arabidopsis, oil palm, wheat, and lily. Identical amino acids are shaded in black and gray. e Interaction of the CBD of LlWRKY39 and LlCaM3 as determined by BIFC. Bar = 50 μm Page 5 of 14 Ding et al. Horticulture Research (2021) 8:36 cytoplasmic and nuclear protein39. Here, we found that the ﬂuorescence of the LlWRKY39-GFP fusion protein appeared only in the nucleus (Fig. 2b), which might explain why the ﬂuorescence emitted by LlWRKY39 interacting with LlCaM3 appeared only in the nucleus. Similarly, ﬁreﬂy luciferase complementation imaging (FLC) assays also showed that LlWRKY39 interacted with LlCaM3 (Fig. 2c). However, compared with that of homologous genes, the CBD of LlWRKY39 is not highly conserved (Fig. 2d). Therefore, we sought to determine whether the interaction between LlWRKY39 and LlCaM3 depends on this domain. We truncated LlWRKY39 into two parts, one segment with the CBD and one without the CBD. Overexpression of LlWRKY39 increases the thermotolerance of transgenic plants In a transient expression assay, the overexpression of LlWRKY39 in lily leaves did not affect their relative ion leakage under normal growth conditions. However, the relative ion leakage of ‘white heaven’ leaves in the control was signiﬁcantly higher than that of leaves transformed with LlWRKY39 under HS, indicating that the transient expression of LlWRKY39 can protect the cells of lily from high temperature (Fig. 4a, b). Four homozygous T3 transgenic Arabidopsis lines were identiﬁed (Supple- mentary Fig. S4), and three transgenic Arabidopsis lines (OE-2, OE-4, and OE-5) were selected for BT and AT analyses. The survival rate of the WT plants was sig- niﬁcantly lower than that of the three transgenic lines under both HS conditions (Fig. 4d, e), which showed that the overexpression of LlWRKY39 can increase the BT and AT of transgenic Arabidopsis. In addition, the expression levels of HS-inducible genes AtHSFA1, AtHSFA2, AtHSP101, AtDREB2A, AtMBF1c, AtGolS1, and LlCaM3–LlWRKY39 interaction depends on Ca2+ To determine whether the LlCaM3–LlWRKY39 inter- action depended on Ca2+, we conducted a transient FLC assay with N. benthamiana leaves and CaCl2 and calcium Fig. 3 Effects of CaCl2 and EGTA treatment on the interaction between LlWRKY39 and LlCaM3 in N. benthamiana leaves. a LUC bioluminescence in N. benthamiana leaves coinﬁltrated with mixed bacterial solutions of LlWRKY39-cLUC and LlCaM3-nLUC by different treatments. b, d LUC bioluminescence intensity was quantiﬁed using Andor Solis v15 software. c LUC bioluminescence in N. benthamiana leaves coinﬁltrated with mixed bacterial solutions of LlWRKY39-nLUC and LlCaM3-cLUC by different treatments. Values represent the mean ± SD of three independent experiments. Signiﬁcant differences are indicated by Student–Newman–Keuls test Fig. 3 Effects of CaCl2 and EGTA treatment on the interaction between LlWRKY39 and LlCaM3 in N. benthamiana leaves. a LUC bioluminescence in N. benthamiana leaves coinﬁltrated with mixed bacterial solutions of LlWRKY39-cLUC and LlCaM3-nLUC by different treatments. b, d LUC bioluminescence intensity was quantiﬁed using Andor Solis v15 software. c LUC bioluminescence in N. benthamiana leaves coinﬁltrated with mixed bacterial solutions of LlWRKY39-nLUC and LlCaM3-cLUC by different treatments. Values represent the mean ± SD of three independent experiments. Signiﬁcant differences are indicated by Student–Newman–Keuls test Ding et al. Horticulture Research (2021) 8:36 Page 6 of 14 Fig. 4 (See legend on next page.) Fig. 4 (See legend on next page.) Page 7 of 14 Ding et al. Horticulture Research (2021) 8:36 (see ﬁgure on previous page) Fig. 4 Thermotolerance assay in lily and transgenic Arabidopsis lines. a The expression of LlWRKY39 in transient-overexpressing lily. b Relative ion leakage (%) of lily leaves under RT and HS (42 °C, 2 h). Each bar indicates the mean ± SD of three repeated experiments (t test, P < 0.05). c Phenotypes of one-week-old seedlings before exposure to HS. d Phenotypes of one-week-old seedlings after the BT test. left: schematic representations of BT; right: survival rate of BT. e Phenotypes of one-week-old seedlings after the AT test. left: schematic representations of AT; right: survival rate of AT. A bar shows the mean of three independent experiments (P < 0.05, t test). f Relative expression levels of HS-inducible genes in the transgenic plants. Each bar indicates the mean ± SD of three repeated experiments. Signiﬁcant differences are indicated by t test (P < 0.05 and P < 0.01) AtWRKY39 increased signiﬁcantly in transgenic Arabi- dopsis (Fig. LlWRKY39 activates the expression of LlMBF1c LlWRKY39 activates the expression of LlMBF1c MBF1c is an extremely conserved transcriptional coactivator that plays an important role in the HSR22,23. Given that the overexpression of LlWRKY39 can increase the expression of AtMBF1c in Arabidopsis (Fig. 4f), Agrobacterium-mediated transient transformation was performed with lily leaves to verify whether the same regulation mode exists in lily. The overexpression of LlWRKY39 in lily activated the expression of LlMBF1c (Fig. 5a), although LlWRKY39 showed no transactivation activity in yeast cells (Supplementary Fig. S6). In addition, LlMBF1c and LlWRKY39 shared similar expression pat- terns under HS (Supplementary Fig. S7). To further identify the regulatory mechanism between LlWRKY39 and LlMBF1c, we isolated and analyzed the promoter of LlMBF1c. The LlMBF1c promoter contained various cis- elements, such as drought responsiveness elements (MYB2AT); light responsiveness elements (I-BOX and GATA-BOX); CCAAT-BOX, the binding site of the WRKY TF family (W-box); and others (Supplementary Table S2). Then, we conducted a yeast one-hybrid assay to conﬁrm that LlWRKY39 binds to the W-box element in the promoter region (–500 to –486 bp) of LlMBF1c. The yeast cells cotransformed with LlWRKY39 and the W-box element of the LlMBF1c promoter survived on SD med- ium without Leu, Trp, and His (SD-LWH), even in the presence of 75 mM 3-amino-1,2,4-triazole (3-AT), which implies that LlWRKY39 has a high afﬁnity for the W-box LlCaM3 negatively affects LlWRKY39 in the transactivation of LlMBF1c To understand how the LlWRKY39–LlCaM3 interac- tion affects the function of LlWRKY39, we performed a dual-luciferase reporter assay. The ratio of LUC/REN when coexpressing LlWRKY39 together with LlCaM3 was signiﬁcantly lower than that when only LlWRKY39 was expressed (Fig. 6b), which suggests that the LlWRKY39- LlCaM3 interaction represses the activation ability of LlWRKY39 for its target genes. LlCaM3–LlWRKY39 interaction depends on Ca2+ 4f), which might facilitate the increase in thermotolerance of transgenic lines. Unexpectedly, the expression of AtHSFB2A, a transcriptional repressor40, was also signiﬁcantly induced. In addition, the expression of AtHSP70, AtAPX1, AtAPX2, and AtHSFA3, which are the downstream genes of AtHSFA1, AtHSFA2, and AtDREB2A41–43, did not change signiﬁcantly, suggesting that LlWRKY39 also participated in other pathways to control the expression of these genes. We also measured the expression level of genes such as LlHSFA1, LlHSFA2, and LlDREB2B in transiently overexpressed LlWRKY39 lily leaves. The qRT-PCR results showed that the expression levels of the LlHSFA1, LlHSFA2, and LlDREB2B genes were enhanced in lily leaves over- expressing LlWRKY39 (Supplementary Fig. S5). element (GTCAA). However, when the GTCAA sequence was mutated to TTCAC, LlWRKY39 failed to bind to it (Fig. 5c), implying that the G and A residues in GTCAA are essential for the recognition and combination of LlWRKY39. To further verify these results, we performed an electrophoretic mobility shift assay (EMSA). As shown in Fig. 5e, the LlWRKY39-HIS complex bound to the W- box element in the LlMBF1c promoter and produced a mobility shift, which implies that LlWRKY39 binds to the LlMBF1c promoter via the W-box to regulate its expres- sion. The effector-reporter assay showed that the N. benthamiana leaves cotransformed with control combi- nations emitted a very low LUC signal, but the signal increased signiﬁcantly after cotransformation with LlWRKY39 and proLlMBF1c-LUC, and the ratio of LUC/ REN was also signiﬁcantly higher than that of the control (Fig. 5g, h). Therefore, these results suggest that LlWRKY39 can activate the expression of LlMBF1c and that this activation may be achieved by directly binding the LlMBF1c promoter. Discussion Calcium, a universal secondary messenger, is one of the main signal transducers and regulators in plant cells44–47. The transient changes in cytosolic Ca2+ concentration are called Ca2+ signatures, and Ca2+ sensors respond to these changes by activating or inactivating target proteins to participate in speciﬁc biochemical processes and regulate gene expression48. Most Ca2+ sensors are proteins with one or more EF-hands that have Ca2+-binding helix-turn- helix structures49. Ca2+ sensors with EF-hands are roughly classiﬁed into two groups: sensor responders and sensor relays50,51. CaMs are a group of well-characterized Ca2+ sensor relays that do not have any functional Ding et al. Horticulture Research (2021) 8:36 Page 8 of 14 Fig. 5 LlWRKY39 activates the expression of LlMBF1c. a Transient expression of LlWRKY39 in lily increased the expression of LlMBF1c. Each bar indicates the mean ± SD of three repeated experiments. Signiﬁcant differences between the control and transient overexpression plants were determined by t test (P < 0.05). b The sequence of the LlMBF1c promoter and its W-box mutated version; the red label indicates the W-box element. c Yeast one-hybrid assay. The interaction between LlWRKY39 and W-box elements was determined in SD-LWH medium with different concentrations of 3-AT. d Schematic diagram of the LlMBF1c promoter; the probe sequence is shown below the diagram. e LlWRKY39-HIS complex binds to the W- box in EMSA. The term 250× indicates the usage of excess unlabeled probe as a competitor, and “+” and “−” indicate its presence and absence, respectively. f The schematic diagram of the effector and reporter. The 836-bp fragment of the LlMBF1c promoter was used in this assay. g Bright ﬁeld and dark ﬁeld images of N. benthamiana leaves in the transient expression assays. h The ratio of LUC/REN. 1: mixed bacterial solutions of pGreenII-62- SK and pGreenII-0800-LUC (2:1); 2: mixed bacterial solutions of pGreenII-62-SK-LlWRKY39 and pGreenII-0800-LUC (2:1); 3: mixed bacterial solutions of pGreenII-62-SK and pGreenII-0800-proLlMBF1c-LUC (2:1); 4: mixed bacterial solutions of pGreenII-62-SK-LlWRKY39 and pGreenII-0800-proLlMBF1c-LUC (2:1). Each bar indicates the mean ± SD of three repeated experiments (P < 0.05, t test) Fig. 5 LlWRKY39 activates the expression of LlMBF1c. a Transient expression of LlWRKY39 in lily increased the expression of LlMBF1c. Each bar indicates the mean ± SD of three repeated experiments. Signiﬁcant differences between the control and transient overexpression plants were determined by t test (P < 0.05). Discussion In addition, although AtHSFA1, AtHSFA2, and AtDREB2A were induced in the transgenic plants, the expression of their target genes41,43, AtHSP70, AtAPX1, AtAPX2, and AtHSFA3, was not affected (Fig. 4f), which also suggests that another negative pathway exists in the LlWRKY39- mediated HSR to sustain the balance in the HSR. Among the detected genes, an important MBF1, AtMBF1c, was signiﬁcantly induced in transgenic Arabidopsis. MBF1 proteins are highly conserved transcriptional coactivators that can build bridges between TFs and transcriptional regulation machinery22,61–63. MBF1c is a critical heat- response regulator in the HSR and functions upstream of SA, trehalose, and ethylene signaling to participate in the establishment of thermotolerance22,23. The homolog of MBF1c in lily, LlMBF1c, can be activated by LlWRKY39 by directly binding the W-box element in the prompter of LlMBF1c (Fig. 5), which suggested that LlWRKY39 might be an upstream regulator of LlMBF1c. domains; however, when they bind to Ca2+, they can activate or inactivate interacting proteins by relaying the signal52. AtWRKY7 was the ﬁrst WRKY TF to be reported to interact with CaM in a Ca2+-dependent manner by binding a short conserved motif (VSSFK[K/R]VISLL in the C-region), which is also called a CBD domain53. AtWRKY7 belongs to the WRKY group IId subfamily, and all members of this subfamily interact with Ca2+/CaM in Arabidopsis53. Here, we identiﬁed a new WRKY IId pro- tein, LlWRKY39, from lily, which has a similar primary motif (HARF:RTGHARFRR[A/G]P) that is the distinctive characteristic of WRKY group IId TFs28. Additionally, LlCaM3, which is reportedly associated with HS in lily39, interacts with LlWRKY39 by binding the CBD in a Ca2+- dependent manner (Figs. 2 and 3). Ca2+-dependent CBD motifs are normally grouped into two categories, namely, motifs 1-5-10 and 1-8-14, the numbers of which imply the positions of conserved hydrophobic residues54. The CBD of AtWRKY7 does not belong to either of the two classical CBD motifs; however, AtWRKY7 is a Ca2+-dependent CBP53. LlWRKY39 may be similar to AtWRKY7, with a CBD that is not consistent with either of the two major classes (Fig. 2d). In this study, the interaction between LlWRKY39 and LlCaM3 depends on the CBD in the N- region of LlWRKY39, although it is not completely con- sistent with the CBD of the WRKY IId TFs in Arabidopsis (Fig. 2c, d), which suggests that the basic CBD can interact with the CaM. Discussion b The sequence of the LlMBF1c promoter and its W-box mutated version; the red label indicates the W-box element. c Yeast one-hybrid assay. The interaction between LlWRKY39 and W-box elements was determined in SD-LWH medium with different concentrations of 3-AT. d Schematic diagram of the LlMBF1c promoter; the probe sequence is shown below the diagram. e LlWRKY39-HIS complex binds to the W- box in EMSA. The term 250× indicates the usage of excess unlabeled probe as a competitor, and “+” and “−” indicate its presence and absence, respectively. f The schematic diagram of the effector and reporter. The 836-bp fragment of the LlMBF1c promoter was used in this assay. g Bright ﬁeld and dark ﬁeld images of N. benthamiana leaves in the transient expression assays. h The ratio of LUC/REN. 1: mixed bacterial solutions of pGreenII-62- SK and pGreenII-0800-LUC (2:1); 2: mixed bacterial solutions of pGreenII-62-SK-LlWRKY39 and pGreenII-0800-LUC (2:1); 3: mixed bacterial solutions of pGreenII-62-SK and pGreenII-0800-proLlMBF1c-LUC (2:1); 4: mixed bacterial solutions of pGreenII-62-SK-LlWRKY39 and pGreenII-0800-proLlMBF1c-LUC (2:1). Each bar indicates the mean ± SD of three repeated experiments (P < 0.05, t test) Ding et al. Horticulture Research (2021) 8:36 Page 9 of 14 Fig. 6 Effect of LlCaM3 on the activation activity of LlWRKY39 at the promoter of LlMBF1c in N. benthamiana leaves according to a dual- luciferase reporter assay. a The construction of the effect vectors and reporter vector. b Ratio of LUC/REN. Mixed bacterial solutions of effectors and reporter cultures (3:1). Each bar indicates the mean ± SD of three repeated experiments (P < 0.05, t test) Fig. 6 Effect of LlCaM3 on the activation activity of LlWRKY39 at the promoter of LlMBF1c in N. benthamiana leaves according to a dual- luciferase reporter assay. a The construction of the effect vectors and reporter vector. b Ratio of LUC/REN. Mixed bacterial solutions of effectors and reporter cultures (3:1). Each bar indicates the mean ± SD of three repeated experiments (*P < 0.05, t test) the HS-responsive genes, namely, AtHSFA1, AtHSFA2, AtDREB2A, AtMBF1c, AtHSP101, and AtGolS1, were sig- niﬁcantly upregulated in the transgenic lines (Fig. 4f). Their elevated expression may contribute to an increase in thermotolerance since all of these genes play positive roles in thermotolerance22,34,55–57. Notably, the basal expression of AtWRKY39 was also upregulated in the transgenic lines, possibly because of the self-activation of endogenous AtWRKY39. Discussion W-box elements in the promoters of stress- induced genes and WRKY genes indicate that WRKY TFs can also be self-regulated, e.g., WRKY33. The expression level of AtWRKY33 is usually low in healthy plants, but when plants are exposed to environmental stimuli, acti- vated AtWRKY33 induces its own expression to generate a feedback mechanism for the rapid and strong induction of AtWRKY33 target genes that respond to biotic or abiotic stresses58–60. Nevertheless, HSFB2A, a transcriptional repressor40, is also induced, which is speculated to be a mechanism that maintains the balance in the HSR. In addition, although AtHSFA1, AtHSFA2, and AtDREB2A were induced in the transgenic plants, the expression of their target genes41,43, AtHSP70, AtAPX1, AtAPX2, and AtHSFA3, was not affected (Fig. 4f), which also suggests that another negative pathway exists in the LlWRKY39- mediated HSR to sustain the balance in the HSR. Among the detected genes, an important MBF1, AtMBF1c, was signiﬁcantly induced in transgenic Arabidopsis. MBF1 proteins are highly conserved transcriptional coactivators that can build bridges between TFs and transcriptional regulation machinery22,61–63. MBF1c is a critical heat- response regulator in the HSR and functions upstream of SA, trehalose, and ethylene signaling to participate in the establishment of thermotolerance22,23. The homolog of MBF1c in lily, LlMBF1c, can be activated by LlWRKY39 by directly binding the W-box element in the prompter of LlMBF1c (Fig. 5), which suggested that LlWRKY39 might be an upstream regulator of LlMBF1c. the HS-responsive genes, namely, AtHSFA1, AtHSFA2, AtDREB2A, AtMBF1c, AtHSP101, and AtGolS1, were sig- niﬁcantly upregulated in the transgenic lines (Fig. 4f). Their elevated expression may contribute to an increase in thermotolerance since all of these genes play positive roles in thermotolerance22,34,55–57. Notably, the basal expression of AtWRKY39 was also upregulated in the transgenic lines, possibly because of the self-activation of endogenous AtWRKY39. W-box elements in the promoters of stress- induced genes and WRKY genes indicate that WRKY TFs can also be self-regulated, e.g., WRKY33. The expression level of AtWRKY33 is usually low in healthy plants, but when plants are exposed to environmental stimuli, acti- vated AtWRKY33 induces its own expression to generate a feedback mechanism for the rapid and strong induction of AtWRKY33 target genes that respond to biotic or abiotic stresses58–60. Nevertheless, HSFB2A, a transcriptional repressor40, is also induced, which is speculated to be a mechanism that maintains the balance in the HSR. Discussion In the present study, the induction of LlWRKY39 expression by HS implies that LlWRKY39 might partici- pate in the regulation of HSR (Fig. 1d). Therefore, we identiﬁed the function of LlWRKY39 in the HSR process. The overexpression of LlWRKY39 increased the thermo- tolerance of lily and Arabidopsis (Fig. 4b, d, e). These results were consistent with those of previous studies on AtWRKY39 in Arabidopsis, in which the overexpressing plants showed greater thermotolerance during both seed germination and seedling growth than wrky39 mutants or wild-type plants under HS34. In the current study, some of The proteins of the CaM-binding subgroup, AtWRKY7, AtWRKY11, and AtWRKY17, of the WRKY group IId Page 10 of 14 Page 10 of 14 Ding et al. Horticulture Research (2021) 8:36 Ca2+/CaM played an important role in inhibiting excessive activation of LlWRKY39-mediated thermotolerance to sustain balance in the HSR. Additional examples show that MBF1 proteins can reduce tolerance to stresses, although MBF1 transcription cofactors often play positive roles against many stresses. For example, in pepper, CaMBF1 transcript levels are dramatically decreased in response to salt or cold stress, which suggests that MBF1 family factors have a negative effect on the stress response66. In addition, AtMBF1 genes can relieve abscisic acid (ABA)-dependent inhibition of germination, which implies that MBF1 pro- teins negatively regulate the ABA-dependent response to some extent19. ABA signaling can also be involved in the HSR67,68. In this study, the interaction between LlCaM3 and LlWRKY39 weakened the transcriptional activity of LlMBF1c activated by LlWRKY39 to a certain extent, suggesting that this feedback regulation balances the HSR to prevent the damage caused by excessive activation responses. There may be a similar regulatory mechanism in the downstream genes of LlMBF1c because some genes that negatively regulate thermotolerance in transgenic lines were upregulated, e.g., HSFB2A, and the expression of downstream HSP genes was not changed, e.g., HSP70, implying the existence of negative feedback regulation in the LlWRKY39-mediated HSR. proteins, play negative roles in the plant basal defense response64,65. wrky7 mutants exhibited increased resistance to Pseudomonas syringae 1, whereas plants overexpressing AtWRKY7 displayed a higher sensitivity to the pathogen by repressing the expression of SA-regulated defense genes or repressing weakly activated jasmonic acid (JA) signaling64. Similarly, wrky11 mutants showed increased resistance to P. Materials and methods Plant materials and growth conditions Plant materials and growth conditions The lily hybrid Lilium longiﬂorum ‘white heaven’ used in this experiment was cultured on Murashige and Skoog (MS) medium. Arabidopsis thaliana (Col-0) and N. ben- thamiana were grown in potting medium. All plant materials were cultured in a growth room at 22 °C with a 16-h photoperiod. Fig. 7 A proposed working model of LlWRKY39-mediated HSR under HS. When lily suffers from high temperature, LlWRKY39 is Fig. 7 A proposed working model of LlWRKY39-mediated HSR under HS. When lily suffers from high temperature, LlWRKY39 is rapidly induced, and its protein directly binds to the W-box element on the promoter of LlMBF1c to activate its expression. The feedback regulation mechanism also begins to respond, and Ca2+ ﬂows into the cell to bind and activate LlCaM3; then, LlCaM3 interacts with LlWRKY39 to inhibit its activation-inducing function promoting the expression of downstream genes, e.g., LlMBF1c, which might contribute to the prevention of the side effects caused by excessive activation to sustain balance in the HSR under HS. When lily suffers from high temperature, LlWRKY39 is rapidly induced, and its protein directly binds to the W-box element on the promoter of LlMBF1c to activate its expression. The feedback regulation mechanism also begins to respond, and Ca2+ ﬂows into the cell to bind and activate LlCaM3; then, LlCaM3 interacts with LlWRKY39 to inhibit its activation-inducing function promoting the expression of downstream genes, e.g., LlMBF1c, which might contribute to the prevention of the side effects caused by excessive activation to sustain balance in the HSR Discussion syringae 1, and wrky11 and wrky17 double mutants exhibited a higher increase in resistance, which was due to the decreased JA levels and the downregulation of JA- responsive genes65. In contrast to previous studies, LlWRKY39 activated the expression of the downstream gene LlMBF1c. Additionally, the interaction between LlWRKY39 and LlCaM3 repressed the expression of LlMBF1c activated by LlWRKY39 (Fig. 6), suggesting that Fig. 7 A proposed working model of LlWRKY39-mediated HSR under HS. When lily suffers from high temperature, LlWRKY39 is rapidly induced, and its protein directly binds to the W-box element on the promoter of LlMBF1c to activate its expression. The feedback regulation mechanism also begins to respond, and Ca2+ ﬂows into the cell to bind and activate LlCaM3; then, LlCaM3 interacts with LlWRKY39 to inhibit its activation-inducing function promoting the expression of downstream genes, e.g., LlMBF1c, which might contribute to the prevention of the side effects caused by excessive activation to sustain balance in the HSR In conclusion, LlWRKY39 may act as a downstream component of the CaM-mediated calcium signaling pathway that lies upstream of LlMBF1c in the HSR. Considering these ﬁndings, we propose a simpliﬁed working model that may shed light on the mechanisms of the LlWRKY39-mediated HSR (Fig. 7). When lily is exposed to high temperature, LlWRKY39 is rapidly induced, which directly activates the expression of LlMBF1c and participates in the establishment of the HSR. Simultaneously, the feedback regulation mechanism also begins to respond, and Ca2+ enters the cell to activate LlCaM3, which upon interaction with LlWRKY39, can contribute to the prevention of the side effects caused by excessive activation to sustain balance in the HSR. CaCl2 and EGTA treatment The bacterial solutions of pCAMBIA1300-LlWRKY39 and pCAMBIA1300 (control) were collected by cen- trifugation and resuspended in the same buffer as described for the CaCl2 and EGTA treatment. The resuspended bacterial solutions were placed in the dark for 5 h and inﬁltrated into the leaves of ‘white heaven’. After 72 h, the inﬁltrated leaves were harvested for qRT- PCR analysis. The bacterial solutions (LlWRKY39-cLUC with LlCaM3-nLUC and LlWRKY39-nLUC with LlCaM3- cLUC) were collected by centrifugation and resuspended in buffer (10 mM MgCl2, 200 mM acetosyringone (AS), 10 mM 2-morpholino ethanesulfonic acid (MES), pH 5.6) to a ﬁnal OD600 of 1.0. Then, the resuspended bacterial solutions were placed in the dark for 3 h. To induce transient expression, 1-cm diameter samples were cut from N. benthamiana leaves with a puncher. Then, the samples were placed into the bacterial suspension and inﬁltrated under vacuum for 10–15 min until the samples became transparent. After the vacuum was released, the samples were placed on medium (0.4% agar) in the dark. After 24 h, the samples were transferred to medium (0.4% agar) with CaCl2 (20 mM) or EGTA (20 mM) and incu- bated for 24 h. The samples on medium (0.4% agar) were Transactivation activity assay of LlWRKY39 in yeast Transactivation activity assay of LlWRKY39 in yeast The ORF of LlWRKY39 was inserted into the pGBKT7 vector using speciﬁc primers (Supplementary Table S3). The pGBKT7 vector, pGBKT7-GAL4, and pGBKT7- LlWRKY39 were transformed into yeast strain AH109. The transformed yeast cells were incubated on SD med- ium lacking Trp at 30 °C for 3 days. Positive clones were selected on SD medium (lacking Trp and His) containing 3-AT at 30 °C for 3 days. Heat stress treatment of lily To detect gene expression levels under HS, one-month- old ‘white heaven’ plants were exposed to 37 °C for 0, 0.5, 1, 3, 6, and 12 h. Heat treatment was applied in a tem- perature incubator (GZL-P80-A, Nanjing, China) without light. Samples of leaves were harvested for qRT-PCR analysis after heat treatment. Molecular cloning and sequence analysis of LlWRKY39 Total RNA was extracted from the leaves of ‘white heaven’ with TRIzol reagent according to the manu- facturer’s instructions (Invitrogen, USA). cDNA was bio- synthesized by a reverse transcription system (TaKaRa, Page 11 of 14 Ding et al. Horticulture Research (2021) 8:36 used as controls. The ﬂuorescence signal was observed with a luminometer (PIXIS1024B, China). The LUC activity measurement was performed using Andor Solis v15 software as described in a previous study69. Japan). Primers for gene cloning are listed in Supple- mentary Table S3. The physicochemical properties of LlWRKY39 were estimated by EXPASY (http://web. expasy.org/compute_pi/, default setting). Protein interaction assays y In the BIFC assay, the ORFs without stop codons of LlWRKY39 and LlCaM3 were ampliﬁed and inserted into pSPYCE (M) and pSPYNE173 vectors using a recombinant ligase (Vazyme, Nanjing, China). The reconstructed vectors and empty vectors were transformed into Agrobacterium tumefaciens strain GV3101. The combination of bacterial solutions (YCE:YNE:P19 = 3:3:1) was coinﬁltrated into the leaves of N. benthamiana. After 48 h, a confocal laser- scanning microscope (LSM800, Zeiss, Germany) was used to observe the ﬂuorescence signal. In the FLC assay, the ORFs without stop codons of LlWRKY39 and LlCaM3 were cloned into pCAMBIA1300-nLUC vectors, and the ORFs with stop codons of LlWRKY39 and LlCaM3 were cloned into pCAMBIA1300-cLUC vectors using recombinant ligase (Vazyme, Nanjing, China). The reconstructed vectors were transformed into A. tumefaciens strain GV3101. The cor- responding combination of bacterial solutions (cLUC:nLUC = 1:1) was inﬁltrated into the leaves of N. benthamiana. After 48 h, the ﬂuorescence signal was observed with a luminometer (PIXIS1024B, China). For a yeast two-hybrid assay, the split-ubiquitin vectors of pPR3-N and pBT3-STE were used. The ORF of LlCaM3 was inserted into pPR3-N, and the fragments (1 to 60; 1 to 45) of LlWRKY39 were cloned into pBT3-STE. The empty vectors pPR3-N and pBT3-STE were used as negative controls. The corre- sponding plasmids were cotransformed into yeast strain NMY51. The interaction was identiﬁed by spot assay on Leu-, Trp-, His-, and Ade-deﬁcient SD medium with 3-AT (20 mM). All the primers used for vector construction are shown in Supplementary Table S3. The ORF without the stop codon of LlWRKY39 was inserted into a pCAMBIA1300-GFP vector to generate a fusion protein (LlWRKY39-GFP). The primers used for vector construction are shown in Supplementary Table S3. The empty vector pCAMBIA1300-GFP was used as the control. The plasmids pCAMBIA1300-GFP and pCAMBIA1300-LlWRKY39-GFP were transformed into A. tumefaciens strain GV3101, and then, the bacterial solution was inﬁltrated into N. benthamiana. A confocal laser-scanning microscope (LSM800, Zeiss, Germany) was used to observe the ﬂuorescence. Promoter activity analysis of LlWRKY39 The isolated 737-bp fragment of the LlWRKY39 pro- moter was fused to a pGreenII-0800-LUC vector using a recombinant ligase (Vazyme, Nanjing, China). The con- structed vector and an empty vector (control) were transformed into A. tumefaciens strain GV3101 (pSoup), which was used to transform N. benthamiana leaves. After 48 h, one-half of the leaves with inﬁltrated material were treated at 37 °C for 2 h, and allowed to recover from HS at 22 °C for 12 h; then, the ﬂuorescence signal was observed with a luminometer (PIXIS1024B, China). A luciferase reporter assay system (Promega, USA) was used to measure the activity of ﬁreﬂy luciferase (LUC) and Renilla luciferase (REN). The primers for vector con- struction are shown in Supplementary Table S3. Gene expression analysis in lily Total RNA was extracted using TRIzol as described above. A HiScript II Kit with gDNA Eraser (Vazyme, Nanjing, China) was used for cDNA biosynthesis with an oligo dT primer. qRT-PCR was performed using a 20-μL reaction system. Lily 18S rRNA was used as the endo- genous gene. Three independent technical replicates were performed for each of three biological replicates. The Page 12 of 14 Page 12 of 14 Ding et al. Horticulture Research (2021) 8:36 recovered for 2 h at 22 °C, and then treated again at 45 °C (shown in Fig. 4e). After these treatments, the plants were placed in the culture room for a 7-day recovery, and the phenotypes and survival rates of the seedlings were recorded. One-week-old seedlings of the transgenic and WT plants were collected to determine the expression levels of heat-related genes: AtHSFA1 (AT4G17750), AtHSFA2 (AT2G26150), AtHSFA3 (AT5G03720), AtHSFB2A (AT5G62020), AtDREB2A (AT2G40340), AtWRKY39 (AT3G04670), AtAPX2 (AT3G09640), AtAPX1 (AT1G07890), AtGolS1 (AT2G47180), AtMBF1c (AT3G24500), AtHSP70 (AT3G12580), and AtHSP101 (AT1G74310). AtActin2 (AT3G18780) was used as the endogenous gene. The primers used for qRT-PCR are shown in Supplementary Table S3. relative expression level was calculated with the 2–ΔΔCt method70,71. The primers for qRT-PCR are shown in Supplementary Table S3. Isolation and analysis of promoter sequences The genomic DNA of ‘white heaven’ was extracted using a plant DNA extraction kit (Zomanbio, Beijing, China) following the manufacturer’s instructions. The promoter was isolated using hi-TAIL PCR72. PLACE databases (http://www.dna.affrc.go.jp/PLACE/) were used to analyze the cis-elements in the promoters. Yeast one-hybrid assay A fragment (-500 to -486) of the LlMBF1c promoter with three repeats was cloned into pHis2.1 to obtain the pHis2.1–3 × W-box; two sites of this fragment (Fig. 5b) were mutated and cloned into pHis2.1 to generate pHis2.1–3 × mW-box. LlWRKY39 was ampliﬁed and inserted into the pGADT7 vector. Different combinations of plasmids were cotransformed into yeast strain Y187 to identify positive clones. SD medium lacking Trp, Leu, and His was used to detect possible interactions. The primers used for vector construction are listed in Supplementary Table S3. Luciferase reporter assay Plant transformation and generation of transgenic lines It is difﬁcult to obtain lily transgenic plants since a stable genetic transformation system of lily has not yet been established. Therefore, the model plant A. thaliana (Col-0) was used to verify the biological function of LlWRKY39 under HS. The bacterial solution of pCAMBIA1300-LlWRKY39 was collected by centrifuga- tion and resuspended in a sucrose solution (5%). Arabi- dopsis plant genetic transformation was performed according to the ﬂoral dip method73. The harvested seeds were selected on MS medium containing 30 mg/L hygromycin until the T3 generation. One-week-old seedlings were collected to extract RNA for RT-PCR identiﬁcation of the transgenic lines. The 836-bp fragment of the LlMBF1c promoter was cloned into pGreenII-0800-LUC to obtain the reporter vector. The ORF of LlWRKY39 was cloned into a pGreenII-62-SK vector to generate the effector vector. These vectors were transformed into A. tumefaciens strain GV3101 (pSoup). The mixed bacterial solution of the TF and promoter cultures (2:1) for the induction analysis was used to inﬁltrate N. benthamiana leaves. 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https://openalex.org/W2988750847	https://link.springer.com/content/pdf/10.1007%2F978-3-030-20900-1_3.pdf	English	null	Agricultural Inputs and Their Energy Costs 1900–2010	Environmental history	2,019	cc-by	16,398	© The Author(s) 2020 M. González de Molina et al., The Social Metabolism of Spanish Agriculture, 1900–2008, Environmental History 10, https://doi.org/10.1007/978-3-030-20900-1_3 3.1 Comments on Methodology According to our methodological proposal, inputs from outside the agricultural sector constitute the Imports (I) in the MEFA methodology. These Imports added to Domes- tic Extraction (DE) determine domestic consumption (DC) after deduction of Exports (E). Although we considered both, human work and animal traction, to calculate the EROIs, they are not taken into account here because they can be deﬁned as ﬂows that circulate within the agroecosystem and do not, therefore, come from outside. We thus developed a time series on the use of external inputs from 1900 to 2008. Their type and quantity were obtained mainly from agricultural statistics supplemented with technical reports as well as research studies and were based on the assumption that growth rates were constant during the years for which we lacked data. Yearbook data included fertilizers since 1933, tractors and other agricultural machines since 1955, fuels since 1960, pesticides since 1933 and greenhouses and tunnels from 1975 onwards. Pesticide data from 1950 to 1980 were expressed in monetary terms, and we converted them into weights using deﬂation data from Carreras and Tafunell (2005). Fertilizers in 1900–1922 were estimated from the data compiled by Gallego Martinez (1986) and Mateu Tortosa (2013). Fuel consumption data for 1950 and 1990–2008 were retrieved from Spanish statistics (MI 1961a, b; MINETUR 2015) and from FAOSTAT (FAO 2016) for the years 1970–1980. Fuel consumption from 1900 to 1940 was estimated based on the machinery’s installed capacity. Electricity consumption data for 1950 were obtained from the INE (1960), from Carpintero and Naredo (2006) for 1960, from FAOSTAT (FAO 2016) for 1970–1980 and from MINETUR (2015) for 1990 onwards. The consumption of electricity before 1950 was estimated assuming that agricultural electricity represented the same proportion of total electricity consumption in Spain as in 1950. Corominas data (2010) was used to take into account upstream electricity consumption in the irrigation. The surface areas represented by each type of irrigation were taken from MAGRAMA (2015a). The ofﬁcial machinery data in the ﬁrst half of the twentieth century was complemented by data from Martinez-Ruiz (2000). We considered that 97% of the greenhouses belonged to the “‘Almería vineyard’ ‘type and 3% were of the’ ‘Glass greenhouse’” type (MAGRAMA 2008). The data are presented at selected decadal intervals, that is, 1900, 1910, 1922, 1933, 1940, 1950, 1960, 1970, 1980, 1990, 2000, and 2008, as we did for biomass production. Chapter 3 Agricultural Inputs and Their Energy Costs 1900–2010 The socio-metabolic transition to industrial society led to displacing agriculture as the main source of energy and materials and generalizing the use of fossil fuels and minerals (Krausmann and Haberl 2002; Fischer-Kowalski and Haberl 2007; Kraus- mann et al. 2008; Kuskova et al. 2008; Infante-Amate et al. 2015). This transition also affected the agricultural sector itself, which underwent big quantitative and qualita- tive changes in its technical means of production. These technological changes are usually associated with corresponding increases in agricultural production (increase in land productivity) and in the reduction of human labor (increase labor productiv- ity) (Boserup 1981; Giampietro et al. 1999; Fischer-Kowalski et al. 2014). As we have seen in Chap. 2, Spanish agroecosystems experienced signiﬁcant intensiﬁca- tion and productive specialization. Given that land uses were segregated, that many connections between them broke down, and technological innovation orientations (Fernández Prieto 2001), agroecosystems increasingly needed inputs imported from outside the agricultural sector. Organic inputs produced in the farms themselves and in the local environment, such as manure and animal traction, were replaced by large quantities of inorganic inputs powered and manufactured with fossil fuels, including synthetic fertilizers and pesticides, machinery, fuel and electricity (Guzmán Casado and González de Molina 2009). In this chapter, we quantify the inputs used in Spanish agriculture since the begin- ning of the twentieth century. The aim is to examine the evolution of the social fund element, i.e., the technical means of production (TMP), during the industrialization process. TMP eventually not only had a decisive role in maintaining productive activ- ity but productive activity ended up being dependent on it. The objective is not that of examining technical changes in agriculture, which goes beyond the scope of this research, but to evaluate the magnitude of the changes and their consequences for the functioning of Agrarian metabolism (AM). Although we have been expressing ﬂows in tons of materials, in this chapter, we chose to express them in units of energy to give a fuller account of the developments. In Chap. 6, where we analyze the structure and functioning of agrarian metabolism by relating both the funds and the ﬂows from which they originate, we will express inputs in tons to maintain metric consistency. 69 © The Author(s) 2020 M. González de Molina et al., The Social Metabolism of Spanish Agriculture, 1900–2008, Environmental History 10, https://doi.org/10.1007/978-3-030-20900-1_3 3 Agricultural Inputs and Their Energy Costs 1900–2010 70 3.1 Comments on Methodology Where possible, the decennial values were deﬁned based on 5-year aver- ages. The series combines raw data taken directly from historical sources, estimates made by other researchers and our own estimates. Since ofﬁcial statistics are pub- lished annually, we show long-term trends for some years as we believe they are of interest. We divided the inputs into industrial (chemical fertilizers, machinery, etc.) and non-industrial (biomass) inputs. The energy we calculated for industrial inputs is embodied energy, that is, the sum of gross energy of the input plus the energy needed for its production and distribution. The embodied energy of industrial inputs evolved over time, following varying trends in production energy efﬁciency and the delivery of inputs. We developed a working document that describes the embod- 71 Comments on Methodology 3.1 ied energy (and its components) in agricultural inputs over the 1900–2010 period, together with theoretical and methodological considerations (Aguilera et al. 2015). Given that the data in this working document are provided in a 10-year format and do not always correspond to data for Spain, missing data were estimated by linear interpolation. To estimate the machinery’s embodied energy, we took into account the installed power (MW) of the machinery, the years of manufacture of the “mix” of machinery and the actual replacement rate in the year studied (estimated according to records and annual census). Estimated embodied energy in the electricity was based on the Spanish electric mix and thermal power plant efﬁciency (Bartolomé- Rodríguez 2007; UNESA 2005; MINETUR 2016; REE 2012), supplemented with data on the embodied energy of fuels, taken from Aguilera et al. (2015). In terms of non-industrial inputs, the energy contained in the net imported biomass (seeds and feeds) was the gross energy of the different products, calculated using conversion factors included in Guzmán et al. (2014). The energy cost of transport taken from Aguilera et al. was added to it (2015). The energy required to produce the biomass was not taken into consideration, to avoid double accounting problems, since this cost must be attributed to the agroecosystems of origin. g y g The embodied energy metric would be equivalent to the concept of “cumulative energy demand” used in life cycle assessments as well as to the concept of “en- ergy intensity” used in some energy studies. All embodied energy components are expressed in terms of gross energy. 3.1 Comments on Methodology Energy requirements refer to the energy used in the production of a given input. They are divided into direct and indirect energy requirements. The direct energy requirements refer to the gross energy of the fuels used directly in the production process. Indirect energy requirements include all the remaining processes necessary for input production and its use on the farm, includ- ing the production and transport of fuel, production, and transport of raw materials, energy integrated into buildings and equipment and transportation of ﬁnished prod- ucts to the farm. Worthy of note, only physical processes were included. For the analysis of the agricultural systems’ energy inputs, we considered it more appropri- ate to use gross energy (GE), rather than net energy, since the former reﬂects total energy contained in the input. In addition, agricultural energy products were almost always expressed in gross energy values, as in our review of the energy content of biomass products and residues (Guzmán et al. 2014). Therefore, we also used GE values in our analysis of the embodied energy of agricultural inputs. On the other hand, we did not apply any quality correction factor to the caloriﬁc value of the different fuels. Changes in energy efﬁciency in the manufacturing and operation of inputs were taken into account, especially industrial ones. To our knowledge, however, these changes in efﬁciency have barely been considered in historical analyses of agrarian systems. Only studies based on monetary data systematically take these changes into account (e.g., Cleveland 1995; Cao et al. 2010). The studies by Pelletier et al. (2014) on egg production in the USA and Pellegrini and Fernandez (2018) on global energy use in agriculture are two of the few examples of studies accounting for temporary changes in the energy efﬁciency of agricultural inputs from a life cycle analysis perspective. 72 3 Agricultural Inputs and Their Energy Costs 1900–2010 The general trend has conisted in signiﬁcant technological improvements in energy efﬁciency in the production of most agricultural inputs, such as nitrogen and phosphate fertilizers, steel for the production of machinery, etc. (Smil 1999, 2013; Jenssen and Kongshaug 2003; Ramirez and Worrell 2006; Dahmus 2014). Over some periods, such as during the 1980s energy crisis, this trend intensiﬁed due to the increase in energy prices and concerns about the security of energy supplies (Bhat et al. 1994). 3.2 Mechanical Traction In our categorization, traction includes the energy used to perform mechanical work contained in agricultural tasks, excluding the pumping of water for irrigation, and also exluding the work performed by draft animals, which is based on internal energy from the agroecosystem. Therefore, it includes the work accomplished by self-propelled machines in the ﬁeld, such as tillage, sowing or harvesting, but also the tasks that were performed outside the farm, such as threshing. Quantifying the total energy and environmental impacts of mechanical traction requires determining the amount of fuels used for direct and indirect energy consumption as well as the embodied energy of manufacturing and machinery maintenance. Additional data on animal traction and greenhouse gas emissions from animal and mechanical traction is provided in Aguilera et al. (2019a). 3.1 Comments on Methodology However, some agricultural inputs, which in the beginning required little energy due to the ease of extraction, have ended up demanding higher consumption levels for their extraction and reﬁning (Meadows et al. 1972; Gutowski et al. 2013). Despite technological improvements, the energy efﬁciency of raw material produc- tion may have decreased. This is the case, for example, of oil and gas production in the USA (Hall et al. 2009, 2014), and generally worldwide (Gagnon et al. 2009; Hall et al. 2014), where energy return on investment (EROI) is already decreasing. An exhaustive historical compilation of embodied energy coefﬁcients has been carried out for the main agricultural inputs (Aguilera et al. 2015). The calculations described in this chapter are based on this latter compilation. 3.2.1 Machinery In this section, we reconstruct the time series of the most important parameters to estimate energy and environmental impacts associated to farm machinery production and maintenance in Spain. In our case, this means reconstructing, for each type of farm machine, the annual census, the number of annual registrations in the census and removals from it, the average useful life, and the weight of new, removed and average machines of the census. The machine types studied are locomobiles, threshers, trac- tors, harvesters, other motors (static), tillage machinery and other farm implements. 3.2 Mechanical Traction 73 The number of motorized farm machines is reported yearly by ofﬁcial statistics since 1970 (MA 1973; MAPA 1997; MAGRAMA 2012), and each 5 years since 1955 (MA 1973), although Martinez-Ruiz (2000) provides an annual series since 1955. We also have machine numbers in 1932 (MAIC 1932) and 1947 (Martinez- Ruiz 2000). Martinez-Ruiz (2000) provides threshers and locomotive numbers in 1900. We assumed that the ﬁrst tractors were registered in 1902 and the ﬁrst self- powered harvesters and 1-axis tractors (rototillers) in 1940. We estimated values in the missing years assuming constant growth rates between the two closest years with available data. We also assumed that there was no growth during the Civil War period (1936–1939). The number of machine registrations each year is provided since 1970 (MAPA 1997; MAGRAMA 2012). There is no available information regarding yearly removals of farm machinery from the census. Therefore, we estimated this value as the census of a given year minus the sum of registrations of that year plus the cen- sus of the previous year. We estimated registrations and removals in years previous to 1970 assuming the same removal rate, with respect to the census, as the average of the ﬁrst 20 years with available data (1970–1989). In some speciﬁc years, the census and the yearly registration values do not match, i.e., when the change in the census between 2 years is higher than reported registra- tions in that year. This means that registrations are too low even assuming that there were no removals in that year. In those cases, we have assumed that there were no removals, and the number of registrations was equaled to the difference between the census of the previous and the current year. Moreover, in 1980 there was an unusual peak in the census, followed by the continuation of a fairly stable trend. 3.2.1 Machinery From 308 locomobiles, 300 threshers and 300 “other engines” at the beginning of the century, to 538 locomobiles, 5062 threshers, 5312 3 Agricultural Inputs and Their Energy Costs 1900–2010 74 0 20 40 60 80 1900 1910 1922 1933 1940 1950 1960 [Thousands of units] Other engines Tractors Threshers RotoƟllers Harvesters Locomobiles 0 250 500 750 1000 1250 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units] Tractors RotoƟllers Other engines Harvesters Locomobiles Threshers (a) (b) Graph 3.1 Historical evolution of the number of motorized machines in Spanish agriculture, 1900–1960 (a) and 1900–2008 (b), in thousands of units 0 20 40 60 80 1900 1910 1922 1933 1940 1950 1960 [Thousands of units] Other engines Tractors Threshers RotoƟllers Harvesters Locomobiles (a) (a) 0 250 500 750 1000 1250 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units] Tractors RotoƟllers Other engines Harvesters Locomobiles Threshers (b) Graph 3.1 Historical evolution of the number of motorized machines in Spanish agriculture, 1900–1960 (a) and 1900–2008 (b), in thousands of units (b) Graph 3.1 Historical evolution of the number of motorized machines in Spanish agriculture 1900–1960 (a) and 1900–2008 (b), in thousands of units “Other engines” and 4084 tractors in 1932. Despite the impact of the Civil War and the post-war period, by 1947 the number of tractors had doubled, and in 1955 there were 27,671 units. Thus, the data shows that locomobiles never grew too high above the levels of the nineteenth century, while most of the growth in fam machinery until almost 1930 was dominated by threshers and “other engines”, followed by tractors, which became dominant in the 1950s. The number of tractors and harvesters kept growing until the end of the period in 2010, up to 1,049,950 and 60,263, respectively (Graph 3.1b). As we will see later, this is not due to ever-increasing mechanization of Spanish agriculture but to the lack of removals from the census. p g The number of registrations peaked in the late 1970s in the case of tractors and rototillers (Graph 3.2a and b, respectively), and in the late 1960s in the case of harvesters (Graph 3.2c). However, removals from the census never matched registra- tions, implying the continuation of the growth in census numbers (Graph 3.2c) even decades after the peak in annual registrations. 3.2.1 Machinery We corrected the census value in that year assuming that it followed a constant growth rate. There is no information on the total number of harvesters registered each year, only on the number of cereal harvesters. Likewise, the number of total harvesters is reported in 1990 for the ﬁrst time. We used the ratios of total harvesters to cereal harvesters from the census, in those years where data is available, to estimate the total number of harvesters registered (in the whole series) and censed (in years previous to 1990). Total harvesters represented 105% of cereal harvesters in 1990 and 115% in 2011. The ratio of 1990 was used for the pre-1990 series. The census of “other engines” (excluding irrigation) is only available for a few years along the whole studied period: 1932 (MAIC 1932), 1962 (INE 1963), 1973 (MA 1973), 1980, 1985 and 1988–1993 (MAPA 1997). We assumed constant growth rates in the missing years. In 1900, we assumed the same number of other engines as threshers, in line with the proportion between these two types of machines in the 1932 census. The number of threshers is provided in 1900, 1947, 1955–1975 (Martinez-Ruiz 2000), 1932 (MAIC 1932) and 1976–1981, 1983 and 1986 (INE 1997). We estimated the missing years assuming constant growth rates. There is high uncertainty in the evolution in the number of machines during the ﬁrst half of the twentieth century (Graph 3.1a). However, we can observe a substantial growth between 1900 and 1932. 3.2.1 Machinery This implies that the average age of registered farm machinery was also growing. 3.2 Mechanical Traction 75 -5 5 15 25 35 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units / year] RegistraƟons Removals -5 0 5 10 15 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units / year] RegistraƟons Removals -1 0 1 2 3 4 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units / year] RegistraƟons Removals (a) (b) (c) Graph 3.2 Historical evolution of the annual number of registrations and removals of tractors (a), rototillers (b) and harvesters (c) from the agricultural census, 1900–2008, in thousands of units per year (three-year moving average) 75 3.2 Mechanical Traction -5 5 15 25 35 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units / year] RegistraƟons Removals (a) -5 0 5 10 15 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units / year] RegistraƟons Removals (b) (b) 1970 1980 2000 1960 1950 2008 -1 0 1 2 3 4 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units / year] RegistraƟons Removals (c) (c) Graph 3.2 Historical evolution of the annual number of registrations and removals of tractors (a), rototillers (b) and harvesters (c) from the agricultural census, 1900–2008, in thousands of units per year (three-year moving average) 76 3 Agricultural Inputs and Their Energy Costs 1900–2010 We estimated the average life expectancy of machinery assuming that the machines removed in a given year were the oldest ones in the census (Graph 3.1). In the period previous to 1970, when there were no registrations data, and thus the uncertainty of removals was very high, we assumed a constant life expectancy equal to the average of the ﬁrst 20 years with available data (1970–1989). In the case of threshers, locomotives and other engines we do not have registrations and removals data during the whole studied period. In those cases, we assumed a constant life expectancy of 20 years. Graph 3.3 shows a large increase in the estimated life expectancy of farm machin- ery in Spanish agriculture in the period (1970–2010). 3.2.1 Machinery 0 10 20 30 40 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Years] Tractors Harvesters RotoƟllers Other engines Graph 3.3 Historical evolution of the life expectancy of tractors, rototillers, and harvesters, 1970–2010, in years Graph 3.3 Historical evolution of the life expectancy of tractors, rototillers, and harvesters, 1970–2010, in years Graph 3.3 Historical evolution of the life expectancy of tractors, rototillers, and harvesters 1970–2010, in years Graph 3.3 Historical evolution of the life expectancy of tractors, rototillers, and harvesters, 1970–2010, in years 77 3.2 Mechanical Traction 3.2 Mechanical Traction 77 0 25 50 75 100 125 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] Harvesters Tractors Locomobiles RotoƟllers Other engines Graph 3.4 Historical evolution of the average rated power per machinery unit, 1900–2010, in HP/unit 0 25 50 75 100 125 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] Harvesters Tractors Locomobiles RotoƟllers Other engines Graph 3.4 Historical evolution of the average rated power per machinery unit, 1900–2010, in HP/unit Graph 3.4 Historical evolution of the average rated power per machinery unit, 1900–2010, in HP/unit 3.2.1 Machinery Some reports support this assumption showing that 31% of harvesters were more than 15 years old in 1990 (Pérez-Minguijón 1992), 55% of the tractors were more than 15 years old in 2006 (ANSEMAT 2006), and 47% of them were more than 20 years old in the same year (MAPA 2007). The age of the machines is typically inversely related to their yearly usetime.Thus,harvesterslessthan5yearsoldwereused38daysperyear,whilethose older than 15 years were used 22 days per year on average in Spain in 1990 (Pérez- Minguijón 1992). Thus, the increase in apparent life expectancy does not necessarily mean that the machines are used for an increasingly longer period. Instead, the most probable cause is that old tractors with very little use or even not used anymore are not removed from the census (Pérez-Minguijón 1992, 1999). This possibility is sup- ported by the fact that diesel fuel consumption has not grown signiﬁcantly since the mid-1970s (Sect. 3.2.2), despite installed tractor power has quadrupled in the same period and speciﬁc fuel consumption of new tractors has only decreased by 33% in that period (Aguilera et al. 2015). Moreover, speciﬁc reports which have studied the real use of farm machines further support this argument. For example, a study by MAPA (2007) found that 14.5% of registered tractors were not being used. Thus, the overestimation of the census data implies an underestimation of removals and thus an increase in apparent life expectancy according to our calculations. (Graphs 3.4, 3.5 and 3.6). 3.2.2 Fuels Direct energy consumed in traction includes all agriculture fuels reported by ofﬁcial agricultural statistics except those employed in irrigation and in modern livestock production (particularly for heating). We have estimated fuel energy employed in irrigation in Sect. 3.3. Regarding fuel consumption in livestock production, we have assumed that all non-liquid fuels, except coal during the ﬁrst half of the twentieth century (which was used by threshers and locomobiles), were used in this activity. In the ﬁrst part of this section, we show our reconstruction of total fuel consumption in Spanish agriculture. Then, we segregate traction, irrigation, and livestock fuel consumption. The ﬁrst ofﬁcial published data on fuel consumption in Spanish agriculture refers to 1950 and 1951 (MI 1961a, b). In 1978, the Anuario de Estadística Agraria report (MA 1978) included an annual series of fuel consumption in Spanish agriculture starting in 1958. Another series, also by MA, includes gasoline, diesel, and oil fuel consumption from 1960 to 1977 (MA 1966, 1970, 1975a, b, 1976, 1977). FAOSTAT also reports agricultural fuel consumption data in Spain, starting in 1970 (FAO 2016). The last ofﬁcial series is released by the Spanish Ministry of Industry and starts in 1990 (MINETUR 2015). On the other hand, scholars studying energy balances have also compiled historical sources to estimate fuel consumption in Spain. The ﬁrst of these works, by Naredo and Campos (1980), reports fuel consumption data, distinguishing diesel fuel and other fuels, in 1950, 1951, 1977, and 1978. A more recent work (Carpintero and Naredo 2006) reports total fuel consumption from 1960 to 2000, based on OECD energy balances. The latter reference is also the source of FAOSTAT data. The studies mentioned above employ different units for reporting fuel consump- tion, including kcal, toe (Mg oil equivalent), tce (Mg coal equivalents), liters, and joules. Moreover, some of them express the data as net energy and other as gross energy. 3.2.2 Fuels We have attempted to harmonize the data expressing all of them as TJ gross 3 Agricultural Inputs and Their Energy Costs 1900–2010 78 g p gy 0 20 40 60 80 100 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] RegistraƟons Census average Removals 0 5 10 15 20 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] Census average RegistraƟons Removals 0 50 100 150 200 250 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] RegistraƟons Census average Removals (a) (b) (c) Graph 3.5 Historical evolution of the rated power of the average machines, registered machines and removed machines, including tractors (a), rototillers (b), harvesters, (c) and other engines (d), 0 20 40 60 80 100 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] RegistraƟons Census average Removals (a) 0 20 40 60 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] Census average Removals 0 5 10 15 20 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] Census average RegistraƟons Removals 250 (b) (c) 0 20 40 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP Removals 0 5 10 15 20 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] Census average RegistraƟons Removals (b) 0 5 10 15 20 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] Census average RegistraƟons Removals (b) (b) 0 50 100 150 200 250 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] RegistraƟons Census average Removals (c) 0 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 Graph 3.5 Historical evolution of the rated power of the average machines, registered machines and removed machines, including tractors (a), rototillers (b), harvesters, (c) and other engines (d), in HP/unit (three-year moving average) Graph 3.5 Historical evolution of the rated power of the average machines, registered machines and removed machines, including tractors (a), rototillers (b), harvesters, (c) and other engines (d), in HP/unit (three-year moving average) 3.2 Mechanical Traction 79 0 5 10 15 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] RegistraƟons Census average Removals (d) Graph 3.5 (continued) 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [GW] Other Harvesters Tractors Locomobiles Total tracƟon power 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [MW] Other Harvesters Tractors Locomobiles Total tracƟon (a) (b) 79 3.2 Mechanical Traction 0 5 10 15 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [HP / unit] RegistraƟons Census average Removals (d) Graph 3.5 (continued) 190 191 192 193 194 195 196 197 198 199 200 200 Graph 3.5 (continued) 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [GW] Other Harvesters Tractors Locomobiles Total tracƟon power 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [MW] Other Harvesters Tractors Locomobiles Total tracƟon (a) (b) Graph 3.6 Historical evolution of the installed traction power in Spanish agriculture, 1900–2010. 3.2.2 Fuels Total installed power, in GW (a) and gross annual registrations, in MW/year (b) Graph 3.5 (continued) p 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [GW] Other Harvesters Tractors Locomobiles Total tracƟon power 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [MW] Other Harvesters Tractors Locomobiles Total tracƟon (a) (b) Graph 3.6 Historical evolution of the installed traction power in Spanish agriculture, 1900–2010. Total installed power, in GW (a) and gross annual registrations, in MW/year (b) 0.0 10.0 20.0 30.0 40.0 50.0 60.0 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [GW] Other Harvesters Tractors Locomobiles Total tracƟon power (a) (b) 19 19 19 19 19 19 19 19 19 19 20 20 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [MW] Other Harvesters Tractors Locomobiles Total tracƟon (b) (b) Graph 3.6 Historical evolution of the installed traction power in Spanish agriculture, 1900–2010. Total installed power, in GW (a) and gross annual registrations, in MW/year (b) Graph 3.6 Historical evolution of the installed traction power in Spanish agriculture, 1900–2010. Total installed power, in GW (a) and gross annual registrations, in MW/year (b) 3 Agricultural Inputs and Their Energy Costs 1900–2010 3 Agricultural Inputs and Their Energy Costs 1900–2010 80 Table 3.1 Energy conversion units of fossil fuels Density Litres/ton GCV NCV GCV NCV kg/m3 GJ/t GJ/t GJ/litro GJ/litro Kerosene 802.6 1246 46.2 43.9 37.1 35.3 Motor gasoline 740.7 1350.1 47.1 44.8 34.9 33.1 Gas /dieseloil 843.9 1185 45.7 43.4 38.5 36.6 Naphta 690.6 1448 47.7 45.3 33 31.3 LPG 522.2 1915 50.1 46.2 26.2 24.1 Natural Gas 799.6 1250.6 50.4 45.4 40 36.3 Source IEA (2015) Table 3.1 Energy conversion units of fossil fuels energy. Energy conversion units employed are shown in Table 3.1. The results of the comparison are shown in Graph 3.7. In Graph 3.7 we can observe varying levels of coincidence in reported fuel con- sumption in Spanish agriculture between the compared sources. Some of the differ- ences might be explained by differences in energy conversion coefﬁcients (between different units and net to gross energy). However, the differences between sources 0 20 40 60 80 100 120 140 1950 1953 1956 1959 1962 1965 1968 1971 1974 1977 1980 1983 1986 1989 1992 1995 1998 2001 2004 2007 2010 [PJ / year] Naredo and Campos, 1980 Carpintero and Naredo, 2006 MI, 1960 MINETUR, 2015 FAO, 2016 MA, 1966-1977 MA, 1978 Selected series Graph 3.7 Historical evolution of fuel consumption in Spanish agriculture according to different sources, 1950–201, in petajoules [PJ / year] 1950 1953 1956 1959 1962 1965 1968 1971 1974 1977 1980 1983 1986 1989 1992 1995 1998 2001 2004 2007 2010 Graph 3.7 Historical evolution of fuel consumption in Spanish agriculture according to different sources, 1950–201, in petajoules Graph 3.7 Historical evolution of fuel consumption in Spanish agriculture according to different sources, 1950–201, in petajoules Graph 3.7 Historical evolution of fuel consumption in Spanish agriculture according to different sources, 1950–201, in petajoules 3.2 Mechanical Traction 81 3.2 are often not ﬁxed, implying that there are other factors responsible for the observed differences. We constructed an own series (dotted line in Graph 3.7) prioritizing the national ofﬁcial sources, followed by international ofﬁcial sources. We completed the series in some periods with data of missing fuel types from secondary sources (research articles and monographs). We have tried to include the least number of conversions between energy units and types, and we have discarded the data not coherent with the other series. In particular, FAOSTAT (FAO 2016) data from 1970 to 1989 has been complemented with data from Spanish reports. 3 Agricultural Inputs and Their Energy Costs 1900–2010 Our series starts with 1951 and 1952 data from Naredo and Campos. Then, we assume a constant growth rate in fuel consumption until 1958, when data from MA (1978) starts. From 1970 to 1974 we take diesel and LPG data from FAOSTAT, but gasoline and other liquids data from MA (1978). From 1975 to 1979 we take all data from FAOSTAT, but estimate other liquids consumption by interpolating MA (1978) value in 1969 with FAOSTAT value in 1980. From 1980 to 1989, all values are from FAOSTAT. From 1990 to 2013, all values are from MINETUR (2015), the ofﬁcial Spanish statistics, which is very similar to FAOSTAT for most fuel types, although there are signiﬁcant differences in some speciﬁc fuels types and periods, such as fuel oil from 1990 to 2003 (Graph 3.8). 3 Agricultural Inputs and Their Energy Costs 1900–2010 Moreover, FAO data does not include renewable fuels reported by 0% 20% 40% 60% 80% 100% 120% 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 [% FAO / MINETUR] Natural gas LPG Total Diesel Fuel oil Graph 3.8 Comparison of FAO (2016) and MINETUR (2015) data of fuel consumption in Spanish agriculture, by type of fuel, 1990–2012 (FAO values expressed as % of MINETUR values) [% FAO / MINETUR] Diesel Graph 3.8 Comparison of FAO (2016) and MINETUR (2015) data of fuel consumption in Spanish agriculture, by type of fuel, 1990–2012 (FAO values expressed as % of MINETUR values) 3 Agricultural Inputs and Their Energy Costs 1900–2010 82 0 20 40 60 80 100 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Other renewables Biogas Biomass Natural Gas Diesel Fuel oil Gasoline Coal LPG Total Graph 3.9 Historical evolution of fuel consumption in Spanish agriculture, by fuel type, 1900–2008, in petajoules/year 0 20 40 60 80 100 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Other renewables Biogas Biomass Natural Gas Diesel Fuel oil Gasoline Coal LPG Total Graph 3.9 Historical evolution of fuel consumption in Spanish agriculture, by fuel type, 1900 2008 in petajoules/year Graph 3.9 Historical evolution of fuel consumption in Spanish agriculture, by fuel type 1900–2008, in petajoules/year Graph 3.9 Historical evolution of fuel consumption in Spanish agriculture, by fuel type, 1900–2008, in petajoules/year MINETUR (2015), such as biomass and biogas, nor renewable thermal energy such as solar thermal and geothermal. In Graph 3.9, we show the selected annual series of fuel consumption from 1958 to 2012. For constructing the full 1900–2008 decadal series, we had to estimate fuel con- sumption in the pre-1950 period. To our knowledge, there is a lack of published ofﬁcial statistics or estimations made by scholars on fuel consumption in Spanish agriculture before 1950. In our estimation, we assume that fuel consumption is pro- portional to the installed power of machinery, taking 1950–1951 data as a reference for fuel consumption. We distinguished the relative proportions of the different types of fuel based on qualitative information. For example, Naredo and Campos report that in 1948 diesel engines still represented only 20.6% of total installed capacity of agricultural machinery. 3 Agricultural Inputs and Their Energy Costs 1900–2010 According to Martinez-Ruiz (2000), 40% of the tractors still had petrol engines in 1955, and the majority of them in previous periods. We assumed that all new tractors from 1955 onwards used diesel fuel. This means that in 1960 gasoline would represent about 8% of fuel consumption in agriculture. This ﬁgure somewhat contradicts the annual data reported by MA (1978), which indicates that gasoline consumption from 1958 to 1961 was 0, while fuel oil represented 10–25% and diesel fuel 75–90%. In fact, null or almost null gasoline consumption values during the whole annual series from different sources in the 1958–2011 period also contradict the fact that many types of common agricultural small machines (such as chainsaws or weeding machines) employ gasoline. Therefore, we do not know whether gasoline amount is included within other types of fuel, or it is just not reported (the most probable). This reveals two transitions in the major types of fuels used during the studied period, from coal to gasoline, during mainly the 1920s, and from gasoline to diesel, during the 1950s. It also shows the appearance in the last decades of gas products employed for heating, which signiﬁcantly contributed to the increase in total fuel consumption in Spanish agriculture. The distinction between the different uses of fuels is shown in Graph 3.10. 83 3.3 Irrigation 83 0% 20% 40% 60% 80% 100% TracƟon fuels Livestock fuels IrrigaƟon fuels Graph 3.10 Historical evolution of fuel consumption in Spanish agriculture in selected time steps, by activity type, 1900–2008, expressed as %. Own estimation (see text) Graph 3.10 Historical evolution of fuel consumption in Spanish agriculture in selected time steps by activity type, 1900–2008, expressed as %. Own estimation (see text) 3.3.1 Irrigation Systems We have estimated the surface area of each type of irrigation, including surface, sprinkle and trickle irrigation systems. The ofﬁcial statistics include an additional category named “intermittent irrigation” (“riego eventual”), which includes surface irrigation systems where water is only applied on certain occasions. This category has been equaled to one-third of surface irrigation systems for the estimation of the effectively irrigated surface area. Our estimation of the evolution of the surface area of each type of irrigation at the beginning of the twentieth century was based on two government reports (MAICOP 1904; MF 1918). We have used MAICOP (1904) data for our 1900 time-step, MF (1918) data for our 1922-time step and the average between the two of them for the 1910-time step. We have found no other data distinguishing each type of irrigation until the 1962 Agrarian Census (INE 1963). Thus, the values of the intermediate time steps (1933, 1940 and 1950) have been linearly interpolated. Up to 1960-time step, the only two irrigation categories were surface and intermittent surface irrigation. As we have data of total irrigation area for all time steps, we estimated intermittent surface irrigation values as explained above, while surface irrigation area values were calculated as the difference between total irrigation and intermittent irrigation. Sprinkle irrigation data for the 1960–1990 period was obtained from Calatayud and Martínez-Carrión (2005). We assumed that intermittent surface irrigation disappeared from 1970 onwards. Drip irrigation area data at the national level starts with 1989 data from Calatayud and Martínez-Carrión (2005). We used that value for our 1990-time step and assumed that drip irrigation area in 1980 was 1/10 of the area in 1989, based on the approximate growth rate of Murcia region from 1975 to 1992 (Calatayud and Martínez-Carrión 2005). Data for 2000-time step was taken from 2003 report by the Spanish Ministry of Agriculture (MAPA 2003), based on a 2002 survey. Data for 2008-time step was taken from 2015 report by the Spanish Ministry of Agriculture (MAGRAMA 2015a), based the average of 5 surveys performed from 2006 to 2010. We can observe different stages in the evolution of irrigated area in Spain (Graph 3.12). During the beginning of the twentieth century, only surface irriga- tion systems existed. The majority of them, almost 0.89 million hectares (Mha), had constant irrigation, while 0.33 Mha were irrigated only intermittently. 3.3 Irrigation Irrigation inputs include infrastructure, on-farm energy use (energy consumption by water pumps), off-farm energy use (energy consumed in desalination and diver- sion channels) and indirect energy use (energy required for the production of fuels and electricity). Additional information on irrigation inputs and their C footprint is provided in Aguilera et al. (2019b). In the last decades, starting with the “Tajo-Segura” water diversion in 1979 and continuing with other diversions and desalination projects in the last two decades, there has been an increase of high-energy consuming water sources (Graph 3.11). 0 200 400 600 800 1000 [Hm3 / year] Reuse DesalinaƟon Diversions Graph 3.11 Historical evolution of agricultural water use from unconventional sources, 1900–2008, in hm3/year. Source Corominas (2010) and own estimations (see text) DesalinaƟon Graph 3.11 Historical evolution of agricultural water use from unconventional sources, 1900–2008, in hm3/year. Source Corominas (2010) and own estimations (see text) 3 Agricultural Inputs and Their Energy Costs 1900–2010 84 Thus, all the increase in water availability for irrigation has implied higher energy requirements and environmental costs, a trend which is enhanced by the increase in the average depth of wells. There is a lack of data of average values of this factor at the national level in Spain, but regional data of Comunidad Valenciana in East Spain shows an increase from 23 to 87 meters between 1940 and 1970 (Calatayud and Martínez-Carrión 2005). 3.3.1 Irrigation Systems Thus, our corrected irrigated area series starts at just 1.00 Mha, and grows signiﬁcantly until 1922 (1.24 million hectares). Then, the share of intermittent irrigation starts grow- ing, making the corrected series to drop even if the sum of constant and intermittent 3.3 Irrigation 85 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Millons of ha] Trickle Sprinkler Surface, intermiƩent Surface EﬀecƟvely irrigated Graph 3.12 Historical evolution of irrigated area in Spain, 1900–2008, in hectares. Own estimation (see text) 85 85 3.3 Irrigation Graph 3.12 Historical evolution of irrigated area in Spain, 1900–2008, in hectares. Own estimation (see text) surface irrigation areas still grows until 1940. The Autarky period (1940–1950) was associated to a reduction of both the gross and the corrected total irrigated area, the latter reaching 1.12 Mha. In 1950 starts a strong growth trend. The corrected irrigated area doubles in just 2 decades, and reaches 3.18 Mha in 1990. Growth continues until the present, although the growth rate has slowed down in the last decades. The ﬁrst stage of the growth period was based on the expansion of constant surface irrigation area, while sprinkler irrigation expanded from 1960 to 1990 and trickle irrigation from 1980 onwards. In 1980, the surface irrigation area peaks at 2.30 Mha, and in the last time step it has dropped to half of this value (1.09 Mha), while trickle irrigation area reaches 1.54 Mha. This trend has continued until 2014, when surface irrigation dropped below 1 Mha and trickle irrigation covered 1.76 Mha. 3.3.2 Installed Mechanical Power Data on the number of irrigation engines was gathered from different ofﬁcial pub- lications, namely MF (1918) for 1916, MA (1933) for 1932, and MAPA (1980) for 1955–1980 (Graph 3.13). For the estimation of the number of irrigation engines in our selected time steps (Graph 3.14), we assumed a constant growth rate between 1900 and 1933, based on the observed growth rate between 1916 and 1932. For the period after 1980, in the case of combustion engines, we assumed the same growth rate as in the 1970–1980 period, while the number of electric engines was based on the evolution of electricity consumption in agriculture. The number of installed engines (Graph 3.13) grew from about 1000 units in 1916 (JCA 1918) to 264,000 units in 1995 (MAPA 1997), reaching nearly 279,000 units in 2008. Most of the irrigation water in the early twentieth century was directly supplied by gravity from rivers, springs, and reservoirs through channels and acequias. Water 3 Agricultural Inputs and Their Energy Costs 1900–2010 86 0 50 100 150 200 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units] Combuson Electric Graph 3.13 Historical evolution of the number of irrigation engines, 1900–2008, in thousands of units. Own estimation 0 50 100 150 200 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Thousands of units] Combuson Electric [Thousands of units] Electric 1960 Graph 3.13 Historical evolution of the number of irrigation engines, 1900–2008, in thousands o units. Own estimation 0 2 4 6 8 10 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [Kw / unit] CombusƟon Electric Total Graph 3.14 Historical evolution of the average rated power of irrigation engines, 1900–2008, in KW /unit. Own estimation (see text) Graph 3.14 Historical evolution of the average rated power of irrigation engines, 1900–2008, in KW /unit. Own estimation (see text) Graph 3.14 Historical evolution of the average rated power of irrigation engines, 1900–2008, in KW /unit. Own estimation (see text) elevation was only required in about 8 and 9% of the irrigated surface in 1904 and 1918, respectively (MAICOP 1904; MF 1918, respectively). In 1916, pump engines wereusedinabout 31%of this surfacerequiringwater elevation, whilemanual pumps (13,000 units) were used in 2% of this surface, water pumping windmills (3,000 units) in 3%, and water wheels (48,000 units) in 64% (MF, 1918). 3.3.2 Installed Mechanical Power The number of water pumping windmills doubled up to 7000 units in 1932 (MAIC 1932), but there is no data on the continuation of the trend. The number of waterwheels had grown to 73,000 units in 1932 (MAIC 1932), and to 85,000 in 1962 (INE 1963), and there is no more data after that year. There is information on the installed power of irrigation engines from 1955 to 1979 (MAPA 1980). The evolution of average engine power in the previous and later periods was estimated based on the evolution of the rated power of average traction 87 3.3 Irrigation 0 500 1,000 1,500 2,000 2,500 3,000 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [MW] Electric CombusƟon Total Graph 3.15 Historical evolution of the total installed power of irrigation engines, 1922–2008, in GW. Own estimation (see text) Graph 3.15 Historical evolution of the total installed power of irrigation engines, 1922–2008, in GW. Own estimation (see text) machinery (see Sect. 2) (Graph 3.14). The total installed power of irrigation engines (Graph 3.15) was calculated by multiplying the number of engines by their average rated power. The rated power of irrigation engines shows signiﬁcant growth, more than dou- bling during the studied period (Graph 3.14). Most of the growth is observed during the 1950–1980 period when ofﬁcial statistics are available. These numbers are sub- stantially lower than those of the Comunidad Valenciana region of Spain in the 1940–1970 period, where the average power of irrigation pumps was 21 KW/unit in 1940 (Calatayud and Martínez-Carrión 2005). But, according to the same source, the average power had grown up to 57 KW/unit in 1970, thus showing a similar growth pattern as in Spain as a whole. The installed power for irrigation grew from 14 to 2496 MW in the 1922–2008 period (Graph 3.15). Electric engines represented around half of the installed power up to 1940, when heat engines started increasing their share up to nearly 90% in 1980. After that year trend reversed, and electric engines represented over 50% of the installed power in 2008. 3.3.3 Fuels Fuel consumption in irrigation (Graph 3.16) was estimated based on the num- ber of irrigation combustion engines, assuming a ﬁxed fuel consumption rate per engine installed power of 7.0 GJ/KW, which is the consumption rate in 1995, the only year with available data on fuel consumption in irrigation (Corominas 2010). The distribution between coal and liquid fuels was based on the relative share of steam engines and internal combustion engines. In 1916 and 1932 the historical sources (respectively, MF 1918; MA 1933) provide speciﬁc data of engine fuel types. We 3 Agricultural Inputs and Their Energy Costs 1900–2010 88 0 2 4 6 8 10 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Coal Oil Total Graph 3.16 Historical evolution of direct fuel consumption in irrigation, 1900–2008, in petajoules. Own estimation (see text) Graph 3.16 Historical evolution of direct fuel consumption in irrigation, 1900–2008, in petajoules. Own estimation (see text) took the 1916 value for 1910 and 1922, and the 1932 value for 1933 and 1940. We assumed that the relative share of oil fuels in 1900 was half of that in 1916. Engines using coke oven gas, which were relatively common in the early twentieth century, have been included within the “oil” category. After 1950, we assumed steam engines to be phased out. Total fuel consumption increased from 0.5 TJ in 1900 up to a peak 8450 TJ in 1990 and slowly declined after that year, reaching 7993 TJ in 2008. Coal dominates fuel consumption in the early twentieth century. Despite our estimation coal consumption remains practically ﬂat from 1922 to 1940, its relative share drops dramatically from 1922 to 1932, due to the sharp increase in internal combustion engines. 3.3.4 Electricity We found various sources reporting agriculture electricity use in different periods. The National Statistics Institute (INE) reports electricity consumption in agriculture from1938to1958(INE1960),whileanotherofﬁcialstudyfromtheIndustryMinistry (MI) provides a series from 1945 to 1959 (MI 1961a, b). The data in INE report is expressed as MWh, while the data in MI report is expressed as tons of coal equivalents (tce). If we express them as TJ, using the coefﬁcient reported in MI document of 29.3 GJ/tce, we get much higher values from MI than for INE (Graph 3.9). However, the trends are identical from 1945 to 1955. On the other hand, INE data is similar to 1950 data reported by Naredo and Campos (1980), but show a drop in electricity consumption in 1956–1958 that is not supported by historical data, and which does not ﬁt well with the following data series starting in 1960 (Carpintero and Naredo 3.3 Irrigation 89 2006). Thus, we made the exercise of recalculating MI values using a modiﬁed GJ/tce coefﬁcient, equaling its data with those of INE (1960) in 1945. The results are shown in Graph 3.16. We can observe that the modiﬁed MI data series (MI-modiﬁed) matches well all overlapping INE series except the period 1956–1958. It also matches well Carpintero and Naredo (2006) series starting in 1960, which in turn matches FAOSTAT data starting in 1970. Therefore, we used INE (1960) data from 1939 to 1945, the modiﬁed MI data series from 1945 to 1959, Carpintero and Naredo (2006) data from 1960 to 1969, FAOSTAT (FAO 2016) data from 1970 to 1989, and IDAE (2015) for 1990–2013. Graph 3.17a shows the disparity between different published statistics of electric- ity consumption in Spanish agriculture during the mid-twentieth century, which is solved by our corrected estimation of MI data. 3.3.4 Electricity Graph 3.17b shows our estimation of 0 1 2 3 4 5 6 1,939 1,942 1,945 1,948 1,951 1,954 1,957 1,960 1,963 1,966 1,969 1,972 1,975 [PJ / year] FAO, 2016 Carpintero & Naredo, 2006 MI, 1961-original MI, 1961-modiﬁed INE, 1960 0 5 10 15 20 25 1,898 1,904 1,910 1,916 1,922 1,928 1,934 1,940 1,946 1,952 1,958 1,964 1,970 1,976 1,982 1,988 1,994 2,000 2,006 2,012 [PJ / year] INE, 1960 MI, 1961-modiﬁed Carpintero & Naredo, 2006 FAO, 2016 IDAE Proposed series (a) (b) Graph 3.17 Historical evolution of on-farm electricity use in Spanish agriculture according to various sources, 1939–1976 (a) y 1898–2014 (b), in petajoules/year 0 1 2 3 4 5 6 1,939 1,942 1,945 1,948 1,951 1,954 1,957 1,960 1,963 1,966 1,969 1,972 1,975 [PJ / year] FAO, 2016 Carpintero & Naredo, 2006 MI, 1961-original MI, 1961-modiﬁed INE, 1960 (a) (a) 0 5 10 15 20 25 1,898 1,904 1,910 1,916 1,922 1,928 1,934 1,940 1,946 1,952 1,958 1,964 1,970 1,976 1,982 1,988 1,994 2,000 2,006 2,012 [PJ / year] INE, 1960 MI, 1961-modiﬁed Carpintero & Naredo, 2006 FAO, 2016 IDAE Proposed series (b) 0 5 10 15 20 25 1,898 1,904 1,910 1,916 1,922 1,928 1,934 1,940 1,946 1,952 1,958 1,964 1,970 1,976 1,982 1,988 1,994 2,000 2,006 2,012 [PJ / year] INE, 1960 MI, 1961-modiﬁed Carpintero & Naredo, 2006 FAO, 2016 IDAE Proposed series (b) (b) 1,898 1,904 1,910 1,916 1,922 1,928 1,934 1,940 1,946 1,952 1,958 1,964 1,970 1,976 1,982 1,988 1,994 2,000 2,006 2,012 Graph 3.17 Historical evolution of on-farm electricity use in Spanish agriculture according to various sources, 1939–1976 (a) y 1898–2014 (b), in petajoules/year 3 Agricultural Inputs and Their Energy Costs 1900–2010 90 annual electricity consumption from 1898 to 2013. The estimated direct electricity consumption in agriculture grew 10-fold in two decades, from 7 TJ at the beginning of the twentieth century to 70 TJ in 1922. By 1955, it had grown ten-fold again, at 656 TJ, and again in 1980, at 7646 TJ. It peaked in 2007 at 20,279 TJ and dropped down to 13,924 in 2013, to grow again to 18.6 PJ in 2016. We have also estimated off-farm (“upstream”) electricity consumption for agri- culture in our selected time steps (Graph 3.18b and c). This electricity is used for obtaining unconventional water sources, including desalination and water pumping in channels and diversions. 3.3.4 Electricity We multiplied the quantity of water coming from these sources calculated in Sect. 3.1 by energy consumption coefﬁcients from Coromi- nas (2010), of 1.2, 3.7 and 0.25 KWh/m3 for diversions, desalination and reuse, respectively. The results are shown in Graph 3.18a. The electricity employed to obtain irrigation water from unconventional sources (Graph 3.18a) rose from 1092 TJ after the construction of Tajo-Segura diversion in 1979, to 3716 TJ in 2008 when other diversions and many desalination plants were operating. Irrigation electricity grew from 7 TJ in 1900 to 223 TJ in 1933, 473 TJ in 1950 and 23,099 TJ in 2008. A large share of the growth in the last decades has been due to the rise in off-farm electricity use. Overall, direct energy consumption in irrigation in Spain grew from 8 TJ in 1900 to 31,091 TJ in 2008. Despite most energy consumption was electricity at the beginning of the twentieth century, oil fuels consumption rapidly expanded since 1933, and dominated the use of energy in irrigation during the middle decades of the century. Fuel consumption, however, peaked in 1980, while electricity continued growing until the end of the studied period, in line with the growth in electric engines numbers and installed power. In addition, off-farm electricity consumption was also boosted in the last decades, with the expansion of technologies such as desalination and diversion channels. 3.4 Fertilizers We have reconstructed the consumption of mineral fertilizers in Spain in terms of nutrients on an annual basis, including synthetic fertilizers, saltpeter, and guano. This estimation is based on historical data expressed in total fertilizers amount for the period 1900–1940 and expressed as nutrients during the 1945–2010 period. The ﬁrst period up to 1940 is composed mainly by statistical data for all types of fertilizers in some single years (namely 1907, 1908, 1919, 1920, 1928, 1930–1935, 1939 and 1940) and a continuous data series of the apparent consumption of total fertilizers from 1898 to 1940 (excluding the 1936–1938 Civil War period). In addition, we have continuous series of ammonium sulphate and sodium nitrate consumption from 1900 to 1914. 3.4 Fertilizers In addition, he compiled the foreign trade fertilizer data from the nineteenth century up to the Spanish Civil War (1936). These statistics include information of different items with various levels of aggregation, but the only item that could be studied separately during the whole series is ammonium sulphate. In addition, Mateu (2013) reconstructed sodium nitrate (saltpeter) imports (which can be equaled to apparent consumption) from 1901 to 1914. Therefore, the missing gaps of individual types of fertilizers up to 1914 were estimated using total apparent consumption of fertilizers in the given year, minus ammonium sulphate and sodium nitrate, multiplied by the average of the share of each individual type of fertilizer in the closest years where data is available. For example,superphosphateconsumptionin1912wasestimatedsubtractingammonium sulphate and sodium nitrate consumption to total fertilizer apparent consumption in 1912 and multiplying the result by the average of the percentage represented by superphosphate in total fertilizer consumption minus ammonium sulphate and sodium nitrate in 1907, 1908, 1919 and 1920. Potassium sulphate and potassium chloride consumption values in 1920 were much higher than the values of close years, so we considered them outliers and assumed that production was the same as in 1919. Data for the 1941–1944 period was estimated by exponential interpolation using 1940 and 1945 data. Data from 1945 onwards were taken directly from the selected Spanishgovernmentofﬁcialstatistics(AnuariodeEstadísticaAgraria reports,MAIC 1928, 1930, 1931; MA 1933, 1934, 1935, 1939, 1940, 1973; MAPA 1990, 1999; MARM 2010; MAGRAMA 2013a, b, c). We only altered this ofﬁcial series in 1957–1964, when we added potassium sulphate data gathered from a National Statis- tics Institute publication (INE 1965). As we mentioned above, the data up to 1940 is expressed as gross tons of fertil- izers. Therefore, we calculated nutrients using coefﬁcients (Table 3.2). Most of the coefﬁcients were taken from Aguilera et al. (2015). In the cases where no data, or ranges instead of single values, were provided in Aguilera et al. (2015), we took the values from Gallego (1986). We also took fertilizers mixtures nutrient contents from Gallego (1986), who estimated them as the average of nutrients contents. Moreover, we estimated guano nutrient contents based on Wikipedia (2015) and Smil (2001), taking into account that guano nitrogen content in the twentieth century should be in the lower side of its historical range, as richer sources had already been depleted (Table 3.2). Graph 3.19a shows the evolution of the amounts of nitrogen consumed. 3.4 Fertilizers Our main data source is the work by Gallego (1986), who compiled and systematized historical statistics of fertilizers consumption in Spain during the ﬁrst half of the twentieth century, including data from Alonso de Illera for 1907 and 1908, data from Junta Consultiva Agronómica government agency from 1919 and 1920, 91 91 3.4 Fertilizers 3.4 Fertilizers 91 0 1 2 3 4 [PJ / year] Reuse DesalinaƟon Diversions 0 5 10 15 20 25 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Electricity upstream Electricity on farm 25 30 35 80% 100% ] Electricity upstream Electricity on farm (a) (b) (c) 0 1 2 3 4 [PJ / year] Reuse DesalinaƟon Diversions (a) 0 1 2 3 4 [PJ / year] Reuse DesalinaƟon Diversions 0 5 10 15 20 25 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Electricity upstream Electricity on farm 0 5 10 15 20 25 30 35 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Electricity upstream Electricity on farm Coal Oil Total direct (a) (b) (c) Graph 3.18 Direct energy consumption for irrigation in Spanish agriculture including upstream electricity consumption from unconventional water sources 1980–2008 (a), total electricity con- sumption 1900–2008 (b) and total direct energy use 1900–2008 (c), in petajoules/year (a) DesalinaƟon 0 5 10 15 20 25 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Electricity upstream Electricity on farm (b) (b) 0 5 10 15 20 25 30 35 0% 20% 40% 60% 80% 100% 1900 1910 1922 1933 1940 1950 1960 1970 1980 1990 2000 2008 [PJ / year] Electricity upstream Electricity on farm Coal Oil Total direct (c) Graph 3.18 Direct energy consumption for irrigation in Spanish agriculture including upstream electricity consumption from unconventional water sources 1980–2008 (a), total electricity con- sumption 1900–2008 (b) and total direct energy use 1900–2008 (c), in petajoules/year Graph 3.18 Direct energy consumption for irrigation in Spanish agriculture including upstream electricity consumption from unconventional water sources 1980–2008 (a), total electricity con- sumption 1900–2008 (b) and total direct energy use 1900–2008 (c), in petajoules/year 3 Agricultural Inputs and Their Energy Costs 1900–2010 92 and from the Anuario de Estadística Agraria reports from 1928 to 1940. 3.4 Fertilizers We can observe an upward trend during the pre-Civil War period (1898–1935, Graph 3.19a), when consumption grew 26-fold. This growth was only brieﬂy interrupted in 1918 by the effect of the First World War. The dominant N fertilizer during this period was ammonium sulphate, which was obtained mainly through recovery from coke oven gases. Imported Chilean nitrate (and lately also calcium nitrate) also played a role in the supply of mineral N in Spain, while guano was already almost exhausted by the beginning of the century. 3.4 Fertilizers 93 Table 3.2 Nutrient contents of fertilizers employed in Spain, percentage of nutrient N (%) P2O5 (%) K2O Source Ammonia 82.0 0.0 0.0 Aguilera et al. (2015) Ammonium Nitrate 35.0 0.0 0.0 Aguilera et al. (2015) Ammonium Sulfate 21.0 0.0 0.0 Aguilera et al. (2015) Calcium-Ammonium Nitrate 25.0 0.0 0.0 Aguilera et al. (2015) Calcium Nitrate 16.0 0.0 0.0 Aguilera et al. (2015) Urea 46.0 0.0 0.0 Aguilera et al. (2015) Potassium Nitrate 13.5 0.0 45.0 Gallego (1986) Complex NPK fertilizers 15.0 15.0 15.0 Aguilera et al. (2015) Mono Ammonium Phosphate 11.0 52.0 0.0 Aguilera et al. (2015) Di Ammonium Phosphate 18.0 46.0 0.0 Aguilera et al. (2015) Ammonium phosphate* 14.5 49.0 0.0 Aguilera et al. (2015) Phosphate rock 0.0 32.0 0.0 Aguilera et al. (2015) Triple Superphosphate 0.0 48.0 0.0 Aguilera et al. (2015) Single superphosphate 0.0 18.0 0.0 Gallego (1986) Slag 0.0 16.7 0.0 Gallego (1986) Complex PK fertiizers 0.0 22.0 22.0 Aguilera et al. (2015) Muriate of potash (potassium chloride) 0.0 0.0 60.0 Aguilera et al. (2015) Sulfate of potash 0.0 0.0 50.0 Aguilera et al. (2015), INE (1965) Sodiumnitrate 15.0 0.0 0.0 Smil (2001) Mixtures Dynamic coefﬁcient Gallego (1986) Cianamida de cal 20.0 0.0 0.0 Smil (2001) Kainita 0.0 0.0 18.0 Gallego (1986) Silvinita 0.0 0.0 18.0 Gallego (1986 Guano 10.0 10.0 2.5 Wikipedia (2015), Smil (2001) The post-Civil War period (Graph 3.19b) started with stagnant use of N fertilizers, which only surpassed pre-war levels by the mid-1950s. This stage corresponds to the Autarky period of Franco’s dictatorship and was followed by an exponential growth up to 1979. Growth slowed down onwards, with signiﬁcant variability between years, but still continued up to 2000, when N fertilizer consumption peaked at 1279 Gg N. After that year, N fertilizer consumption fell to a minimum of 740 Gg N in 2008, and partially recovered afterwards, reaching 962 Gg in 2013. 3.4 Fertilizers The N fertilizer mix was dominated by ammonium sulphate until the late-1960s, when it was surpassed by calcium-ammonium nitrate. Urea and complex fertilizers became major N sources in the 1970s and now represent the majority of the N fertilizer mix, together with calcium-ammonium nitrate. 3 Agricultural Inputs and Their Energy Costs 1900–2010 94 0 10 20 30 40 50 60 70 80 90 1898 1900 1902 1904 1906 1908 1910 1912 1914 1916 1918 1920 1922 1924 1926 1928 1930 1932 1934 [Gg N / year] Other Ammonium sulphate Calcium Cyanamide Chilean nitrate Guano 0 200 400 600 800 1,000 1,200 1,900 1,910 1,922 1,933 1,940 1,950 1,960 1,970 1,980 1,990 2,000 2,008 [Gg N / year] Ammonium nitrate Other NPK Urea Calcium-ammonium nitrate Ammonium sulphate Calcium Cyanamide Chilean nitrate Guano (a) (b) Graph 3.19 Nitrogen fertilizer consumption in Spain, 1898–1935 (a) and 1900–2013 (b), in Gg N/year 0 10 20 30 40 50 60 70 80 90 1898 1900 1902 1904 1906 1908 1910 1912 1914 1916 1918 1920 1922 1924 1926 1928 1930 1932 1934 [Gg N / year] Other Ammonium sulphate Calcium Cyanamide Chilean nitrate Guano (a) (a) 0 200 400 600 800 1,000 1,200 1,900 1,910 1,922 1,933 1,940 1,950 1,960 1,970 1,980 1,990 2,000 2,008 [Gg N / year] Ammonium nitrate Other NPK Urea Calcium-ammonium nitrate Ammonium sulphate Calcium Cyanamide Chilean nitrate Guano (b) Graph 3.19 Nitrogen fertilizer consumption in Spain, 1898–1935 (a) and 1900–2013 (b), in Gg N/year (b) Graph 3.19 Nitrogen fertilizer consumption in Spain, 1898–1935 (a) and 1900–2013 (b), in Gg N/year Phosphate fertilizer consumption (Graph 3.20a) show a similar pattern than N fertilizer consumption. Phosphate consumption grows by one order of magnitude during the ﬁrst third of the twentieth century (Graph 3.20b). The relative growth is not as high as for N, but initial levels of P consumption are higher. The drop in 1918 is also very evident in this case. Phosphate fertilizer consumption is completely dominated by single superphosphate during this period, with minor amounts of rock phosphate, Thomas meal, mixtures and other (Graph 3.21). Potassium fertilizer consumption trends resemble those of the other nutrients. Growth rates were signiﬁcant during pre-Civil War period and slowed down during the 1940s and 1950s. Potassium chloride, followed by potassium sulphate, was the major K fertilizers until the mid-1960s. Then, the arrival of compound NPK fertilizers meant a boost in potassium fertilizer consumption growth. 3.4 Fertilizers After slowing down in the 1970s due to a decrease in the use of potassium chloride, total potassium consumption grew fast again in the 1980s and 1990s, peaking in 1998 at 356 Mg K2O. 3.5 Crop Protection 95 0 50 100 150 200 250 1898 1900 1902 1904 1906 1908 1910 1912 1914 1916 1918 1920 1922 1924 1926 1928 1930 1932 1934 [Gg P2O5 / year] Other Superphosphate Thomas meal Rock phosphate Guano 0 100 200 300 400 500 600 700 1,900 1,910 1,922 1,933 1,940 1,950 1,960 1,970 1,980 1,990 2,000 2,008 [Gg P2O5 / year] Guano Other Compound Thomas meal Superphosphate Rock phosphate (a) (b) Graph 3.20 Phosphate fertilizer consumption in Spain, 1898–1935 (a) and 1939–2013 (b), in Gg P2O2 /year 95 3.5 Crop Protection 95 0 50 100 150 200 250 1898 1900 1902 1904 1906 1908 1910 1912 1914 1916 1918 1920 1922 1924 1926 1928 1930 1932 1934 [Gg P2O5 / year] Other Superphosphate Thomas meal Rock phosphate Guano (a) 0 100 200 300 400 500 600 700 1,900 1,910 1,922 1,933 1,940 1,950 1,960 1,970 1,980 1,990 2,000 2,008 [Gg P2O5 / year] Guano Other Compound Thomas meal Superphosphate Rock phosphate (b) Graph 3.20 Phosphate fertilizer consumption in Spain, 1898–1935 (a) and 1939–2013 (b), in Gg P2O2 /year Graph 3.20 Phosphate fertilizer consumption in Spain, 1898–1935 (a) and 1939–2013 (b), in Gg P2O2 /year 3.5.1 Pesticides There is very limited information on the amounts of pesticides employed in Spain during the studied period. The available statistics are fragmentary both temporally and in terms of a full accounting of pesticide products. Moreover, only recently they havebeenexpressedasactivematterweight.Theﬁrstavailabledata,fromtheAnuario de Estadística Agraria (MA 1930, 1931, 1933, 1934, 1935, 1939, 1940) reported sulphur, copper sulphate and iron sulphate. An annual series expressed as monetary value of main pesticide categories (including insecticides and acaricides, fumigants, nematicides, fungicides, herbicides, veterinary products, and other) and covering the 1950–1967 period is reported by the 1973 Anuario de Estadística Agraria report (MA 3 Agricultural Inputs and Their Energy Costs 1900–2010 96 0 5 10 15 20 25 30 1898 1900 1902 1904 1906 1908 1910 1912 1914 1916 1918 1920 1922 1924 1926 1928 1930 1932 1934 [Gg K2O / year] Compound Potassium chloride Guano Potassium sulphate Other 0 100 200 300 400 500 1,900 1,910 1,922 1,933 1,940 1,950 1,960 1,970 1,980 1,990 2,000 2,008 [Gg K2O / year] Compound Potassium sulphate Potassium chloride Guano Other (a) (b) Graph 3.21 Potassium fertilizer consumption in Spain, 1898–1935 (a) and 1939–2013 (b), in Gg K2O /year 0 5 10 15 20 25 30 1898 1900 1902 1904 1906 1908 1910 1912 1914 1916 1918 1920 1922 1924 1926 1928 1930 1932 1934 [Gg K2O / year] Compound Potassium chloride Guano Potassium sulphate Other (a) 0 100 200 300 400 500 1,900 1,910 1,922 1,933 1,940 1,950 1,960 1,970 1,980 1,990 2,000 2,008 [Gg K2O / year] Compound Potassium sulphate Potassium chloride Guano Other (b) Graph 3.21 Potassium fertilizer consumption in Spain, 1898–1935 (a) and 1939–2013 (b), in Gg K2O /year Graph 3.21 Potassium fertilizer consumption in Spain, 1898–1935 (a) and 1939–2013 (b), in Gg K2O /year 1973), and this series is extended up to 1993 by the 1994 edition of that report (MAPA 1994). In 1955, starts another series including the quantities of pesticide products grouped by active matter (including DDT, lindane, malathion, nitro compounds, arsenic compounds, cyanuric compounds, and many other) but reported as total weight, not active matter weight. This series ﬁnishes in 1968 and is published in three reports (INE 1960, 1965, 1970). Last, the available data in FAOSTAT (FAO 2016) starts in 1990, groups pesticides by main categories (including insecticides, fungicides herbicides and other and it is expressed as active matter weight. 3.5.1 Pesticides We estimated the trends in the use of pesticides (sulfur and copper pesticides) from 1900 to 1920 taking the 1933 value as a reference and assuming that the rate of change was similar to that of total synthetic fertilizers during each interval (see Fertilizers section). In 1933 and 1940 we took the reported values of the ofﬁcial statistics, assuming that only sulfur and copper pesticides were used. There is very high uncertainty in the estimation of pesticide use from 1940 to 1990. The conversion of the monetary value series to physical units is hindered by the lack of information of pesticide prices. On the other hand, the conversion of the total weight series to active matter weight requires to know the average richness of each type of pesticide. If we 3.5 Crop Protection 97 3.5 estimate pesticide use based on 1990 inﬂation-adjusted (inﬂation data from Carreras de Odriozola and Tafunell Sambola 2006) monetary value series we get much lower pesticide consumption values than using the total weight series taking Naredo and Campos (1980) approach of assuming 40% active matter weight (Graph 3.22a). In fact, this assumption is really uncertain, as active matter content of commercial pesticides can vary from less than 1% to almost 100%, with very large variations between different commercial products even within a single active matter compound. Moreover, this series shows a sudden drop in the late 1960s that is not supported by the historical sources, and if we examine the trends of the speciﬁc compounds, we 1,955 1,956 1,957 1,958 1,959 1,960 1,961 1,962 1,963 1,964 1,965 1,966 1,967 1,968 [Gg Ac ve er / year] DDT DDT Lindane Endrin Malathion Pelitre NicoƟne sulfate Dinitro compounds Arsenic compounds Cyanuric compounds 0 15 30 45 60 [Gg AcƟve maƩer / year] AcƟve maƩer- based SyntheƟc pesƟcide (selected series) Total amount- based Price-based (a) (b) Graph 3.22 Historical evolution of synthetic pesticide use in Spain (excluding sulfur and copper pesticides), according to different estimations, 1950–2011, Gg Active matter /year. 3.5.1 Pesticides Price-based series is calculated from MA (1973) and MAPA (1994); total amount-based series is calculated from INE (1960, 1965, 1970), assuming 40% average active matter; active matter series is from FAOSTAT (FAO 2016) 0 15 30 45 60 [Gg AcƟve maƩer / year] AcƟve maƩer- based SyntheƟc pesƟcide (selected series) Total amount- based Price-based (a) (a) AcƟve maƩer- based 1,955 1,956 1,957 1,958 1,959 1,960 1,961 1,962 1,963 1,964 1,965 1,966 1,967 1,968 [Gg Ac ve er / year] DDT DDT Lindane Endrin Malathion Pelitre NicoƟne sulfate Dinitro compounds Arsenic compounds Cyanuric compounds (b) (b) 1,955 1,956 1,957 1,958 1,959 1,960 1,961 1,962 1,963 1,964 1,965 1,966 1,967 1,968 Graph 3.22 Historical evolution of synthetic pesticide use in Spain (excluding sulfur and copper pesticides), according to different estimations, 1950–2011, Gg Active matter /year. Price-based series is calculated from MA (1973) and MAPA (1994); total amount-based series is calculated from INE (1960, 1965, 1970), assuming 40% average active matter; active matter series is from FAOSTAT (FAO 2016) 3 Agricultural Inputs and Their Energy Costs 1900–2010 98 0 15 30 45 [Gg / year] SyntheƟc pesƟ Copper and sul pesƟcides Graph 3.23 Historical evolution of pesticide use in Spain, 1900–2008, Gg /year SyntheƟc pesƟcides Copper and sulphur pesƟcides Graph 3.23 Historical evolution of pesticide use in Spain, 1900–2008, Gg /year ﬁnd very unrealistic oscillations that suggest that those statistics are not very accurate (Graph 3.22b). Therefore, we have selected the estimation based on prices instead of that based on total weight. The selected series in Graph 3.22a suggests that most growth in synthetic pesticide use took place during the 1970s and 1980s decades, being more or less stagnant afterwards.Graph3.22bshowstheerraticbehaviorofindividualpesticidesuseduring the 1955–1968 period according to INE. This lack of consistency has led us to discard this data source (Graph 3.23). Our reconstruction of pesticide use during the twentieth and early twenty-ﬁrst centuries shows two major growth periods, one during the ﬁrst third of the century, with the growth in copper and sulfur pesticides, and another one after the autarky period of Franco’s dictatorship, with the growth in synthetic pesticide use. How- ever, the copper and Sulphur series is only based on published statistics during the 1933–1970 period. Therefore, there is a high uncertainty in this series before and after that period. 3.5 Crop Protection Graph 3.24 Historical evolution of greenhouses, tunnels and mulches surface areas in Spain, 1970–2012, in thousands of hectares iﬁcations of previous reports (MAPA 1988, 1989, 1990, 1992, 1993, 1997, 1999, 2001, 2002, 2004, 2006, MARM 2008, 2010, MAGRAMA 2012, 2013a, b, c). The complete series is shown in Graph 3.24a while Graph 3.24b shows 5-year average data in our selected time steps. According to an ofﬁcial 2008 survey, about 97% of ﬁxed installations are plastic-covered and 3% glass-covered (MARM 2008a, b, c). p g The reported surface areas in 1975 were 310 ha of ﬁxed installation, 45 ha of tunnels and 120 ha of plastic mulches. Maximum surface area of ﬁxed greenhouse installations was reached in 2006, with 52,867 ha, and in 2012 it had dropped to 48,206 ha. Maximum surface area of tunnels was reached in 2006, with 14,621 ha, and maximum area of plastic mulches was reached in 2002, with 116,172 ha. The latter dropped heavily in the following years, down to 44,827 ha in 2012. Thus, the growth in this input was one of the latest among the agricultural inputs employed in Spanish agriculture. It was only possible when the technologies used in the construction of plastic-covered greenhouses and other crop protection techniques used in Spain were mature enough to be economically applied at the large scale. Moreover, greenhouse crop production is largely devoted to off-season and fresh produce for export, which are dependent on well-developed distribution chains that were only present in Spain relatively late. 3.5.2 Greenhouses Greenhouses, tunnels, and plastic mulches now represent an important share of hor- ticultural crop production in Spain. The ﬁrst published data on greenhouse and pro- tected crop areas in Spain is in the 1975 Anuario de Estadística Agraria report (MA 1975a, b). Thereafter, the ofﬁcial statistics offer data on some of these items in 1981 (MAPA 1981), and on all of them in 1984 and 1986 (MAPA 1984, 1986). We esti- mated area values in the middle years of those periods, and in years previous to 1975, assuming constant growth rates. After 1986, protected crops data were published on an annual basis. In some of these years, we have data from the previous year or years. We selected preferably the latest published data, which sometimes had mod- 3.5 Crop Protection 99 0 40 80 120 160 200 1970 1980 1990 2000 2008 [Thousands of ha] Sand mulches PlasƟc mulches Tunnels Fixed installaƟons Graph 3.24 Historical evolution of greenhouses, tunnels and mulches surface areas in Spain, 1970–2012, in thousands of hectares 99 3 Agricultural Inputs and Their Energy Costs 1900–2010 100 Table 3.3 External inputs used by Spanish agriculture (TJ), 1900–2008 1900 1930 1950 1970 1990 2008 Feed 1269.9 2046.8 1119.7 53,402.6 51,822.0 187,841.6 Seeds 314.6 183.6 578.2 0.0 0.0 5335.6 Total non-industrial inputs 1584.5 2230.4 1697.9 53,402.6 51,822.0 193,177.0 Traction 528.9 2131.5 5681.3 81,695.6 103,296.4 129,665.6 Irrigation 995.3 2132.2 5096.0 20,712.0 58,565.5 75,406.9 Chemical fertilizers 1884.1 17,865.7 15,142.6 75,240.8 104,987.8 77,618.1 Crop protection 25.8 794.9 653.5 2384.8 24,369.3 31,135.1 Total industrial inputs 3432.2 22,924.2 26.573.5 180,033.3 291,219.0 313,825.7 Total external inputs 5018.7 25,154.6 28,271.3 233,436.0 343,041.0 507,002.7 1900 = 100 Feed 100 161 88 4205 4081 14792 Seeds 100 58 184 0 0 1696 Total non-industrial inputs 100 141 107 3370 3271 12192 Traction 100 403 1074 15,446 19,530 24,516 Irrigation 100 214 512 2081 5884 7576 Chemical fertilizers 100 948 804 3003 5572 4120 Crop protection 100 3081 2533 9243 94,455 120,679 Total industrial inputs 100 668 774 5245 8485 9144 Total external inputs 100 501 653 4651 6835 10102 Source Author’s own compilation based on statistical sources and the use of coefﬁcients included in Aguilera et al. (2015) orders of magnitude in the period between those two years, two thirds of which were industrial inputs. Industrial inputs and their energy costs grew strongly in the second half of the 20th century, ﬁrst with the use of chemical fertilizers and mechanization, then by irrigation and crop protection. As a result, yields per unit area increased, especially in irrigated areas and in farms using new seed varieties, both hybrid and improved. In fact, the use of seeds particularly increased between 1980 and 2008 with the arrival of industrial germplasm from specialized global companies. The most immediate effect of the application of this land-saving technology was the use of varieties that were more productive than traditional ones under optimum nutrient and moisture supply conditions. But that was not the only effect. It also broke the necessary rotations of traditional management to adapt to the shortage of both nutrients and moisture. Thus, there was an expansion of monocultures and crop rotations determined not by agronomic rationality but by agricultural market demands. Resulting biodiversity reductions favored the appearance of plant plagues and diseases and additional use of phytosanitary products that had until then been quite limited. 3.6 Use of Inputs in the Agricultural Sector (Imports) In the previous sections, we saw how the growth of external inputs was unstoppable, especially since 1960, making it possible to intensify and specialize production as described in the previous chapter. Table 3.3 shows all the inputs used from 1900 to 2008, distinguishing between industrial and biological inputs, as well as their different behavior throughout the period. The use of inputs generally increased by two 3 Agricultural Inputs and Their Energy Costs 1900–2010 3 Agricultural Inputs and Their Energy Costs 1900–2010 These kinds of chemical remedies generated a vicious circle in which orders of magnitude in the period between those two years, two thirds of which were industrial inputs. Industrial inputs and their energy costs grew strongly in the second half of the 20th century, ﬁrst with the use of chemical fertilizers and mechanization, then by irrigation and crop protection. As a result, yields per unit area increased, especially in irrigated areas and in farms using new seed varieties, both hybrid and improved. In fact, the use of seeds particularly increased between 1980 and 2008 with the arrival of industrial germplasm from specialized global companies. The most immediate effect of the application of this land-saving technology was the use of varieties that were more productive than traditional ones under optimum nutrient and moisture supply conditions. But that was not the only effect. It also broke the necessary rotations of traditional management to adapt to the shortage of both nutrients and moisture. Thus, there was an expansion of monocultures and crop rotations determined not by agronomic rationality but by agricultural market demands. Resulting biodiversity reductions favored the appearance of plant plagues and diseases and additional use of phytosanitary products that had until then been quite limited. These kinds of chemical remedies generated a vicious circle in which Use of Inputs in the Agricultural Sector (Imports) 101 3.6 the breaking of trophic chains (the use of insecticides led to the disappearance of beneﬁcial insects that control insect plagues), along with the progression of crops and homogeneous varieties over large stretches of land, made it necessary to increasingly use these substances to control pests and diseases. This item visibly grew the most over time, despite not exceeding 6% of total energy expenditure in 2008. In the next chapter, we will see the effects of mechanization on human work, including the elimination of animal labor. The use of mechanical traction became widespread between the sixties and seventies and never stopped growing, even in the midst of the economic-ﬁnancial crisis. Irrigation followed a similar course: at ﬁrst, irrigation was linked to large hydraulic works and later to so-called “irrigation modernization”, consisting in the ever-increasing role of groundwater elevation and pressurized irrigation networks requiring a high energy use. To ﬁnish, we have to mention that coinciding with the livestock production process described above, feed grew spectacularly, multiplying by a factor of 121 since 1900. 3 Agricultural Inputs and Their Energy Costs 1900–2010 102 References Aguilera E, Guzmán GI, Infante-Amate J, Soto D, García-Ruiz R, Herrera A, Villa I, Torremocha E, Carranza G, González de Molina M (2015) Embodied energy in agricultural inputs. Incorporating a historical perspective. Sociedad Española de Historia Agraria. DT-SEHA 1507 Aguilera E, Guzmán GI, González de Molina M, Soto D, Infante-Amate J (2019a) From animals to machines. The impact of mechanization on the carbon footprint of traction in Spanish agriculture, 1900–2014. 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J Ind Ecol 18:883–897 FAO (2016) Database for food and agriculture. Rome. Available in: http://faostat3.fao.org/browse R/*/E Fernández Prieto L (2001) El cambio tecnológico en la historia agraria de la España contemporánea Hist Agrar 24:59–86 Fischer-Kowalski M, Haberl H (2007) Socioecological transitions and global change: Trajectories of social metabolism and land use. Institute of Social Ecology. Vienna (Austria). 3 Agricultural Inputs and Their Energy Costs 1900–2010 In 2008, it accounted for 38% of the energy value of all inputs used in the agricultural sector. This phe- nomenal growth of inputs generally explains Spanish agriculture’s loss of efﬁciency, as we will see in Chap. 5. Graph 3.25 clearly shows the enormous amount of energy originating from outside the agricultural sector that was necessary to inject into agroecosystems to maintain the continuous growth of agricultural production. While biomass DE grew by 38%, the use of external energy multiplied by a hundred. In 1900, industrial inputs from outside the agroecosystem and used for production rep- resented only 14.5% of total invested energy; in 2008, that percentage had risen to 62%, and if we add the feed from Latin America, it reached 99.4% of total energy invested in agricultural production; that is, practically all the energy invested came from outside the agricultural sector. The socio-economic consequences of this are described in the following chapter. 0 50000 100000 150000 200000 250000 300000 350000 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2008 [Tj] PesƟcides Chemical ferƟlisers IrrigaƟon TracƟon Graph 3.25 Energy value of the inputs used in Spanish agriculture, in TJ. Source see Table 3.3 Graph 3.25 Energy value of the inputs used in Spanish agriculture, in TJ. Source see Table 3.3 Graph 3.25 Energy value of the inputs used in Spanish agriculture, in TJ. Source see Table 3.3 3 Agricultural Inputs and Their Energy Costs 1900–2010 3 Agricultural Inputs and Their Energy Costs 1900–2010 References Edward Elgar Publishing Fischer-Kowalski M, Krausmann F, Pallua I (2014) A sociometabolic reading of the Anthropocene: modes of subsistence, population size and human impact on earth. Anthropocene Rev 1(1):8–33 G N H ll CAS B i k L (2009) A li i i ti ti f t Gagnon N, Hall CAS, Brinker L (2009) A preliminary investigation of energy return on energy investment for global oil and gas production. Energies 2:490–503 Á Gallego D (1986) La producción agraria de Álava, Navarra y La Rioja desde mediados del siglo XIX a 1935. Universidad Complutense de Madrid, Madrid Giampietro M, Bukkens SGF, Pimentel D (1999) General trends of technological changes in agri culture. 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https://openalex.org/W4328096286	https://editoraintegrar.com.br/publish/index.php/rems/article/download/3677/434	Portuguese	null	ACOMPANHAMENTO DA ENFERMAGEM NA SAÚDE DO TRABALHADOR	Revista Multidisciplinar em Saúde	2,023	cc-by	3,507	Citação: ç MAGALHAÃES, B. M. S. et al. As consequências do exercício físico na função cognitiva do idoso com doença de alzheimer. MAGALHAÃES, B. M. S. et al. As consequências do exercício físico na função cognitiva do idoso com doença de alzheimer. MAGALHAÃES, B. M. S. et al. As consequências do exercício físico na função cognitiva do idoso com doença de alzheimer. Revista Multidisciplinar em Saúde, v. 4, n. 1, 2023. https://doi org/10.51161/integrar/rems/3677 Palavras-chave: Saúde do Trabalhador, Atuação da Enfermagem, Saúde Ocupacional, Funções da enfermagem. Data de Aceite: 10/03/2023 1Docente do Curso Técnico de Enfermagem, Sistema de Ensino Gaúcho/Seg. Av. Dorival Cândido Luz de Oliveira, 4490 - COHAB A, Gravataí - RS, 94050-000. 2 Docente do Curso de Enfermagem, Centro Universitário Ritter dos Reis/ UniRitter, Porto Alegre, RS. Rua Orfanotrófio 555, Porto Alegre, RS, CEP: 90840-440. Data de Publicação: 17/03/2023 Revisador por: Bruna Aparecida Lisboa Maria Aurea Soares de Oliveira Revisador por: Bruna Aparecida Lisboa Maria Aurea Soares de Oliveira *Autor correspondente: Diego Silveira Siqueira, diegosilveirasiqueira@gmail.com Silveira Siqueira, diegosilveirasiqueira@gmail.com Artigo de Revisão Artigo de Revisão RESUMO RESUMO Introdução: O trabalho gera e modifica as condições de viver, adoecer e morrer dos indivíduos, considerado um dos principais determinantes sociais que impactam a saúde. De acordo com o Ministério da Saúde, atenção à saúde do trabalhador é estabelecer novas formas de compreensão da relação saúde do trabalhador entre o trabalho e o processo saúde/doença. Objetivo: Discutir a atuação do profissional de enfermagem no serviço de saúde ocupacional nos diferentes níveis de atenção. Métodos: Trata-se de uma revisão narrativa da literatura, tendo como base a busca de artigos científicos indexados as bases de dado na Biblioteca Virtual de Saúde (BVS), utilizando como descritores: saúde do trabalhador, atuação da enfermagem, saúde ocupacional, funções da enfermagem. Realizada a busca foram encontradas 100 produções científicas, aplicando filtros e critérios estabelecidos de inclusão e exclusão, após ficaram 10 artigos que compuseram o estudo. Resultados: Após buscas de artigos e análises, elucida nessa pesquisa duas categorias, sendo elas, a primeira categoria Saúde do Trabalhador e Políticas Públicas, e a segunda categoria o enfermeiro e sua contribuição na saúde do trabalhador. Conclusões: Evidenciou a atuação do Enfermeiro do Trabalho na Serviço de Saúde Ocupacional nos diferentes níveis de atenção, trazendo conhecimentos a respeito da área de Saúde do Trabalhador focando o campo da Enfermagem. Sendo assim, o Enfermeiro do Trabalho deve trazer em sua “bagagem” um rol de conhecimentos tanto na área de saúde, como na área administrativa, já que a necessidade de gestão é extremamente relevante no desempenho das suas atividades. DOI: 10.51161/integrar/rems/3677 Editora Integrar© 2023. Todos os direitos reservados. Keywords: Worker’s Health, Nursing Practice, Occupational Health, Nursing Functions. Keywords: Worker’s Health, Nursing Practice, Occupational Health, Nursing Function ABSTRACT ABSTRACT Introduction: Work generates and modifies the conditions of living, falling ill and dying of individuals, considered one of the main social determinants that impact health. According to the Ministry of Health, worker health care is to establish new ways of understanding the worker’s health relationship between work and the health/disease process. Objective: To discuss the role Rev. Multi. Saúde, v.4. n.1 Rev. Multi. Saúde, v.4. n.1 Santos; Siqueira, 2023 Santos; Siqueira, 2023 of nursing professionals in occupational health services at different levels of care. Methods: This is an integrative review of the literature, based on the search for scientific articles indexed the databases of data in the Virtual Health Library (VHL), using as descriptors: worker health, nursing practice, occupational health, nursing functions. The search was carried out, 100 scientific productions were found, applying filters and established inclusion and exclusion criteria, after 10 articles that comprised the study. Results: After searching for articles and analyses, it elucidates in this research two categories, being the first category Worker’s Health and Public Policies, and the second category the nurse and his contribution to workers’ health. Conclusions: Evidenced the work of the Occupational Health Nurse in the Occupational Health Service at different levels of care, bringing knowledge about the area of Occupational Health focusing on the field of Nursing. Thus, the Work Nurse must bring in his “baggage” a list of knowledge both in the health area and in the administrative area, since the need for management is extremely relevant in the performance of their activities. 1 INTRODUÇÃO O trabalho gera e modifica as condições de viver, adoecer e morrer dos indivíduos, considerado um dos principais determinantes sociais que impactam a saúde. A saúde do trabalhador se caracteriza por uma abordagem multidisciplinar e intersetorial das ações, com foco na promoção da saúde, prevenção de agravos e atenção curativa do trabalhador e, tem como principais eixos, as causas ou os determinantes dos agravos, a exposição aos riscos e os danos ou as consequências (MARZIALE, 2010). De acordo com o Ministério da Saúde, atenção à saúde do trabalhador é estabelecer novas formas de compreensão da relação saúde do trabalhador entre o trabalho e o processo saúde/doença. Propõe ainda, práticas de atenção à saúde dos trabalhadores e intervenção nos ambientes de trabalho aliada ao saber do trabalhador sobre seu ambiente de trabalho e suas vivências das situações de desgaste e reprodução (BRASIL, 2002). O Sistema Único de Saúde reconhece a saúde do trabalhador nos ambientes e processos de trabalho, as condições para eventos agressivos à saúde “de quem trabalha” na perspectiva epidemiológica. Os profissionais de saúde não se restringem a atender o lesionado individualmente, mas busca quantificar o número de pessoas expostas à insegurança e qualificar essas condições para posteriores mudanças (BRASIL, 2004). Desse modo, segurança do Trabalho pode ser definida como a ciência que, por meio de metodologias e técnicas apropriadas estuda as possíveis causas de acidentes do trabalho objetivando a prevenção de sua ocorrência, cujo papel é assessorar o empregador para preservação da integridade física e mental dos trabalhadores e a continuidade do processo produtivo (VOTORANTIM, 2005). Entretanto, muitos trabalhadores encontram-se expostos a diversos riscos ambientais, físicos, mentais, riscos de acidentes e doenças ocupacionais e muitas vezes enfrentam situações laborais inapropriadas. Seja qual for à ótica em que se discuta se faz necessário o envolvimento do setor de saúde visando interceptar, prevenir, controlar e erradicar as causas de doenças e morte de profissionais relacionados ao trabalho em que o trabalho/ocupação é provocador ou agravador de distúrbios e de doenças pré-existentes (BRASIL, 2004). Aspecto de grande relevância apontado para discussão é a atuação do processo de enfermagem dentro da saúde do trabalhador em distintos ambientes de trabalho em todas as empresas, fato que 143 ID: 3824 Rev. Multi. Saúde, v.4. n.1 Rev. Multi. Saúde, v.4. n.1 Santos; Siqueira, 2023 favorece algumas dificuldades, como as relações de trabalho e as ações conjuntas, citadas pelos próprios enfermeiros do trabalho. 1 INTRODUÇÃO Tal forma de estruturação leva à fragmentação do olhar sobre a saúde e segurança do trabalhador e, consequentemente, à ruptura da integralidade na atenção. É preciso que as empresas e os profissionais transcendam esta visão dicotomizada e implementem programas de saúde e segurança que incluam a promoção, proteção e recuperação da saúde do trabalhador de forma coletivizada e com abordagem transdisciplinar e intersetorial (MENDES; WUNSCH, 2007). Desse modo, a escolha do tema surgiu, primeiramente, a partir do interesse em estudar a atuação do enfermeiro do trabalho nos diferentes níveis de atenção no campo da saúde do trabalhador. Esta pesquisa pretende ainda agregar conhecimentos há quem aderir a leitura, a respeito da inserção, das funções e atuações do enfermeiro na saúde do trabalhador. Diante do exposto, este estudo tem por objetivo de discutir através da literatura publicada a atuação do profissional de enfermagem no serviço de saúde ocupacional nos diferentes níveis de atenção. 2 MATERIAL E MÉTODOS Trata-se de uma revisão narrativa da literatura, que é definida como um método que proporciona a síntese de conhecimento e a incorporação da aplicabilidade de resultados de estudos significativos na prática (SOUZA; SILVA; CARVALHO, 2010). Para a composição da pesquisa, foi realizado um levantamento de artigos disponibilizados online na Biblioteca Virtual de Saúde (BVS), para a busca foram utilizados seguintes Descritores em Ciências da Saúde (Decs): Saúde do Trabalhador, Atuação da Enfermagem, Saúde Ocupacional. Os critérios de inclusão foram artigos em português e inglês, disponíveis na íntegra de Janeiro de 2008 a setembro de 2022, período da busca foi em outubro de 2022 que possuíssem os descritores selecionados. O critério de exclusão que não elencassem a proposta de busca do estudo. Foi utilizado o Portal Regional da BVS que é espaço de integração de fontes de informação em saúde que promove a democratização e ampliação do acesso à informação científica e técnica em saúde na América Latina e Caribe (AL&C). É desenvolvido e operado pela BIREME em 3 idiomas (inglês, português e espanhol). E possui várias bases de dados indexadas. As variáveis de interesse selecionadas foram: título do artigo, assunto principal e ano de publicação. 3 RESULTADOS E DISCUSSÃO Após buscas de artigos e análises deles, elucida nessa pesquisa duas categorias, sendo elas, a primeira categoria Breve Histórico da Enfermagem do Trabalho , e a segunda categoria O enfermeiro e sua contribuição na saúde do trabalhador. 3.1 Breve Histórico da Enfermagem do Trabalho A essência do processo de trabalho na enfermagem é a prestação de cuidados humanos e o desenvolvimento de promoção, prevenção de doenças e acidentes, e a reabilitação da saúde. O campo de atuação está mais abrangente a cada dia e dispõe de áreas além de hospitalares, área de pesquisa, empresas, indústrias e usinas onde a atuação é como enfermeiro do trabalho (SILVA et al, 2011). Essa atuação como enfermeiro ocupacional é resultado do processo evolutivo iniciado na Inglaterra Essa atuação como enfermeiro ocupacional é resultado do processo evolutivo iniciado 144 ID: 3824 144 ID: 3824 Santos; Siqueira, 2023 Rev. Multi. Saúde, v.4. n.1 Rev. Multi. Saúde, v.4. n.1 no final do século XIX. No final da década de 60, os países industrializados viveram um grande movimento social mundial, com a discussão de saúde e segurança no trabalho (SILVA, 2005). Conforme Silva et al (2011), o primeiro curso de pós-graduação em enfermagem do trabalho, no Brasil, aconteceu no ano de 1974, no estado do Rio de Janeiro, quando ocorreu a inclusão da equipe de saúde ocupacional. A partir dessa data a enfermagem do trabalho passou a ser reconhecida como uma função específica dentro das empresas. A Enfermagem é uma das profissões da área de saúde, cuja essência e especificidade é o cuidado ao ser humano, individualmente, na comunidade ou na família, desenvolvendo atividades de promoção, prevenção de doenças, recuperação e reabilitação da saúde, atuando em equipes. Em todo o mundo, a enfermagem, constitui o maior contingente da força de trabalho em cuidados à saúde. No entanto, encontramos os enfermeiros em muitos outros campos, entre os quais destacamos neste estudo, indústrias, empresas e usinas de cana de açúcar, campo onde atuam os enfermeiros do trabalho (DURAN; ROBAZZI; MARZIALE, 2007). No campo da assistência à saúde, a decisão dos enfermeiros, tem sido pouco explorada e, pelas características de autonomia no campo profissional e competência técnica, propicia um novo conceito de enfermagem, e consequentemente maior desenvolvimento profissional (COSTA, 1978). A enfermagem moderna, como profissão cientificamente orientada, deve estar integrada aos padrões normativos técnico-científico e ético-cultural do desempenho do papel profissional. Ela deve ser autoridade no seu campo específico, dentro dos padrões profissionais, o que quer dizer, apenas, que em assuntos de enfermagem, é o enfermeiro aquele que decide (DURAN; ROBAZZI; MARZIALE, 2007). Silva et al (2011) conceitua o enfermeiro do trabalho como o profissional que possui especialização em enfermagem do trabalho e é incumbido de assistir os trabalhadores, promovendo e zelando pela sua saúde, incentivando a prevenção de acidentes e doenças relacionadas ao trabalho e/ou prestando cuidados aos doentes e acidentados. A inserção do enfermeiro do trabalho se deu com as reformas iniciadas no final do século XVIII e desenvolveu-se paralelamente à Revolução Industrial, marco inicial da moderna industrialização, ocorrida entre 1760 e 1830, inicialmente na Inglaterra, estendendo-se mais tarde para a Alemanha, a França e os demais países europeus, onde começaram a se notar com maior evidência os problemas de saúde dos trabalhadores. 3.2 O enfermeiro e sua contribuição na saúde do trabalhador A enfermagem em relação a saúde-trabalho contempla, ao longo da história, avanços e marcos referenciais distintos, envolvendo a Medicina do Trabalho, a Saúde Ocupacional e a Saúde do Trabalhador, sendo esta última a abordagem mais atual e ampla e cujo objeto é o processo saúde e doença, em sua relação com o trabalho, na busca de condições e ambientes saudáveis, contextualizada no momento sócio-político de uma determinada sociedade (MARZIALE, 2010). A capacitação dos profissionais de saúde pode ser considerada como fundamental na assistência aos pacientes, haja vista que estes devem se capacitar para atuar, visando à orientação dos pacientes na forma correta de lavar as mãos, a importância da utilização de máscaras, dentre outros e devem estar preparados para responder os questionamentos, de modo a seguir os protocolos e orientações do Ministério da Saúde (ZOCCHIO, 2012). Assim, segundo a Organização Mundial da Saúde, a principal finalidade dos Serviços de Saúde Ocupacional consiste na promoção de “condições de trabalho que garantam o mais elevado grau de qualidade de vida no trabalho, protegendo a saúde dos trabalhadores, promovendo o seu bem-estar físico, mental e social e prevenindo a doença e os acidentes”. Nomeadamente, pretende-se a sua proteção contra os riscos resultantes da presença de agentes nocivos à sua saúde, colocando e mantendo o trabalhador num emprego que convenha às suas aptidões fisiológicas e psicológicas, isto é, adaptar o trabalho ao homem e cada homem ao seu trabalho. A Saúde do Trabalhador tem como foco a abordagem multidisciplinar e intersetorial de ações na perspectiva da totalidade, com vistas à superação da compreensão e intervenções estanques e fragmentadas; possibilita a participação dos trabalhadores, enquanto sujeitos de sua vida e de sua saúde, capazes de contribuir com seu conhecimento para o avanço da compreensão do impacto do trabalho sobre o processo saúde-doença e de intervir politicamente e promover a saúde do trabalhador (MARZIALE, 2010). De acordo com Hayashide e Buschinelli (2016), a saúde do trabalhador deve ser monitorada diariamente quando este executa a sua atividade laboral, analisando o risco com o intuito de detectar sinais, que possam trazer riscos de acidentes, que podem causar danos a sua saúde ou até a morte. As diretrizes trazidas pela Política Nacional de Saúde do Trabalhador do Ministério da Saúde (BRASIL, 2004) visam à redução dos acidentes e doenças relacionadas ao trabalho, através de ações de promoção, reabilitação e vigilância na área de saúde. ID: 3824 De acordo com Malagutti; Miranda (2010) Em 1830 foi iniciado o primeiro serviço de saúde ocupacional do mundo, através da nomeação do médico Robert Baker, pelo governo britânico. Duas décadas depois, também na Inglaterra, tem início a reforma da Enfermagem, sob a direção da Florence Nightingale, que com sua determinação e grande perspicácia lançou os alicerces da profissão e disciplina da Enfermagem. Apesar de Florence Nightingale ter iniciado a preocupação com a Enfermagem do Trabalho, a real origem ocorreu em 1878, quando a enfermeira inglesa Phillipa Flowerday foi contratada por uma empresa para prestar cuidados aos seus trabalhadores, após a promulgação de uma lei que criou o sistema centralizado de inspeções de fábricas com a chefia de investigações em Londres. Philipa foi, portanto, a primeira enfermeira industrial a ser contratada para um serviço de prestação de cuidados de Enfermagem a trabalhadores (MALAGUTTI; MIRANDA, 2010). Ribeiro (2012) completa que a atenção á saúde do trabalhador, embora campo específico do setor Ribeiro (2012) completa que a atenção á saúde do trabalhador, embora campo específic 145 ID: 3824 Rev. Multi. Saúde, v.4. n.1 Santos; Siqueira, 2023 saúde que busca o objetivo de desenvolver e implementar uma política de saúde dos trabalhadores; promover e proteger a saúde no trabalho; melhorar os serviços de saúde ocupacional e o acesso a eles e elaborar um sistema de vigilância dos riscos no trabalho; precisa, para isso, interligar-se com outros setores. 4 CONCLUSÃO Com este estudo conseguimos alcançar o objetivo em conhecer e analisar a atuação do Enfermeiro do Trabalho na Serviço de Saúde Ocupacional nos diferentes níveis de atenção, trazendo conhecimentos a respeito da área de Saúde do Trabalhador focando o campo da Enfermagem. Neste sentido, é importante destacar que a saúde do trabalhador é uma esfera de intervenção de grande relevância, que considera o ambiente de trabalho como um local estratégico para a prevenção primária dos perigos laborais, a proteção e promoção da saúde dos funcionários, além do acesso aos serviços de saúde no local de trabalho. Dessa forma, o papel do enfermeiro se torna fundamental no contexto das empresas, atuando na preservação da saúde e integridade dos colaboradores, garantindo a conformidade com as diretrizes estabelecidas pelas Normas Regulamentadoras do Ministério do Trabalho e Emprego. O enfermeiro desempenha uma função crucial na promoção do bem-estar dos trabalhadores, oferecendo orientação, supervisão e cuidado para assegurar um ambiente de trabalho seguro e saudável. 3.2 O enfermeiro e sua contribuição na saúde do trabalhador Suas diretrizes compreendem a atenção integral à saúde, a participação popular, o apoio a estudos e a capacitação de recursos humanos. Pensando neste bem estar, faz-se necessária uma atuação contínua e sistemática, ao longo do tempo, no sentido de detectar, conhecer, pesquisar e analisar os fatores determinantes e condicionantes dos agravos à saúde relacionados aos processos e ambientes de trabalho, em seus aspectos tecnológico, social, organizacional e epidemiológico, com a finalidade de planejar, executar e avaliar intervenções sobre esses aspectos, de forma a eliminá-los ou controlá-los, onde se atua a Vigilância em Saúde do Trabalhador (ALEXANDRINO; COUTINHO; SANTOS, 2013). ID: 3824 ID: 3824 146 Santos; Siqueira, 2023 Rev. Multi. Saúde, v.4. n.1 Rev. Multi. Saúde, v.4. n.1 Ribeiro (2012) completa que a atenção à saúde do trabalhador, embora campo específico do setor saúde que busca o objetivo de desenvolver e implementar uma política de saúde dos trabalhadores; promover e proteger a saúde no trabalho; melhorar os serviços de saúde ocupacional e o acesso a eles e elaborar um sistema de vigilância dos riscos no trabalho; precisa, para isso, interligar-se com outros setores. REFERÊNCIAS ALEXANDRINO, F. C; COUTINHO, M. A. A; SANTOS, C. M. F. Atuação do enfermeiro do trabalho no serviço de saúde ocupacional nos diferentes níveis de atenção. Curso de Especialização em Enfermagem do Trabalho do Programa de Pós-Graduação Lato Sensu da Escola Bahiana de Medicina e Saúde Pública, 2013. BRASIL. Ministério da Saúde. Política Nacional de Saúde do Trabalhador. – Brasília, 2004. BRASIL. Ministério da Saúde. Política Nacional de Saúde do Trabalhador. – Brasília, 2004. BRASIL. Departamento de Atenção Básica. Área Técnica de Saúde do Trabalhador. Caderno de saúde do BRASIL. Ministério da Saúde. Política Nacional de Saúde do Trabalhador. – Brasília, 2004. BRASIL. Departamento de Atenção Básica. Área Técnica de Saúde do Trabalhador. Caderno de saúde do trabalhador, nº 5. Brasília; 2002. BRASIL. Ministério da Saúde. Política Nacional de Saúde do Trabalhador. – Brasília, 2004. BRASIL. Departamento de Atenção Básica. Área Técnica de Saúde do Trabalhador. Caderno de saúde do trabalhador, nº 5. Brasília; 2002. BRASIL. Departamento de Atenção Básica. Área Técnica de Saúde do Trabalhador. Caderno de saúde do trabalhador, nº 5. Brasília; 2002. CONFLITO DE INTERESSE Os autores declaram que não há conflito de interesses. CARVALHO, G. M. Enfermagem do Trabalho. São Paulo: EPU, 2001. COSTA, M. J. C. Atuação do enfermeiro na equipe multiprofissional. Rev. Bras. Enferm, Brasília , v. 31, n. 3, p. 321-339, 1978. DURAN, E. C. M; ROBAZZI, M. L. C. C; MARZIALE, M. H. P. Conhecimento de enfermagem em saúde do trabalhador oriundo de dissertações e teses. Revista Gaúcha de Enfermagem, v. 28, n. 3, p.416-23, 2007. HAYASHIDE E BUSCHINELLI. A saúde do trabalhador que executará o trabalho. 2016. Revista Brasileira de Saúde Ocupacional ISSN: 2317-6369 (online) RBSO Disponível em: http://dx.doi. org/10.1590/2317-6369000128615. Acesso em: 26 de Fevereiro de 2023. ID: 3824 147 Santos; Siqueira, 2023 Rev. Multi. Saúde, v.4. n.1 MALAGUTTI, W; MIRANDA, S. M. R. C. Os Caminhos da enfermagem: de Florence à Globalização – São Paulo: Phorte, 2010. MALAGUTTI, W; MIRANDA, S. M. R. C. Os Caminhos da enfermagem: de Florence à Globalização – São Paulo: Phorte, 2010. MARZIALE, M. H. P. Contribuições do enfermeiro do trabalho na promoção da saúde do trabalhador. Acta paul. enferm, vol.23, no.2 - São Paulo-SP- Mar./Apr. 2010. RIBEIRO, M. C. S. Enfermagem e Trabalho. 2 ed. São Paulo: Martinari, 2012. RIBEIRO, M. C. S. Enfermagem e Trabalho. 2 ed. São Paulo: Martinari, 2012. SOUZA, M. T; SILVA, M. D; CARVALHO, R. Revisão integrativa: o que é e como fazer. Einstein (São Paulo), v. 8, n. 1, p. 102-106, Mar. 2010. SILVA, L. A et al. Enfermagem do trabalho e ergonomia: prevenção de agravos à saúde. Rev. enferm. UERJ, Rio de Janeiro, v. 19, n. 2, p. 317-23, abr/jun, 2011. SILVA, S. L. Interações do enfermeiro do trabalho com a saúde do trabalhador em âmbito de prática e assistência de enfermagem - Rio de Janeiro: UFRJ/ EEAN, 2005. Disponível em: http://teses.ufrj.br/ EEAN_d/SergioLimaDaSilva.pdf. ZOCCHIO, Á. Prática da prevenção de acidentes : ABC da segurança do trabalho. 7 ed. São Paulo: Atlas, 2012. ZOCCHIO, Á. Prática da prevenção de acidentes : ABC da segurança do trabalho. 7 ed. São Paulo: Atlas, 2012. ID: 3824 148 148
https://openalex.org/W2169136139	https://digitalcommons.lmu.edu/cgi/viewcontent.cgi?article=1601&context=ce	English	null	The United States: The Role of the Brothers of Holy Cross	Catholic education/Catholic education (Dayton, Ohio. Online)	2,007	cc-by	3,858	Journal of Catholic Education Journal of Catholic Education Volume 11 Issue 2 Article 7 12-1-2007 The United States: The Role of the Brothers of Holy Cross The United States: The Role of the Brothers of Holy Cross Philip C. Armstrong Journal of Catholic Education Journal of Catholic Education Volume 11 Issue 2 Article 7 12-1-2007 The United States: The Role of the Brothers of Holy Cross The United States: The Role of the Brothers of Holy Cross Philip C. Armstrong Journal of Catholic Education Journal of Catholic Education Journal of Catholic Education Journal of Catholic Education Volume 11 Issue 2 Article 7 The United States: The Role of the Brothers of Holy Cross The United States: The Role of the Brothers of Holy Cross Philip C. Armstrong The United States: The Role of the Brothers of Holy Cross The United States: The Role of the Brothers of Holy Cross Philip C. Armstrong Follow this and additional works at: https://digitalcommons.lmu.edu/ce Recommended Citation Recommended Citation Recommended Citation Recommended Citation Armstrong, P. C. (2007). The United States: The Role of the Brothers of Holy Cross. Journal of Catholic Education, 11 (2). http://dx.doi.org/10.15365/joce.1102072013 Armstrong, P. C. (2007). The United States: The Role of the Brothers of Holy Cross. Journal of Catholic Education, 11 (2). http://dx.doi.org/10.15365/joce.1102072013 Armstrong, P. C. (2007). The United States: The Role of the Brothers of Holy Cross. Journal of Catholic Education, 11 (2). http://dx.doi.org/10.15365/joce.1102072013 This Focus Section Article is brought to you for free with open access by the School of Education at Digital Commons at Loyola Marymount University and Loyola Law School. It has been accepted for publication in Journal of Catholic Education by the journal's editorial board and has been published on the web by an authorized administrator of Digital Commons at Loyola Marymount University and Loyola Law School. For more information about Digital Commons, please contact digitalcommons@lmu.edu. To contact the editorial board of Journal of Catholic Education, please email JCE@nd.edu. 218 Catholic Education/December 2007 EARLY 20TH CENTURY EDUCATION IN THE UNITED STATES: THE ROLE OF THE BROTHERS OF HOLY CROSS PHILIP C. ARMSTRONG, C.S.C. Brothers of Holy Cross, Notre Dame, IN PHILIP C. ARMSTRONG, C.S.C. The Congregation of Holy Cross, the founding religious community behind the University of Notre Dame, had its origin in the ranks of diocesan clergy in France. This article chronicles the early history of the congregation, struggles between priests and its brothers, and its movement into the ministry of Catholic education. Catholic Education: A Journal of Inquiry and Practice, Vol. 11, No. 2, December 2007, 218- 225 © 2007 University of Notre Dame. BACKGROUND T he French Revolution bears an ironic responsibility for generating works of charity. To counteract the devastating social effects of that late 18th century uprising, numerous religious communities were founded in France, among them the Congregation of Holy Cross. T In 1820, conditions then permitting, the bishop of Le Mans and several diocesan priests prevailed upon Fr. Jacques François Dujarié, one of their number and a veteran of the difficult days of clerical persecution, to organ- ize some laymen into a teaching force designed to assist in restoring Catholic elementary education to the scattered parishes of the diocese. For 15 years, Dujarié supervised the growth of this small band, known as the Brothers of St. Joseph, and managed, with the aid of one or two among their number and the collaboration of the LaSalle Christian Brothers and the Ploërmel Brothers, to provide them with some fundamental religious training as well as with the minimal academic background needed to begin their apostolic work. Illness, however, forced Dujarié to hand over his fledgling group to someone else. Ready to assist was Fr. Basile-Antoine Moreau, also of Le Mans, who himself had organized a small number of diocesan priests to spe- cialize in preaching missions and substituting where necessary throughout the diocese. Moreau’s dream was to establish not only priests, but brothers and sisters as well, in one united family of apostolic religious. Dujarié’s request was seen as a providential moment, and Moreau readily accepted responsibility for the brothers. Merging them officially in 1837 with the Armstrong/THE ROLE OF THE BROTHERS OF HOLY CROSS 219 priests, Moreau called the group the Association of Holy Cross. In 1857, when through Propaganda Fide papal approbation was granted to the com- munity, the title became the Congregation of Holy Cross. From 1835, Moreau enhanced the religious formation of the brothers and saw to their continuing effectiveness in small parish schools throughout the area. But Moreau’s horizons spread far beyond the diocese of Le Mans in France. He had a zealous missionary spirit that prompted him almost imme- diately to send his best religious to respond to the cries of bishops in four needy areas of the world: Algeria, India, Canada, and the United States. PRESENCE IN ELEMENTARY EDUCATION Beginning that year and expanding its vision as well as its presence over the next 50 years, the small missionary group began apostolic ministry in the south-central area of Indiana, and then a year later in the northern part of the state at the present site of the University of Notre Dame near South Bend. During these 50 years, the brothers spread throughout the Midwestern part of the US and even ventured to the Eastern seaboard, taking on at various times and for various periods responsibility for some 76 elementary schools in 13 states. The need for Catholic elementary education was great in this pioneering era of U.S. development, and the Brothers of Holy Cross were able to respond by reason of their numbers, their sacrificial generosity, and their capability to recruit willing young men not only from among the French immigrant families they came to serve, but from among lads of Irish or German extraction as well. Despite the significant and effective growth of the brothers’ presence during the last half of the 1800s in elementary education, the turn of the cen- tury found them paradoxically closing or withdrawing from almost all of the 76 missions undertaken between 1841 and 1893. BACKGROUND In 1841, he sent a small contingent of six brothers and one priest to assist Bishop Céléstin de la Hailandière of Vincennes, Indiana, in establishing parochial schools and auxiliary services for the diocese. Burns (1912) added, Brothers who teach in the parish schools generally receive from $300 to $400 per year. Notwithstanding this, the per capita saving in their schools is not much if any, above that in the schools of the Sisters. The living expenses of women are not so high as those of men. Nor do men understand as women do the art of economizing. (p. 282) Burns (1912) also noted that “the salary most commonly received by Sisters engaged in parish-school work is $20 per month, or $200 per year” (p. 280). Weber (1961) stated: Burns (1912) also noted that “the salary most commonly received by Sisters engaged in parish-school work is $20 per month, or $200 per year” (p. 280). Weber (1961) stated: The teaching Sisters replaced the Brothers because of the great savings involved. One estimate is that one Brother taught for one-half the cost of a male public school teacher and that a Sister taught for one-half the Brother’s salary. An increase in vocations to the Sisterhood, together with permission for the Sisters to teach boys up to the age of thirteen, also began the decline of the teaching Brothers’ power. (p. 157) The second reason for the decline in the brothers’ presence in primary education was that the University of Notre Dame—from its founding in 1842 a principal focus of apostolic presence for both the priests and brothers of the Congregation of Holy Cross and its motherhouse in the US—required both money and manpower to support its continuing growth and development. Much of the funding and many of the brothers formerly assigned to primary education elsewhere were husbanded by provincial superiors to contribute to the enhancement of the various programs at Notre Dame, including the pri- mary, preparatory, and college levels. O’Dwyer (1937) notes, “Schools were given up for various reasons, not the least among them being the need for more Brothers in various capacities at the expanding University of Notre Dame” (p. 12). Burns (1912) also remarks: Most of these parish schools were eventually relinquished. The chief cause of this was the scarcity of vocations to the teaching brotherhood, together with the remarkable growth of the College of Notre Dame, which necessitated an even greater concentration of the Community at home. (p. REASONS FOR CHANGE Three compelling reasons contributed to this turn of events. The first was that Catholic religious sisters were, in their numbers, becoming available for and engaging in primary education up to the eighth grade for such a meager stipend that men simply could not compete. Burns (1912) noted, A number of the teaching brotherhoods that came to the country during the Immigration Period have grown and prospered; but they have not, generally speaking, kept pace with the growth of the communities of women which were Catholic Education/December 2007 220 established here at the same time. Vocations to the religious life appear to be more plentiful among women than among men. (p. 104) established here at the same time. Vocations to the religious life appear to be more plentiful among women than among men. (p. 104) Burns (1912) added, 104) It must be borne in mind that though the University of Notre Dame has traditionally been associated with the priests of Holy Cross, the brothers, as Armstrong/THE ROLE OF THE BROTHERS OF HOLY CROSS 221 proper to the egalitarian nature of the congregation, have been an integral factor in its founding and staffing—especially during the first 100 of its 160- plus years—in campus roles ranging from professorships to prefecting and teaching on the primary and secondary levels, to clerical skills, and to the most common but necessary maintenance tasks. The brothers were in com- plete control of the “minims” (the primary school section) and the prepara- tory department of the university. It was at great sacrifice to the much need- ed independent development of the society of brothers in the congregation that so many of them were withdrawn from various apostolic commitments and returned to the stateside motherhouse to shore up its human resources and assure its fiscal well-being. A third reason, one not expressed by either Burns (1912) or Weber (1961), contributed to the decline of the brothers in primary education, a rea- son that in hindsight was perhaps fortuitously beneficial. As the 20th centu- ry began, bishops were beginning with some urgency to seek male religious to conduct boys’ secondary school departments in their dioceses. This new and ready market for the brothers’talents thus became a prime focus of apos- tolic activity. Unfortunately, few Holy Cross brothers had as yet been ade- quately prepared to engage in secondary school administration or teaching. Fr. John Zahm, provincial superior in 1905, wrote, We have only to look around to see that, in every country, the Governments are tending to exact official grades of those destined for the mission of teaching. It is prudent to take immediate measures to that effect, so that we may be ready for any eventuality. Besides, if solid piety already possesses...an efficacy so deeply rooted that nothing can take its place, how much more would not the same piety effect, when armed with profound knowledge and requirements, slowly and perfectly acquired, and amply sufficient for all branches of learning, rather than those superficial attainments, vague and disconnected and hastily and incompletely stored in mind. (J. Burns (1912) added, Zahm, Circular Letter, October 15, 1905) This situation was also alluded to by O’Dwyer (1937) in his manuscript, “The First Hundred Years.” This situation was also alluded to by O’Dwyer (1937) in his manuscript, “The First Hundred Years.” The problem of vocation recruitment affected the brothers’ potential for an adequate response to the needs of the dioceses. Burns (1917) stated, It remains that men teachers are, by common consent, preferable for boys of high-school age. We would have a larger number of Brothers’ high schools if we had more teaching Brothers. One of the greatest needs in Catholic educa- tion at the present time is an increase of vocations to the teaching brotherhoods. (p. 89) 222 Catholic Education/December 2007 These three reasons—the emergence of religious sisters, the recall of brothers to Notre Dame, and the urgent request for male religious as second- ary school teachers—precipitated events in the first part of the 20th century that were destined to determine the apostolic directions of the brothers for the next half century and beyond. BROTHER EPHREM O’DWYER Br. Ephrem (Dennis) O’Dwyer was an immigrant, recruited for the com- munity in Ireland by two native Irish Holy Cross religious. He responded positively and accompanied them to Notre Dame in 1907 at the age of 19 and at the height of the crucial period of intracongregational tension. His intelli- gence and shrewdness, along with a bold and energetic aggressiveness, set him apart early as a capable teacher, administrator, and defender of the rights of the brothers. He joined almost at once after his perpetual profession of vows in seeking a resolution to the problems the brothers were encountering. Assigned in the years after his novitiate to work in the few secondary schools among the brothers’ apostolates, he gained experience in both the classroom and office. In 1923, he was given time to complete his undergraduate work at Notre Dame and earn a Master’s degree in education, as part of the effort by the community to foster professionalism among the brothers. It was during this sabbatical that O’Dwyer, responding to the state of Indiana’s recent emphasis on accreditation of teachers, and recognizing the still inadequate organization of the brothers’ talents toward certification, engaged in a study of the heretofore relatively neglected educational records of the brothers at Notre Dame and prepared a roster of men and their status. The result was the immediate accreditation of some brothers and the estab- lishing of concrete educational goals for others toward secondary classroom and administrative licensing. g O’Dwyer (1937) described this period: The General Chapter of 1906 formulated plans for a revival of teacher-training, and the Very Rev. Gilbert Français, Superior General, whole-heartedly support- ed the movement. Improvement began under the provincialship of Fr. Andrew Morrissey. Dujarié Hall was built as a new House of Studies and opened on August 15, 1907. Fort Wayne grade school became a standard high school in 1909 and the next year Holy Trinity in Chicago assumed high school status. The [General] Chapter of 1912 promulgated further decrees for the welfare of the Brothers and the advancement of their educational aspirations. Sacred Heart Preparatory College, Watertown, Wisconsin, was converted into a Juniorate for postulants to the Brotherhood. The provision for high school work at Watertown removed the postulants from Dujarié Hall, making the building a residence for college students only. The Brothers were given direction of Holy Cross College, New Orleans, a preparatory boarding and day school. INTERNAL COMPLICATIONS However clear as these portents seemed, the change was not achieved with- out difficulty, at times verging on catastrophe. Internal tensions concerning equality between the priests and brothers of Holy Cross had by the turn of the century provoked a critical juncture in their relationships. Some brothers, with what they felt to be adequate reason, incited enough rancor among their number to cause dissension and even prompt a formal request to Rome that the brothers and the priests be separated juridically on all levels of govern- ment to permit them freely to seek their own independent ministries. This request was denied, but the Vatican insisted that the clearly valid complaints of both brothers and priests must be effectively addressed at once. Provincial superiors saw the resolution to tensions to be found in the equal but inde- pendent development of the brothers’ capacities to receive higher education and independently to conduct their own institutions. Many of the priests were supportive of the brothers’ attempts at seeking redress, but they also realized that the apostolic development of the priests’ society was in some sense restrained by the needs and concerns of the brothers. So the priests too yearned for some degree of autonomy. General chapters in the early years of the 20th century, therefore, man- dated the establishment of programs to assure the higher education of the brothers and to permit them to be as independent as possible in the operation of the few secondary schools at which they ministered. The latter, however, were diocesan schools, in which bishops habitually placed “business super- intendents” from among their priests, although the principal was a brother. At times, the superintending was more akin to governing, and the brother administrator felt he had little or no authority in his own school. Several brothers influential in implementing the goals of these decisions toward greater independence in apostolic ministry became highly regarded educators and administrators over the years. Among them—and there were many—were Brothers Justin Dwyer, Aidan O’Reilly, Agatho Heiser, Englebert Leisse, and Ephrem O’Dwyer. It is to the latter figure that we are most indebted by reason of his role in promoting the identity, image, and independence of the brothers in the apostolate. Armstrong/THE ROLE OF THE BROTHERS OF HOLY CROSS 223 NEW DIRECTIONS Fr. Gilbert Français, Superior General, wrote in a letter circulated to the con- gregation in 1912 that a new future was open to the brothers. Although they had lost the parochial schools, Catholic high schools had grown and become more indispensable. “From this time forward,” he proclaimed, “the High School is the outstanding vocation of our Brothers!” (as cited in Weber, 1961, p. 165). He felt that instead of being buried in the general works of the congregation, a new field with ample scope for personal initiative and oppor- tunity was opening to the brothers. “In a word, the High School furnishes an element of durable and holy peace between the two branches of our society. Both will hereafter march unitedly forward like two distinct forces that make but one, and that aid each other mutually” (p. 165). Français was overjoyed that in finding a resolution to the issue of apostolic thrust for the brothers, internal congregational tensions could, he hoped, simultaneously be laid to rest. In 1945, the general chapter of the Congregation of Holy Cross took the momentous step of declaring the societies of priests and brothers autonomous and homogeneous on the provincial level, though united still in their constitutions and in a common governmental council on the general level. This move paved the way for virtually independent development in both societies. Responding to critical post-war needs, O’Dwyer, elected to serve as the first provincial of the autonomous province of brothers, had in mind a con- crete plan of expansion and entrenchment for the brothers as initiators and operators of quality institutions of secondary education throughout the United States. In 10 years as provincial, he not only solidified the brothers’ presence in the diocesan owned high schools, but he also opened 15 new institutions, most of them province owned, and managed the subdivision of the one United States brothers’ province into three separate governmental units headquartered in different areas of the country. The impetus of expansion continued beyond O’Dwyer’s term of office, and, according to statistics available in Holy Cross community directories and the Catholic Directory, by 1967 there were 32 secondary schools in the US staffed by the brothers, who numbered over 550 directly involved in those institutions, about 5% of the over 12,000 brothers of all congregations in the US at that time. BROTHER EPHREM O’DWYER The State of Indiana does not claim jurisdiction over private schools; but, when requested, it recognizes and “commissions” private schools which meet the standard of public schools as regards teachers and equipment. Because of college entrance, this recognition is rated as desirable, and has been so consid- ered since the plan first originated in 1913. It has meant much to the Brothers 224 Catholic Education/December 2007 of Holy Cross, for it has provided a sufficient reason for the thorough training of teachers. (p. 13) of Holy Cross, for it has provided a sufficient reason for the thorough training of teachers. (p. 13) NEW DIRECTIONS By contrast, in the early 1990s, some 50 years after the serious effort began to broaden their presence in secondary education, and some 25 years after the peak of their influential presence, the Brothers Armstrong/THE ROLE OF THE BROTHERS OF HOLY CROSS 225 of Holy Cross had remaining only 19 of their secondary schools, and their numbers in them were decreased to fewer than 200, an even smaller percent- age of the equally decimated total number of some 6,000 brothers in the US. g q y , Forced by circumstances out of primary education as the new century dawned, affected by intracongregational tensions and difficulties as efforts were made on all levels to enable the brothers to merge into the secondary educational level of the early 20th century, and impacted by the requests of bishops and pastors attempting to meet the needs of their people in educa- tion, the Congregation of Holy Cross, led by its general and provincial supe- riors and by such notable individuals in that field as Fr. James Burns, Br. Ephrem O’Dwyer, and Fr. William Cunningham, facilitated the transition of the brothers from largely rural or small town Catholic primary educational ministry to that of urban secondary schools. TODAY AND INTO THE FUTURE The presence of the brothers in education remains visible and tangible, but decreasingly so. To offset this phenomenon, the spirit of their educational ministry is being absorbed and perpetuated by those whom the brothers have educated and who have responded to the needs of today by collaboratively assuming the burden of assuring Catholic education for their own children and grandchildren. Thanks to the determination and generous adaptability of the pioneering men of Holy Cross, the congregation has played a significant and effective role in the fostering of Christian education in the United States, a role that continues, if not as noticeably in its physical presence, at least in its perduringly enviable spirit. Philip C. Armstrong, C.S.C., is a staff assistant in the Province Administration/Development Office for the Midwest Province of the Brothers of Holy Cross. Correspondence should be sent to Brother Philip C. Armstrong, C.S.C., Brothers of Holy Cross, P.O. Box 460, Notre Dame, IN 46556-0460. REFERENCES Burns, J. A. (1912). The growth and development of the Catholic school system. New York: Benziger Brothers. g Burns, J. A. (1917). Catholic education: A study of conditions. New York: Longmans, Green. O’Dwyer, E. (1937). The first one hundred years. Bulletin of the Educational Conferences of the Brothers of Holy Cross, 1(8), 12. Weber, R. E. (1961). Notre Dame’s John Zahm: American Catholic apologist and educator. Notre Dame, IN: University of Notre Dame Press. Philip C. Armstrong, C.S.C., is a staff assistant in the Province Administration/Development Office for the Midwest Province of the Brothers of Holy Cross. Correspondence should be sent to Brother Philip C. Armstrong, C.S.C., Brothers of Holy Cross, P.O. Box 460, Notre Dame, IN 46556-0460.
https://openalex.org/W4396671990	https://ojs.unida.ac.id/karimahtauhid/article/download/12570/4798	Indonesian	null	Implementasi Inovasi pembelajaran berbasis Discovery Learning melalui Pemanfaatan Media Pembelajaran secara Efektif di SDN Bojong Kiharib	null	2,024	cc-by-sa	2,558	1Meliani Putri, 2Noviandra Azzahra, 3Wangi Dema Lestari, 4Arini 1Meliani Putri, 2Noviandra Azzahra, 3Wangi Dema Lestari, 4Arini 1Program Studi Pendidikan Sekolah Dasar, Fakultas Agama Islam dan Pendidikan Guru 2Universitas Djuanda, Jln. Tol Ciawi No.1, Ciawi-Bogor, Jawa Barat, Indonesia Email: melianiputri717@gmail.com, AndraAzzahra03@gmail.com, wanidemalestari@gmail.com, arini361@gmail.com 4Arini 1Program Studi Pendidikan Sekolah Dasar, Fakultas Agama Islam dan Pendidikan Guru 2Universitas Djuanda, Jln. Tol Ciawi No.1, Ciawi-Bogor, Jawa Barat, Indonesia Email: melianiputri717@gmail.com, AndraAzzahra03@gmail.com, wanidemalestari@gmail.com, arini361@gmail.com Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. Implementasi Inovasi pembelajaran berbasis Discovery Learning melalui Pemanfaatan Media Pembelajaran secara Efektif di SDN Bojong Kiharib 1Meliani Putri, 2Noviandra Azzahra, 3Wangi Dema Lestari, 4Arini 1Program Studi Pendidikan Sekolah Dasar, Fakultas Agama Islam dan Pendidikan Guru 2Universitas Djuanda, Jln. Tol Ciawi No.1, Ciawi-Bogor, Jawa Barat, Indonesia Email: melianiputri717@gmail.com, AndraAzzahra03@gmail.com, wanidemalestari@gmail.com, arini361@gmail.com ABSTRAK Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. ABSTRAK Penelitian ini memotivasi dalam menginvestigasi implementasi inovasi pembelajaran berbasis Discovery Learning melalui pemanfaatan media pembelajaran secara efektif dan kemajuan model-model pembelajaran yang sesuai dengan abad-21 mengenai digitalisasi. Discovery Learning merupakan pendekatan pembelajaran yang memberikan penekanan pada proses penemuan dan pemahaman konsep oleh siswa melalui eksplorasi mandiri. Media pembelajaran digunakan sebagai sarana untuk mendukung pencapaian tujuan pembelajaran dengan penggunaan infokus dan chorome book juga bisa meningkatkan motivasi belajar peserta didik. Namun sarana prasarana yang tersedia disekolah belum maksimal seperti Kurangnya alat peraga dan Media. Termasuk dari faktor peserta didik yang butuh pendampingan lebih dari guru. Metode yg digunakan merupakan penelitian kualitatif dengan melalui pendekatan studi lapangan yaitu pengumpulan data dengan cara observasi, wawancara dengan guru dan dokumentasi. Serta studi pustaka dengan menelaah buku, literatur, dan jurnal yang terkait. Media pembelajaran yang digunakan mencakup, simulasi, dan materi ajar berbasis teknologi. Hasil penelitian ini membuktian bahwa implementasi inovasi pembelajaran berbasis Discovery Learning efektif dalam menumbuhkan keterlibatan siswa dan pemahaman konsep. Penggunaan media pembelajaran secara tepat dan terarah bisa menumbuhkan semangat dan motivasi belajar siswa meningkat. Oleh karena itu guru perlu memiliki keterampilan dalam mengelola pembelajaran berbasis Discovery Learning dan 3449 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. digitalisasi sehingga tujuan pembelajaran bisa dilaksanakan dengan maksimal oleh peserta didik. digitalisasi sehingga tujuan pembelajaran bisa dilaksanakan dengan maksimal oleh peserta didik. Kata Kunci: Implementasi Inovasi, Discovery Learning, Media Pembelajaran. PENDAHULUAN Dalam merancang pembelajaran yang efektif dan bermakna, guru perlu menguasai model pembelajaran yang tepat agar dapat membantu proses pembelajaran. Keberhasilan pembelajaran dapat dilihat dari hasil belajar peserta didik, yang juga menjadi tolak ukur kinerja guru. Pemilihan model pembelajaran kreatif dan inovatif yang sesuai dengan materi dan tujuan pembelajaran sangat penting agar siswa tertarik dan aktif dalam proses pembelajaran. Menghindari model pembelajaran konvensional yang cenderung teacher-centered dapat mencegah kebosanan siswa dan meningkatkan keterlibatan mereka dalam pembelajaran (Rahmayani, 2019). Observasi dilakukan di SDN Bojong Kiharib, dan hasil membuktikan dalam proses belajar siswa di kelas VI masih rendah, Ketika guru tidak menggunakan Model dan Alat peraga yang sesuai dengan pembelajaran. Siswa sering kesulitan memahami materi pelajaran. Guru perlu memilih cara mengajar yang mudah dimengerti dan menarik perhatian. Dengan adanya suatu inovasi, Model pembelajaran Discovery Learning melibatkan siswa secara aktif dalam mencari dan menemukan informasi, tanpa langsung diberikan hasil akhir oleh guru. Siswa diberi kebebasan untuk mengeksplorasi dan memecahkan permasalahan dengan bimbingan guru. Proses pembelajaran ini mendorong siswa untuk aktif berpartisipasi, mencari informasi, dan berkolaborasi dalam kelompok. Dengan cara ini, pembelajaran tidak hanya berpusat pada guru, tetapi lebih menekankan pada peran siswa dalam memahami dan mengingat materi pelajaran. Model ini diharapkan dapat meningkatkan pemahaman siswa dan membuat pembelajaran lebih berkesan (Rahmayani, 2019). 3450 Guru memegang peran strategis dalam membentuk karakter bangsa dan 3450 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. mengembangkan potensi kreatif siswa. Siswa yang kreatif membutuhkan guru yang juga kreatif, mampu menggunakan pendekatan dan variasi pembelajaran yang berbeda. Untuk mencapai pembelajaran yang efektif, guru perlu memiliki strategi untuk mendorong ekspresi pemikiran, gagasan, dan pendapat siswa, yang menjadi dasar karya mereka. Penggunaan media pembelajaran dapat membantu siswa belajar mandiri dan memotivasi mereka untuk mengevaluasi materi pelajaran. Media pembelajaran juga dapat memberikan umpan balik yang membantu siswa mengatasi kekurangan dalam pemahaman materi. Dengan demikian, peran guru yang kreatif dan penggunaan media pembelajaran yang tepat dapat meningkatkan kualitas pembelajaran dan pengembangan siswa (Khamidah et al., 2019). Pada abad ke-21, perkembangan teknologi yang pesat memengaruhi Sumber Daya Manusia (SDM) dengan peningkatan yang terus menerus (Maulana et al., 2023). Dalam konteks pembelajaran, guru memainkan peran penting dalam membantu siswa meningkatkan moral, kecerdasan, dan mengembangkan berbagai keterampilan esensial seperti berpikir kritis, kreativitas, konstruksi pengetahuan, dan pemecahan masalah. Kemampuan ini dianggap sangat relevan untuk menghadapi tuntutan abad ke-21. PENDAHULUAN Abad ke-21 ditandai oleh kemajuan informasi digital yang signifikan, di mana masyarakat terhubung secara luas melalui teknologi. Oleh karena itu, guru, siswa, dan bahkan orangtua perlu memiliki literasi teknologi dan kemampuan menggunakan media komunikasi. Hal ini diperlukan agar mereka dapat berkomunikasi secara efektif, berpikir kritis, menyelesaikan masalah, dan berkolaborasi dalam lingkungan yang semakin terhubung dan digital. Kesadaran terhadap perkembangan teknologi menjadi kunci untuk menjawab tuntutan zaman ini (Syahputra, 2018). Tantangan dalam implementasi inovasi Keterbatasan Fasilitas Ditemui kendala seperti kurangnya alat peraga dan media pembelajaran dan Keterbatasan Akses terkait keterbatasan akses siswa terhadap perangkat atau konektivitas internet karena didaerah SD Bojong kiharib Ini kurang stabil jaringannya juga menjadi kendala. Karena memang guru itu perlu banyak berinovasi dan mengembangkan proses pembelajaran, contohnya pengembangan media pembelajaran bisa untuk menunjang 3451 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. proses belajar siswa tidak membosankan, dengan cara penggunaan infokus dan chorome book juga bisa meningkatkan motivasi belajar peserta didik. Dalam upaya meningkatkan kompetensi siswa, guru perlu memilih model pembelajaran yang efektif dan mendukung, selain menggunakan media pembelajaran yang sesuai. Untuk mencapai hal tersebut, inovasi diperlukan dalam bentuk pemanfaatan bahan ajar digital dan penerapan model pembelajaran Discovery Learning. Keunikan penelitian ini terletak pada penggabungan bahan ajar digital dengan pendekatan Discovery Learning, yang bertujuan untuk mempercepat dan meningkatkan hasil belajar siswa dalam waktu yang singkat. Pendekatan ini juga dirancang untuk membuat siswa lebih aktif dalam proses pembelajaran. Penggabungan bahan ajar digital dan model Discovery Learning merupakan suatu kebaruan yang diharapkan dapat memberikan kontribusi positif terhadap prestasi belajar siswa. Melalui kombinasi ini, diharapkan siswa dapat mencapai prestasi belajar yang maksimal dengan cara yang lebih interaktif dan efisien. Pendekatan ini mencerminkan upaya guru untuk memanfaatkan teknologi dan metode pembelajaran yang modern guna menciptakan pengalaman belajar yang lebih berdaya guna dan mendukung kemajuan pendidikan. METODE PENELITIAN Penelitian ini yaitu pendekatan kualitatif dengan metode studi lapangan, yang melibatkan pengumpulan data melalui observasi langsung di lokasi, wawancara dengan guru secara langsung, dan dokumentasi di Sekolah Dasar Negri Bojong Kiharib. Selain itu, studi pustaka digunakan untuk menelaah buku, literatur, dan jurnal terkait. Wawancara ini dilakukan untuk memperoleh informasi tentang implementasi inovasi dengan model pembelajaran yang sesuai dengan abad ke 21. Selain itu dokumentasi dilakukan untuk memperoleh data pendukung yang dapat memperkuat hasil wawancara. Data kemudian di analisis secara kualitatif melalui literatur review dan hasilnya disajikan dengan cara naratif deskriptifPendekatan ini memberikan wawasan 3452 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. mendalam tentang bagaimana guru mengimplementasikan inovasi pembelajaran yang sesuai dengan perkembangan zaman. HASIL DAN PEMBAHASAN Berdasarkan hasil temuan observasi peneliti yang dilaksananakan di SD Negri Bojong Kiharib bahwa Kelas 6 telah menerapkan model Pembelajaran yang sesuai dengan tuntutan abad ke-21 bertujuan untuk mempersiapkan siswa agar memiliki keterampilan dan pemahaman yang relevan dengan dunia yang terus berubah dan terkoneksi secara global, Menyelaraskan metode pembelajaran dengan kebutuhan dan perkembangan zaman. Dengan Inovasi seperti model pembelajaran Discovery Learning ini membantu mempersiapkan siswa untuk membentuk pembelajar yang mandiri dan berpikir obyektif atau kritis. sehingga Memperkuat Keterlibatan siswa dan Media belajar digital yang menyajikan informasi secara menarik dan relevan. Discovery melalui Pembelajaran Outdoor dalam pembelajaran IPAS, siswa diajak keluar kelas untuk melakukan penemuan terkait hubungan atau interaksi antar negara. penemuan dilakukan terhadap kegiatan sekitar, seperti penjual dan lalu lintas di sekitar sekolah. Setelah itu adanya sesi Pertanyaan dan Tanggapan Siswa, Guru bertanya kepada siswa setelah pengamatan, siswa menyimpulkan adanya hubungan atau interaksi di lingkungan sekitar mereka dengan ini guru memancing siswa harus memiliki motivasi, semangat berani, tingkat karakter yang tinggi, serta Besar tekad dan tanggung jawab (Sya et al., 2022) Proses kolaboratif dan kegiatan outdoor meningkatkan keterlibatan siswa dalam pembelajaran Aktif karena dengan adanya inovasi discovery memungkinkan Melalui pengalaman langsung, siswa dapat memahami konsep secara lebih mendalam, sedangkan interaksi sosial memungkinkan mereka berkolaborasi, berbagi ide, dan memperkaya pemahaman bersama. Dengan ini Siswa memandang tugas sebagai teladan yang diberikan oleh guru di dalam maupun di luar kelas sebagai suatu contoh atau model. Tugas dianggap sebagai petunjuk yang mengarahkan mereka untuk mencapai pemahaman dan keterampilan tertentu. Bagi siswa, tugas tidak hanya sebagai tanggung jawab akademis, tetapi juga sebagai peluang untuk mengaplikasikan 3453 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. pengetahuan dalam situasi nyata (Kartakusumah et al., 2022). sekolah harus mengoptimalkan sarana dan prasana belajar siswa agar tercapainya pembelajaran yang efektif dan kegiatan yang menunjang pengembangan baik itu sarana maupun tenaga pendidiknya seperti, Pentingnya penambahan fasilitas pembelajaran yang mendukung inovasi terutama dalam hal alat peraga dan ketersediaan wifi untuk mengakses internet, Guru juga perlu mendapatkan pelatihan untuk mengoptimalkan serta mengevaluasi penggunaan teknologi dalam pembelajaran efektif yang menyesuaikannya dengan kebutuhan siswa dan diadakannya supervisi untuk Pengembangan inovasi agar tetap relevan dengan kebutuhan pembelajaran dan teknologi. Penggunaan Chrome book untuk Menunjang Pembelajaran model Discovery Learning: Penggunaan Chrome book untuk Menunjang Pembelajaran model Discovery Learning: Penggunaan Chrome book untuk Menunjang Pembelajaran model Discovery Learning: 1. Sebagai Pencarian Informasi: 1. HASIL DAN PEMBAHASAN Sebagai Pencarian Informasi: - Siswa dapat menggunakan Chromebook untuk mencari berbagai informasi dari internet, buku, dan sumber lainnya. - Meningkatkan kemampuan siswa dalam mencari dan memahami berbagai sumber bacaan. 2. Keuntungan Penggunaan Chromebook Akses Informasi lebih cepat dan luas - Siswa dapat mengakses informasi lebih cepat dan efisien melalui Chromebook. - Penggunaan Chromebook memperluas akses siswa ke informasi dari internet, memberikan keragaman sumber pembelajaran. 3. Peningkatan Literasi Digital: - Penggunaan teknologi mendukung pengembangan literasi digital siswa. - Meningkatkan keterampilan dalam mengelola dan mengevaluasi informasi dari berbagai sumber. 4. Peran Chromebook sebagai Media Pembelajaran Kolaborasi dan Proyek: - Chromebook mendukung kolaborasi antar siswa dan penerapan model pembelajaran berbasis proyek, menciptakan pembelajaran yang lebih aktif dan menunjukkan pentingnya integrasi teknologi dalam Pendidikan. - Chromebook mendukung kolaborasi antarsiswa dan penerapan model pembelajaran berbasis proyek, menciptakan pembelajaran yang lebih aktif. 3454 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. 5. Tujuan Penggunaan Infokus dan Chromebook: 5. Tujuan Penggunaan Infokus dan Chromebook: Penggunaan infokus dan Chromebook bertujuan untuk mencegah kejenuhan siswa terhadap metode pembelajaran konvensional dan untuk Meningkatkan Motivasi. Media tersebut diharapkan dapat meningkatkan motivasi siswa dengan memberikan Penggunaan infokus dan Chromebook bertujuan untuk mencegah kejenuhan siswa terhadap metode pembelajaran konvensional dan untuk Meningkatkan Motivasi. Media tersebut diharapkan dapat meningkatkan motivasi siswa dengan memberikan pengalaman pembelajaran yang lebih dinamis. Adapun Manfaat Infokus dalam Pembelajaran Discovery untuk memvisualisasikan materi secara lebih jelas dan menarik perhatian siswa pada aspek penting dan Kemampuan interaktif infokus memungkinkan guru untuk langsung terlibat dengan materi sehingga meningkatkan partisipasi siswa. Pentingnya Motivasi dalam Pembelajaran menjadi kunci dalam mencegah kecenderungan bosan siswa terhadap proses pembelajaran yang monoton. Guru Perlu Banyak Berinovasi untuk mengadopsi peran sebagai inovator dan kreatif dalam proses pembelajaran untuk menjawab tantangan zaman dan memenuhi kebutuhan siswa yang terus berkembang, seperti Pengembangan Media Pembelajaran Digital untuk mengoptimalkan Teknologi, karena Pengembangan media pembelajaran digital dengan penggunaan infokus dan Chromebook, membantu guru mengoptimalkan teknologi dalam penyampaian materi yang meluas dan membuat pembelajaran lebih menarik bagi siswa. Fasilitas sebagai Pendukung Pembelajaran yang memadai memberikan landasan fisik untuk pelaksanaan inovasi. Guru harus kreatif dalam menciptakan fasilitas yang mendukung pembelajaran, baik itu dalam bentuk lingkungan fisik kelas, perangkat teknologi, atau bahan ajar. Peran seorang guru dalam menyelaraskan model pembelajarannya yaitu sebagai pengatur dan motivator, menentukan pendekatan pembelajaran yang sesuai dengan tujuan pembelajaran yang ingin dicapai dan karakteristik siswa yang berbeda agar bisa menciptakan pengalaman pembelajaran yang lebih bermakna. HASIL DAN PEMBAHASAN Serta Pentingnya Pembelajaran Inklusif untuk Mendukung siswa yang berkebutuhan khusus. Dengan Pendekatan inklusif memastikan bahwa semua siswa, dengan berbagai preferensi belajar, mendapatkan dukungan yang mereka butuhkan untuk mencapai potensi maksimal mereka. Guru yang selalu mengupgrade diri, baik melalui pelatihan, workshop, atau pendidikan 3455 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. lanjutan, akan lebih mudah menyesuaikan diri dengan perubahan kurikulum. Hal ini penting untuk menjaga relevansi dan kualitas pembelajaran. lanjutan, akan lebih mudah menyesuaikan diri dengan perubahan kurikulum. Hal ini penting untuk menjaga relevansi dan kualitas pembelajaran. Keseimbangan Antar Faktor Sinergi Guru, Siswa, dan Kurikulum. Semua elemen harus berjalan seiring, dengan peran Keterlibatan aktif siswa dan bimbingan guru yang efektif bersama-sama membentuk lingkungan pembelajaran yang dinamis dan mendukung, mencapai tujuan pembelajaran yang optimal.Kontribusi Guru dalam Mencapai Tujuan Guru tidak hanya mendukung inovasi dengan merencanakan pembelajaran, tetapi juga dengan menciptakan fasilitas sesuai kebutuhan. Keseimbangan Antar Faktor Sinergi Guru, Siswa, dan Kurikulum. Semua elemen harus berjalan seiring, dengan peran Keterlibatan aktif siswa dan bimbingan guru yang efektif bersama-sama membentuk lingkungan pembelajaran yang dinamis dan mendukung, mencapai tujuan pembelajaran yang optimal.Kontribusi Guru dalam Mencapai Tujuan Guru tidak hanya mendukung inovasi dengan merencanakan pembelajaran, tetapi juga dengan menciptakan fasilitas sesuai kebutuhan. Melalui pemahaman dan implementasi faktor-faktor di atas, guru dapat menjadi agen perubahan yang efektif dalam meningkatkan kualitas pembelajaran dan mencapai tujuan pendidikan yang diinginkan. KESIMPULAN Implementasi inovasi pembelajaran berbasis Discovery Learning di SDN Bojong Kiharib melalui pemanfaatan media pembelajaran efektif memberikan hasil positif. Model pembelajaran tersebut berhasil menyelaraskan metode pembelajaran dengan tuntutan abad ke-21, mempersiapkan siswa sebagai pembelajar mandiri dan kritis. Pemanfaatan media pembelajaran digital efektif menguatkan keterlibatan siswa, menyajikan informasi secara menarik, dan relevan. Discovery Learning dalam pembelajaran outdoor pada mata pelajaran IPAS memberikan pengalaman langsung kepada siswa terkait interaksi antar negara. Meskipun terdapat kendala seperti kurangnya sarana prasarana, tantangan ini dapat diatasi melalui optimalisasi fasilitas dan pelatihan guru. Peran guru sebagai pengatur dan fasilitator sangat krusial dalam kesuksesan inovasi ini. Secara keseluruhan, implementasi Discovery Learning di SDN Bojong Kiharib membawa dampak positif dalam meningkatkan kualitas pembelajaran dan mempersiapkan siswa menghadapi perkembangan zaman. REFERENSI Kartakusumah, B., Sya, M. F., & Maufur, M. (2022). Task and Feedback-Based on English Kartakusumah, B., Sya, M. F., & Maufur, M. (2022). Task and Feedback-Based on English 3456 Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. Karimah Tauhid, Volume 3 Nomor 3 (2024), e-ISSN 2963-590X \| Putri et al. learning to Enhance Student Character. DIDAKTIKA TAUHIDI: Jurnal Pendidikan Guru Sekolah Dasar, 9(1), 1–10. https://doi.org/10.30997/dt.v9i1.4684 Khamidah, N., Winarto, W., & Mustikasari, V. R. (2019). Discovery Learning: Penerapan dalam pembelajaran IPA berbantuan bahan ajar digital interaktif untuk meningkatkan prestasi belajar siswa. JIPVA (Jurnal Pendidikan IPA Veteran), 3(1), 87. https://doi.org/10.31331/jipva.v3i1.770 Maulana, S., Sya, M. F., Mawardini, A., & Ika Tri Yunianika. (2023). YouTube as a Media for Developing English Proficiency: Experiments on Elementary School Students in Indonesia. Jurnal Pendidikan, 24(2), 93–100. https://doi.org/10.33830/jp.v24i2.6150.2023 Rahmayani A L (2019) Pengaruh Model Pembelajaran Discovery Learning dengan Rahmayani, A. L. (2019). Pengaruh Model Pembelajaran Discovery Learning dengan Menggunakan Media Video Terhadap Hasil Belajar Siswa. Jurnal Pendidikan (Teori dan Praktik), 4(1), 59. https://doi.org/10.26740/jp.v4n1.p59-62 Sya, M. F., Anoegrajekti, N., Dewanti, R., & Isnawan, B. H. (2022). Exploring the Educational Value of Indo-Harry Potter to Design Foreign Language Learning Methods and Techniques. International Journal of Learning, Teaching and Educational Research, 21(10), 341–361. https://doi.org/10.26803/ijlter.21.10.19 Syahputra, E. (2018). Pembelajaran Abad 21 Dan Penerapannya di Indonesia. Prosiding Seminar Nasional SINASTEKMAPAN (E-Journal), I, 127. Seminar Nasional SINASTEKMAPAN (E-Journal), I, 127. 3457
https://openalex.org/W2469195175	https://link.springer.com/content/pdf/10.1007%2Fs10610-016-9321-2.pdf	English	null	An Expanding Culture of Control? The Municipal Administrative Sanctions Act in Belgium	European journal on criminal policy and research	2,016	cc-by	11,402	Eur J Crim Policy Res (2017) 23:59–76 DOI 10.1007/s10610-016-9321-2 An Expanding Culture of Control? The Municipal Administrative Sanctions Act in Belgium Elke Devroe1 & Gerben Bruinsma2 & Tom Vander Beken3 Published online: 7 July 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Published online: 7 July 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Published online: 7 July 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract This article provides an in-depth study of the Act on Municipal Administrative Sanctions 1999 (MAS), which is the first major piece of legislation regulating antisocial behaviour in Belgium. MAS provides municipalities with an instrument to sanction antisocial behaviour and conduct perceived to disturb public order. The article uses Garland’s (2001) thesis on the culture of control to analyse whether MAS has led to increased government control and the exclusion of significant groups of the population. The research is based on a multiple case study in which the application of MAS was analysed over a 25-year period of security policies in Belgium (1985–2010). The Act’s implementation was studied in the two Belgian cities of Antwerp and Liège in order to consider the influence of the Flemish government and the Walloon government, respectively, in this policy area. The article uses insights from this comparison to revisit the culture of control thesis and its limitations in understanding the political competition that exists over the formulation of policies on antisocial behaviour. Keywords Policing public space . Incivilities . Police . Administrative sanctions . Culture of control . Antisocial behaviour * Elke Devroe e.devroe@fgga.leidenuniv.nl 1 Institute of Security and Public Affairs, Department of Governance and Global Affairs, Leiden University, Leiden, The Netherlands Introduction In 1999 Belgium introduced the Act on Municipal Administrative Sanctions (MAS), which enabled municipalities to control antisocial behaviour through the imposition of administrative, 1 Institute of Security and Public Affairs, Department of Governance and Global Affairs, Leiden University, Leiden, The Netherlands 1 Institute of Security and Public Affairs, Department of Governance and Global Affairs, Leiden University, Leiden, The Netherlands 2 Netherlands Institute for the Study of Crime and Law Enforcement (NSCR), Amsterdam, The Netherlands 2 Netherlands Institute for the Study of Crime and Law Enforcement (NSCR), Amsterdam, The Netherlands 3 Department of Criminology, Criminal Law and Social Law, Ghent University, Gent, Belgium 3 Department of Criminology, Criminal Law and Social Law, Ghent University, Gent, Belgium E. Devroe et al. 60 rather than criminal, sanctions. To some extent, this framework is comparable to legal initiatives in other countries (see, e.g. Devroe 2012b; Di Ronco and Peršak 2014; Crawford 2008, 2009; De Maillard 2002; Melossi and Selmini 2000; Frevel 2015). This article discusses the origins and development of this approach during the period 1985–2010. The more recent Municipal Administrative Sanctions Act of 2014 is therefore not included within the scope of this research.1 In particular, the article considers the political context of the introduction of MAS, its objectives, underlying policy presumptions and moral entrepreneurs. Furthermore, the implementation of MAS is examined in two case studies, namely, the municipalities of Antwerp and Liège. This article seeks to answer the question: BCan the development of law on antisocial behaviour in Belgium be explained in terms of Garland’s culture of control thesis?^ Garland’s thesis on the sociology of punishment in the Anglo American context, as presented in the book Culture of Control (2001), is used as a starting point for interpreting the origins and subsequent development of policy responses to antisocial behaviour in Belgium, given the influence of this thesis on other accounts of contemporary crime control in Western European countries (Boutellier 2007; Daems 2009; Snacken 2007; Swaaningen 2005). Garland’s (2001) work initiated the debate on the relative importance of social forces associated with the onset of late or postmodernity and of local factors in determining penal policy, also framed as the punitive turn (Daems 2008), penal regression (Radzinowicz 1991), pénalisation du social (Mary 1997, 2005), penal temptation (Wacquant 1998, 2004), new punitiveness (Pratt et al. 2005), actuarial justice (Feeley and Simon 1992, 1994) and risk justice and management (Swaaningen 1996). Introduction In this article Garland’s thesis is used to draw out the broader questions and themes implied by the introduction of MAS, specifically whether the political imperative for authorities to appear tough and present a punitive display, whilst pursuing more pragmatic measures for managing crime and civil unrest works as an explanation of the origins and subsequent application of MAS. The article therefore outlines the potential significance of Garland’s thesis and how it was used to design a multiple case study of the application of MAS in Antwerp and Liège. The results of the case study are then presented, followed by concluding thoughts on the explanatory value and generalizability of Garland’s thesis. 1 See: Devroe 2016; Cops and Pleysier 2014; Bauwens et al. 2016. David Garland’s culture of control We analysed the social and political context of MAS, the Belgian law on antisocial behaviour. Did MAS originate in a fear of crime, as suggested in Garland’s thesis? 1. We analysed the social and political context of MAS, the Belgian law on antisocial behaviour. Did MAS originate in a fear of crime, as suggested in Garland’s thesis? 2. We focused on the moral entrepreneurs of MAS in Belgium: Were they the professional groups who once dominated the policy-making processes but are increasingly disenfranchised as policy comes to be formulated by political action committees and political advisers (Garland 2001, p. 13)? Was MAS the result of existing ideological and reforming political powers or a product of new institutional arrangements developed as problem-solving devices to deal with Bthe practical experience of government agencies and their constituencies^ (Garland 2001, p. 72)? 3. We studied the policy presumptions underlying the regulation of disorder that could be situated between an expressive/symbolic dimension of the criminology of the other (acting out) or denial—both control dimensions (Garland 2001). Are criminologies of everyday life (rational choice, routine activity or opportunity theories) present in the policy assumptions of Belgian policy makers? 4. We wanted to learn more about the objectives of MAS: Are they the result of a new and urgent emphasis upon the need for security (Garland 2001, p. 13), or are they the result of Bmodest improvements at the margin, the better management of risks and resources, reduction of the fear of crime, reduction of criminal justice expenditure, greater support for crime victims, more expressive penal measures [that] have become the new policy objectives, as policy analysts deem it more realistic to deal with the effects of crime rather than address the thing itself^ (Garland 2001, p. 109)? 5. We wanted to understand the implementation of MAS at the local level and the social reactions of local institutions towards antisocial behaviour: Which different antisocial behav- iour measures were taken, and how were they implemented? We sought to analyse Garland’s statement that Bmeasures of expulsion and exclusion go hand in hand with the development of a network of preventative partnerships at local levels by patient, ongoing, low-key efforts to build up the internal controls of neighbourhoods and to encourage communities to police themselves^ (Garland 2001, p. 17), the latter being an element in the adaptive strategy (Swaaningen 2004). David Garland’s culture of control As this article explores governmental regulation of disorder, Garland’s analysis of govern- mental response to late modern issues of insecurity regarding crime is highly relevant. Central to his culture of control thesis is an analysis of governmental response to disorder and, in particular, the policy predicament. This can be observed as a contradictory mix of measures (see also O’Malley 1999), with on the one hand punitive segregation (control and exclusionary measures) and on the other preventative partnerships and increased informal control by the community. This policy of bifurcation contains no polarization (meaning a sensu stricto governmental response) but a simultaneous performance of contradictory tendencies. Garland identifies the crime-prone society as a main cause of this ambivalent reaction, clustering two basic strategies: the adaptive strategy and the sovereign state strategy. In this context Garland adheres to the fear of crime thesis: the adaptive strategy supposes that the government has to An Expanding Culture of Control? 61 adapt to the reality of high crime rates and manage them; the sovereign state strategy denies that the government has lost its grip and constitutes the core of the control theory, with reactions of denial and acting out. Furthermore, he states that the emergence of a culture of control depends on the context of the crime situation and political context, policy assump- tions, moral entrepreneurs (advocates, stakeholders) and objectives and criminological theories behind late modern law enforcement. He identifies the loss of penal welfarism in favour of retribution and deterrence as one of the main causes. The welfare mode became more risk prone and offence oriented, and control theories such as the criminologies of everyday life and the criminology of the other began to dominate criminological discourse. Last but not least, the economic style of reasoning and the neoliberal way of thinking are, according to Garland, the main causes of an expanding culture of control. Despite the fact that Garland does not fully expand on the consequences of his thesis for local security governance, his analysis of preventative partnerships is important for framing the empir- ical results in this article. He places these partnerships within the control framework (Daems 2009) and depicts them as a mix of informal social controls embedded in everyday activities and the interactions of civic society. We conclude this section with five research questions that are addressed in the article: 1. Methods and data collection The research is a multiple case-study design with triangulation of methods. We wanted to obtain an insight into how and why MAS emerged and was implemented (Orum and Feagin 1991), and a detailed understanding of the institutional political contexts, cultures, historical traditions and governmental arrangements (Newburn et al. 2002). As Belgium has a very specific constitutional/legal setting, it is important to examine general theses about crime control, such as Garland’s, with reference to the control policies that apply in the different regions of Belgium [the political specificity of the environment (Mcara 2005)], on the presumption that the politics of this regional, subnational government will help to explain the origins and development of these policies. Belgium has about 11 million residents, multiple language regions (60 % Flemish speakers, 40 % French speakers and a small proportion of German speakers) and five large municipal- ities.2 Since the state reform of 1970, Belgium consists of three linguistic regions, 11 provinces, 27 judicial districts and 589 municipalities. As a consequence of the state reform in 1988, Belgium became a federal state. Given this historical evolution of the Belgian federal state and its division into and enmities between Flemish/Dutch-, German- and French- speaking community councils and the regional governments of Flanders, Wallonia and Brussels-Capital, these regional authorities have developed different approaches to local security (Devroe 2013). The Flemish Region consists of 308 municipalities, the Brussels- Capital Region 19 and the Walloon Region 262, each steered by the mayor who directs the (local) police to act in close contact with the citizens and to practise community policing (Devroe 2013). A key consequence of the 1988 federal constitution has been a power shift from national to regional and municipal authorities (Devroe 2013). Only a few policy domains remained exclusively within federal government competence, such as the maintenance of public order (police) and law enforcement (justice). Competencies relating to social policy— more specifically, education, employment, health, living conditions, culture, housing, spatial planning—became the responsibility of the regional governments. Because the causes of crime are often situated in regional policy domains, we included regional policy issues in our analysis. Whilst federal and regional authorities retain responsibility for formulating stated goals on security policy, policy implementation (policing in action) (Wodak 2011) became the responsibility of the municipalities (Devroe and Ponsaers 2005). David Garland’s culture of control This last question will be answered by analysing the local case studies in Antwerp and Liège. E. Devroe et al. 62 2 Antwerp and Ghent (Flemish speaking), Charleroi and Liège (French speaking), and Brussels (both languages). 3 Renewed by the New Communal Law (Nieuwe Gemeentewet), B.S., 3 September 1988. p p g g p g g 3 Renewed by the New Communal Law (Nieuwe Gemeentewet), B.S., 3 September 1988. ntwerp and Ghent (Flemish speaking), Charleroi and Liège (French speaking), and Brussels (both languages). d b h l b Methods and data collection To draw upon a classic distinction between steering and rowing made in the study of governance (Osborne and Gaebler 1993), regional authorities steer social policy responses (policy formulation, e.g. agenda setting, allocation of resources) to quality of life issues; federal authorities steer law enforcement and maintenance of public order; municipal authorities are obliged to row (i.e. implement) both criminal justice and social policy approaches. Consequently, it is at the municipal level that these competing federal and regional responses are negotiated and then executed (Devroe 2015b). In Belgium, the mayor is an independent political figure in each municipality, and Belgian mayors are—like their French counterparts—in charge of and responsible for public order, public peace, security and public health. The mayor can establish general police regulations in his/her municipality and recruit the public police (called admin- istrative police) to determine and record violations. The Communal Act of 30 April 18363 created an extensive legislative municipal framework to tackle problems of public disorder An Expanding Culture of Control? 63 under the patronage of the federal government. It is important to mention here that from 1991 until 2014 the Socialist Party ruled in a federal coalition. In the period covering MAS’s genesis (1988–1999), all ministers of interior (federal), municipalities (federal) and cities in Flanders and Wallonia (regional ministers) were socialists. under the patronage of the federal government. It is important to mention here that from 1991 until 2014 the Socialist Party ruled in a federal coalition. In the period covering MAS’s genesis (1988–1999), all ministers of interior (federal), municipalities (federal) and cities in Flanders and Wallonia (regional ministers) were socialists. Data processing for this study was oriented around two main topics: (1) the genesis and (2) the implementation of MAS. In case study A, the genesis was analysed at a federal level over a 25-year period (1985–2010). Data collection was organized around the five research questions (supra). Political reactions to increasing problems of urban antisocial behaviour were analysed in a broader historical context of social and governmental changes in security policy over the study period. This historical reconstruction involved 26 semistructured face-to-face interviews4 (Fontana and Frey 1994), with experts (Aberbach and Rockman 2002) from different policy levels. All ministers of interior and justice who served during the research period were interviewed. In 1999, during the presidency of Prime Minister Verhofstadt I, the position of Government Commissioner of Metropolises was introduced. 4 All interviews were completely transcribed, and the 72 interviews have been kept on tape in archives. A list of names of all respondents and their functions has been published (Devroe 2012a). A selection of quotes from the interviewees is used in this article. 5 A list of the documents has been published (Devroe 2012a), and all documents used are stored in archives. 6 This information was used in the analysis, and the source was referred to as Bconfidential document’. 7 All interviews were completely transcribed and kept in the archives. Names of respondents and a list of all documents used in the analysis have been published (Devroe 2012a). 8 Called the BOctopus agreement’ because suddenly the eight political parties all agreed on the Police Reform Act (7 December 1998, B.S. 5 January, 1999). Methods and data collection In 2000 this function was transformed into the Minister of Metropolises, which was installed to steer and finance renovation and social cohesion projects in the five major cities in Belgium. These successive ministers were also interviewed, along with representatives of their cabinets, members of parliament, political advisors, governors and civil servants. We gathered significant informa- tion on how the perceptions of these policy experts led to certain policy assumptions, motivations, basic principles, statements and, finally, actions. We chose a semistructured style of interviewing in order to gather a diversity of significant answers at a sophisticated level. 5 In addition to interviews, an extensive document analysis5 (N: 432) of official and confidential policy documents was carried out. Analysis included governmental programmes and policy plans; national and local security plans; reports of the Council of Ministers; the Cabinet of the Ministers of the Interior, Justice and Metropolises; statistical reports; annual reports; elective programmes; reports and informal notes of regional councils; preparative documents for Acts; personal letters between ministers; and so on. Thanks to ministers’ and cabinet members' assistance, we were able to gather exclusive, confidential6 political material that had not been revealed before (e.g. minutes of meetings in parliamentary committees, informal and formal letters between ministers, minutes of intercabinet meetings). In order to study the implementation processes of the federal MAS and possible diverging policy instructions from regions at the metropolitan level, two nested case studies (based on differences) in Antwerp (Flemish speaking) (case study B) and Liège (French speaking) (case study C) were added to the analysis. Both cities implemented MAS in 2005, so the relevant period for an analysis of the uses of MAS in these cities is 2005–2012. We chose these particular cities in order to identify any intraregional differences originating in linguistic political influences on the one hand (both cities’ political representatives are currently left wing, but 25 % of voters chose the extreme right-wing party Vlaams Blok in Antwerp in 1991; voters in Liège have consistently voted for left-wing representatives) and political culture (Flemish or Walloon) on the other. Although the population size of Antwerp and Liège varies greatly, they both face a massive inflow of commuters (work and tourism) on a daily basis. In both cases, we focused on policy 64 E. Devroe et al. Methods and data collection makers from the particular institutions responsible for shaping and conceptualizing antisocial behaviour strategies and civil servants who are responsible for implementation of MAS. This approach led us to actors in prevention and social welfare and to institutions of law enforce- ment (police, justice). In each city, we held 23 face-to-face interviews7 (in Dutch in Antwerp; in French in Liège) with mayors, public prosecutors, police constables and chief commis- sioners, social workers, wardens in public transport, city units responsible for the prosecution and sanctioning of antisocial behaviour, members of the local council (college of alderman), city wardens, city coachers, mediators, street-based social workers, those piloting neighbourhood projects, etc. These interviews were complemented with an extensive docu- ment analysis for each city, including municipal statistics on antisocial behaviour, policy plans of majors, local security plans, police plans, antisocial behaviour plans, minutes of the city council, activity reports of institutions, reports of city wardens, and so on. MAS genesis Regarding the social and political context of MAS (question 1), most respondents referred to various events and scandals that shook Belgium’s political institutions and challenged their authority to cope with issues of crime and safety in the late 1980s. The 1980s were described as a black page in Belgian history, marked by the extremely violent raids and killings by the Nivelles Gang (Dupont and Ponsaers 1988), terrorist attacks by the communist Cellules Communistes Combattantes and the deaths of 39 football supporters at the 1985 European Cup Final in the Heysel Stadium. In addition to those events, many other incidents during that period cast a shadow over police and the justice system. Respondents also noted a growing concern about the influx of migrants in the larger cities, a rapidly changing demographic situation in the capital cities and the political success of the extreme rightwing party (Vlaams Blok) in Flanders. On 24 November, 1991, Vlaams Blok obtained 6.6 % of the votes, or 12 of 212 seats in Parliament. This day is now referred to as Black Sunday, a day on which more than 100,000 voters formally expressed their lack of trust in the existing political system (Huyse 1996). Considered a state legitimacy problem, this electoral shift stimulated political awareness of problems of social disorder and minor crimes in deprived neighbourhoods (Van Limbergen 1995). The last wave of incidents occurred in the mid to late 1990s with the 1996 arrest of Marc Dutroux, a paedophile who imprisoned and murdered children. His arrest was accompanied by a massive public expression of solidarity with the children’s parents. The White Balloon March in Brussels in 1996, attended by 300,000 people, was a public statement against the incompetence of police and the justice system. In April 1998 Dutroux briefly escaped during a prison transfer, leading to moral panic in the country and resulting in the unavoidable resignation of the Ministers of Interior and Justice. According to many respon- dents, the police reform that was quickly voted in after this escape8 was considered by all political parties to be necessary to the recovery of governmental legitimacy. 65 An Expanding Culture of Control? A second stimulus for MAS’s genesis was the state reform of 1988 (supra). MAS genesis Following this reform, the Minister of Interior, Louis Tobback, called the Cabinet of the Interior Bthe preservation room for grumpy old men with nothing more to decide.^ The election programme of the Socialist Party9 showed, following state reform, that socialist politicians did not want to be constrained by the party's very narrow range of policies, which stressed repression and public order. They expressed a strong belief in the need for tailor-made support for major municipal areas, and wanted to formulate and implement new policy measures that tackled the causes of crime, even though such tasks had, since 1998, been allocated to the regions and so were officially outside their political sphere. Furthermore, the electoral shift to the extreme right led to a political awareness among socialists that something had to be done to equip cities with the right tools to tackle the deterioration and collapse of social cohesion. Prevention measures (situational, but also social) became key strategies in tackling crime and feelings of insecurity. The federal government financed many social projects, including drug rehabilitation programmes, situational prevention projects such as street cleaning and litter removal, neighbourhood centres and street-based social workers. Policy makers at that time were convinced that punitive answers to minor crimes could only exacerbate the issues at hand (De Witte 1988). Political assumptions were closer to those of the Broken Windows theory (Wilson and Kelling 1989), transforming cities in attractive and clean places, than to deterrence and punishment that was oriented towards individuals (mostly young people), as had been the case after the murder of Jamie Bulger in the UK. Belgian laws were not obviously directed at offensive behaviour (especially by young people), which has featured so significantly in policy in the UK. Because antisocial behaviour is legally considered to be a part of public order problems, the debate on antisocial behaviour that characterized UK debates was not seen in Belgian political debates. The Belgian law is called Municipal Administratrive Sanctions, which reflects its focus on public nuisances (trespass, mess/litter, environmental harms—with the mayor being responsible for maintaining health, peace and order in the city) rather than possessing the almost subcriminal character that antisocial behaviour laws have acquired in the UK (Devroe 2016). Document analysis and interviews clearly reveal that socialists’ reactions to police and judicial malaise was inspired by the French politician Bonnemaison,10 to take a preventative approach. 9 De Witte (1988) BSecurity and petty crime’ (Veiligheid en kleine criminaliteit), an electoral document for the municipal elections. 10 A 1982 report Face a la délinquence, prévention, répression, solidarité, written by Gilbert Bonnemaison and 26 municipal mayors, explored crime in metropolitan areas and prevention measures for young people. This report led to the establishment of prevention councils and prevention contracts in France and was the example for the Belgian prevention policy contracts. 11 Minister of the Interior at that time. 12 Directive of the Minister of the Interior of 12 November 1990 to the mayors on the prevention of crime, and Directive of the Minister of the Interior of 30 November 1990 to the mayors on the security and prevention contracts. 12 Directive of the Minister of the Interior of 12 November 1990 to the mayors on the prevention of crime, Directive of the Minister of the Interior of 30 November 1990 to the mayors on the security and prevent contracts. MAS genesis The federal government approved of the intentions of Louis Tobback,11 transcribed in the ministerial declaration BSecurity of the citizen, police and security^ of June 1992. One of the main propositions in this document was extensive funding for crime prevention12 (Glorie 1997, 2004; Willekens 2008). In order to get a new grip on city governance after the state reform, city contracting (like in France; see Roché 2005), was installed. After consulting the five mayors of the major cities (all socialists) about the amount of federal funding, so-called prevention contracts were signed between the Minister of the Interior and those mayors. The investment consisted mainly of personnel: prevention workers were installed who introduced E. Devroe et al. 66 unemployed, low-skilled citizens into entry-level jobs, such as football coaches, stewards, city monitors, park guards, parking-lot guards and other prevention personnel. This was the first occurrence of a cohort of civil surveillance wardens in public spaces executing tasks such as warning, helping citizens with enquiries and noting down complaints about public infrastruc- ture (Devroe 2015a). As most of these workers were located in the same building as the public police housed within police offices, some were—like the public police—employees of the local authority. One year prior to voting on MAS in 1998, a total of 123 million euro was invested in the cities, installing a fragmented body of surveillance wardens on public streets and squares, helping police to keep peace and order. In 2000, the Minister of Metropolises (also socialist) followed his colleague at the interior and invested a significant amount of funding in the five largest cities, more specifically in social cohesion projects,13 resulting in an increasing body of social workers such as street-based workers and mediators in public spaces. Confidential letters between Ministers of the Interior and of Metropolises14 indicate that the positioning of civil wardens in public spaces—an important aspect of the prevention con- tracts—was the essence of what would become MAS the year after. This clearly illustrates the fact that MAS originated as a preventative and not a criminal law enforcement measure (as had the Antisocial Behaviour Orders (ASBOs) in the UK; Matthews et al. 2007; Field 2003). We specify this last statement in the answers to our second and third queries—namely, moral entrepreneurs, policy assumptions and MAS objectives—which we analyse in the following paragraphs. 13 Act of 17 July 2000 on the requirements municipal authorities have to meet in order to get funding stipulated in contracts with the Ministry of Metropolises, B.S. 8 August 2000. 14 The Department of Metropolises was socialist for the entire period, from its establishment in 1999 until 2008 (Ch. Piqué, J. Vande Lanotte: 1999–2003, M. Arena: 2003–2004 and Ch. Dupont 2004–2008). 15 To give a concrete example, when the Socialist Party no longer occupied the position of Minister of Interior, the socialist (French) Minister of Justice (from 2003–2007) became—together with the Flemish socialist Minister of Finance and the French Minister of Metropolises—moral entrepreneurs for the successive MAS Acts of 2004– 2005. MAS genesis Analysis of the extensive body of policy documents dating from the crucial period before the voting for MAS (1999–2005) and the interviews leads to the conclusion that the socialist body of thought (the belief in the feasibility of society, in state engineering) was transferred across key positions, governmental levels (federal and regional) and even across linguistic tensions.15 The in-depth political reconstruction of communication between different stake- holders shows that the sole moral entrepreneurs were the socialists (politicians and cabinet members), installing MAS as a last resort following the preventive financial injection that political party officials had implemented from the early 1990s onwards. Their main objective was to install formal and informal surveillance in public spaces in order to address public feelings of insecurity and to establish what could be called a city code of conduct by implementing MAS. There were no theoretical assumptions (our third question) underlying this decision: the approach to antisocial behaviour arose from the pragmatic experience of politicians, called a rational-within-limits view (Garland 2004). Only the Minister of Metropolises asserted that he had analysed a number of French scientific studies on urban crime problems, such as Bonnemaison. Though the installation of a body of civil city wardens could refer to capable guardianship (Felson 2002) and so fit in the opportunity theory, the MAS approach could not be called a demoralizing effect of situational crime prevention where the new approach does An Expanding Culture of Control? 67 not look to the revival of social controls and self-controls. Instead, it seeks to make a new style of ‘situational engineering’ succeed where ‘social engineering’ failed^ (Garland 1997, p. 189). not look to the revival of social controls and self-controls. Instead, it seeks to make a new style of ‘situational engineering’ succeed where ‘social engineering’ failed^ (Garland 1997, p. 189). The objectives (question 4) of MAS were—after 10 years of federal investment in crime prevention and a penal system that completely ignored sanctioning municipal violations—to counteract impunity by providing municipalities with a legal instrument to sanction incivilities. By broadening city council competences to sanctioning, municipalities were put in control of the entire chain of enforcement policy: establish regulations, prosecute and punish (Van Heddeghem 2006). 16 Decree of the Walloon Government of 15 May 2003 on prevention and proximity in cities and communes in Wallonia, Ministerial Decision, 4 February 2004: 6701. 17 Act of 13 May 1999 on Municipal Administrative Sanctions, B.S. 10 June 1999. MAS genesis As the Flemish government had competence over sanctions and justice but wanted to decrease crime and disorder, the Flemish Minister of Cities (socialist) also invested in contracting (Social Impulse Funding, Sociaal Impulsfonds) in order to install social policies of city renovation and social cohesion projects in exchange for (Flemish) funding. The following quote illustrates the intention: As we had no competences on problems of crime, and we wanted to reorganize crime- prone cities into viable, agreeable spaces, we tried to invest in citizens’ participation. Of course this was done to avoid a conflict between our official (narrow) competencies and our own policy goals. This Flemish money was only meant for the so-called deprived neighbourhoods. So you were obliged to work in certain bad neighbourhoods, which were overpopulated, with a great mix of cultures and a lot of integration problems and sociodemographically and economically floating beneath the societal surface. And those you have to renovate, so you organize a competition to find out what people think it is important to have in public space (civil servant of the Flemish cabinet). In 2003, almost 7 years after the Flemish investments, Wallonia16 was also able to provide funding for Walloon cities following the signing of contracts between the cities and the Walloon executive that guaranteed the use of prevention-based approaches. This gave a great boost to Flanders, providing infrastructure improvements that were supported by local citizens, and contributed to a reduction in feelings of insecurity (Devroe 2012b). Therefore, if MAS is viewed in isolation, its objectives can be considered to be Bmodest improvements at the margin, the better management of risks and resources, reduction of the fear of crime, reduction of criminal justice expenditure, greater support for crime’s victims^ (Garland 2001, p. 109). We now elaborate on MAS itself. Regulating antisocial behaviour: MAS Tackling antisocial behaviour is a municipal matter in Belgium, directed by the mayor and executed by the public police. This practice, together with the strict division between admin- istrative (public order) and judicial police, is particular to the Belgian constitutional setting stemming from a history of local autonomy (Devroe and Ponsaers 2013). MAS17 provides local councils with an appropriate instrument enabling them to swiftly and forcefully tackle antisocial behaviour on municipal land. As the Belgian legislator considers antisocial behav- iour to be an element of disturbing public order, MAS was embedded in the existing Municipal Act of 1999, creating three innovations: (1) the ability to close down establishments and E. Devroe et al. 68 suspend licences, (2) enlargement of the notion of public order to antisocial behaviour18 and (3) competence for the mayor and alderman to sanction (with fines up to 340 euro) infringe- ments of municipal regulations. MAS combines administrative measures (briefly, measures taken by the mayor, such as closing down bars and discotheques, withdrawal of licences, issuing fines) and administrative sanctions imposed by the city council (mainly fines). Before the amendment in 2004, municipal authorities could only react to the types of behaviour for which neither the federal nor the regional parliament has provided penalties. In other words, municipalities could only respond to new types of antisocial behaviour that had not already been addressed by existing legislation.19 In practical terms, local administrative sanction legislation proved to be an Bempty barrel^ for municipalities. As any overlap with existing provisions elsewhere was formally excluded, local councils did not have much room to manoeuvre (Vander Beken and Vandeviver 2014). Moreover, the Act of 1999 allowed municipalities to draft their own laws but not to appoint designated enforcement staff. Antisocial behaviour could only be established by a report made by police (Devroe 2008). Before MAS, sanctioning antisocial behaviour was the responsibility of the judicial authority (police courts), creating considerable congestion in the public prosecutor’s daily workload. For political reasons—namely the belief in tailor-made local governance with a strong emphasis on municipal autonomy—MAS was meticulously amended by the federal parliament in 2004, leading to two new acts in 2004. In the preparation of this act we noticed—for the first time—a strong voice (Minister of Justice L. Onkelinx, also socialist) underlining the need for manda- tory mediation before punishment. 18 Art. 135 par. 2 al 7 MAS. 19 This means not only federal legislation but also regional decrees on environmental issues, town and country planning, construction companies, residential inspection, etc. 20 Parliament, Chamber 1, 2002–2003, number 2366/1 and 2367/1, 3–5. 18 Art. 135 par. 2 al 7 MAS. Regulating antisocial behaviour: MAS These acts treat minors (aged 16–17) as eligible for sanctioning using MAS, but only after mandatory mediation (Vander Beken 2005). This revised act combines administrative sanctions with criminal procedures in a particular way. In order to hand over sanctioning competencies to the municipality, a series of violations were depenalized. These violations were no longer delimited by other legislation and could be integrated into municipal procedures. A second group of criminal offences are referred to as mixed violations, i.e. behaviour that qualifies as both a criminal offence and an offence written down in municipal regulations. These are not depenalized, but the municipal council is at liberty to issue a pecuniary fine of up to 250 euro for these offences in its byelaws, as the existing penalties provided under the penal code remain untouched when the public prosecutor decides to pursue a case. The penalties already included in the penal code are in turn divided into two separate procedural settlements in which the official police report is required to be sent to the public prosecutor’s office in all cases. With regard to the first series of criminal offences (the so-called lighter-mixed violations), administrative pecuniary fines can only be imposed when the public prosecutor does not communicate his position in the case to the civil servant within 1 month of receiving the police report. If the public prosecutor has allowed this term to expire without making his or her position known, offences under this first series of criminal offences can only and exclusively be punished administratively, with the local council at liberty to take up the prosecution of this offence. Of course, an individual cannot be punished twice for the same offence (non bis in idem).20 An Expanding Culture of Control? 69 The second series of criminal offences (the so-called more serious mixed violations) results in penalisation under traditional criminal justice, unless the public prosecutor’s office indicates that it would be more appropriate to impose a pecuniary fine. With regard to this series of criminal offences, the municipal civil servant can only impose an administrative pecuniary fine if the public prosecutor has advised him/her within a preset timespan that the violation shall not be pursued any further and that he/she feels it would be appropriate to impose an administrative penalty. MAS implementation in two cities Regarding the fifth research question on the mix of informal social controls embedded in the everyday activities within preventive partnerships at local levels, analysis of 46 interviews and numerous documents led to some specific results. Questions such as: BWhat are the rules of discourse and action that organise the diverse practices that made up this field?^ BHow are these rules and these practices related to those of other social domains, such as welfare, politics or the economy?^ BWhat role do these practices play in the governance of late modern society?^ (Garland 2001, p. 2) were analysed in both cities. The way in which MAS was implemented was reviewed—whether top down, meaning mandatory as imposed by city hall, or bottom up, originating in small initiatives within civic society. During the empirical search, attention was paid to the ways of thinking that underlie the programs (mentalities), the organisational forms used to implement those ways of thinking (institutions), the techniques used to turn mentalities into action (technologies) and, of course, results (practices) (Johnston and Shearing 2003, p. 13). Results show a great variety in both cities. In Antwerp, we noticed a very enthusiastic and immediate application of MAS, welcomed by city hall as an excellent opportunity to govern the city, with extra tools for the mayor to execute local security policy. Wardens were able to initiate antisocial behaviour reports, and the administrative staff had the authority to sanction. The local police code was completely overhauled, with administrative sanctions for many minor offences ranging from traffic offences; to antisocial violations at markets, restaurants, bars and sports events; to begging and drinking in public spaces; and hanging out, insulting and intimidating others. Under the socialist mayor P. Janssens, a special city department called Living Together was created, which initiated surveillance and control, street-based social work and educational programmes. The whole security chain, from prevention to mediation, to prosecution and sanctioning, was coordinated by one director in what was called an integrated approach. As had been found when assessing the objectives question, socialist ministers intended to treat MAS as a final option after preventive measures; handing out fines was seen as a final stage when prevention approaches had failed. Mediation is in place for young people. 21 Act dated 20 July 2005 establishing various provisions—matters pertaining to the civil service and urban policy—municipal administrative penalties (art. 21–22), B.L.G, 29 July 2005. Regulating antisocial behaviour: MAS If the public prosecutor fails to communicate his/her position, and the provided time period has expired, the local council is no longer at liberty to act in an administrative sense, which means the violation must be settled in a criminal justice context. It is up to each municipality to draft a list of violations that are punishable by MAS. The 2005 revision of the act21 made it possible for municipal officers to draft reports and sanction all municipalities’ regulations, in addition to the public police. MAS implementation in two cities According to the mayor of Antwerp, the director of the Living Together department and civil servants working in city hall, the aim of mediation is to change the attitudes and conduct of young people who exhibit 70 E. Devroe et al. antisocial behaviour by providing a variety of programmes and services (educational, aggres- sion management, sports facilities, assistance at school, etc.) in order to avoid sanctions (fines). Some respondents saw this as a positive early intervention; others saw it as subjecting the young people to intrusive control in the name of diversion or prevention, forcing them into welfare. In Antwerp, young people who are following the programme in order to avoid sanctions may be subject to net-widening effects, because what was considered normal adolescent behaviour of short duration before MAS is now labelled as a problem that requires special attention and care, and—if a young person does not complete a prevention pro- gramme—is regarded as worthy of a fine. antisocial behaviour by providing a variety of programmes and services (educational, aggres- sion management, sports facilities, assistance at school, etc.) in order to avoid sanctions (fines). Some respondents saw this as a positive early intervention; others saw it as subjecting the young people to intrusive control in the name of diversion or prevention, forcing them into welfare. In Antwerp, young people who are following the programme in order to avoid sanctions may be subject to net-widening effects, because what was considered normal adolescent behaviour of short duration before MAS is now labelled as a problem that requires special attention and care, and—if a young person does not complete a prevention pro- gramme—is regarded as worthy of a fine. Antwerp uses the bargaining model of contracting that is widely used in Anglo Saxon countries, showing characteristics of a multiagency approach with fixed horizontal networks. The preventative partnerships are a core aspect of antisocial behaviour governance, linking actors from different backgrounds and policy domains in one network, and considering the creation of personal dossiers of young offenders to be a policy priority for the whole city. These networks are not mandatory in the way that Crime and Disorder Reduction Partnerships are in the UK (Edwards and Prins 2014) but can be understood within a broader tendency towards horizontal security governance instead of top-down government. In Liège we found a completely different interpretation of MAS. MAS implementation in two cities As policy decisions there are based on the Canadian approach, there is a fundamental belief in the importance of high- profile community-oriented policemen. City wardens cannot issue fines; they only assist the police in monitoring signs of disorder. The policy assumption is that that there is no need to create a kind of municipal criminal justice system when a transparent legal justice system is already in place. In the view of the mayor, the chief of police and many other experts responsible for public order and antisocial behaviour, warnings (oral or written) from the police are the best remedy against frequent disorder, and there is no need for extra fines. Therefore, the Liège municipal regulation code remained limited to violations at specific markets, festivities and sports events. Mayoral measures (see above) are not included in the code and cannot be executed, nor are exclusion measures such as street bans or night curfews. According to respondents, too many prohibitions would hinder inhabitants and restrict their privacy. Analyses showed a total rejection of the use of mediation as a tool to avoid fines. Once a case is registered and proven, a fine is given, even after (successful) mediation. In compar- ison with Antwerp, the act of 1999 is rarely applied, the police code was not substantially revised, and punishment of antisocial behaviour by the classic law enforcement agencies of police and justice continued as before. Liège City Hall carries out specific projects (e.g. street cleaning, targeted alcohol control, free bus passes) and connects actors on an occasional basis for a more integrated approach. These connections, however, are not integrated into a specific framework and often dissolve following the project. Collaboration is based on the initiative and willingness of participants, as there are no official agreements. Tasks related to welfare and social cohesion (completed by social workers) are clearly distinguished from tasks involving prevention and sanctioning (executed by police and justice). Both cities illustrate a bottom-up approach, initiated from a small number of ideas that took shape and were formed in a later phase, confirming Garland’s statement that BThey subsequently became de facto strategies attracting widespread support and multiple ex post facto rationales, the plan followed the practice rather than the other way around^ (Garland 2001, p. 105). It is clear from this study that Antwerp implemented MAS wholeheartedly and within the broader objectives of an integrated security policy. Conclusion and discussion As difficult as a comparison and the translation of criminological concepts can be (Edwards and Hughes 2013; Edwards et al. 2013; Nelken 2015), our research shows the importance of empirical research on control theories in different contexts and on different phenomena. In this article we broadened Garland’s insights on a phenomenon that he did not analyse empirical- ly—namely, issues of public order in public space. As this space became scarce (Hope 2000) the quality of life discourse gained importance (Crawford 1999, 2001; Edwards and Hughes 2002), putting the regulation of disorder in the political spotlight. The Crime and Disorder Act in the UK, regulating public behaviour and mostly targeting individuals who disturb public order, was introduced (1998) before the culture of control (Garland 2001) came about. The Criminal Justice and Public Order Act (1994), introduced by Michael Howard (Home Secretary under a Tory government) aimed to criminalise individuals with an unconventional lifestyle, such as gypsies, squatters and ravers (Hughes 2007); the 1994 New Labour policy plan BA Quiet Life: Tough Action on Criminal Neighbours^ (Squires and Stephen 2005) tempted us to broaden Garland’s insights into this public order domain by piloting extensive empirical research with more than 400 policy documents and 72 in-depth expert interviews. Regulating public space is an important task of mayors in most Western countries and can lead to exclusionary mechanisms (Young 2007, 2009). A digression on this important semiauton- omous field (Bourdieux) of control in public space in Garland’s culture of control would have been valuable, even more so since stringent modes of control in this area were already installed in the UK before the publication of his book in 2001. Applying Anglo Saxon penal insights to the continental setting of a civil-law (federal) country with a complex constitutional setting was a challenging enterprise. We believe this multiple case study research design was justified in relation to the proposition that politics matters (Stone 2008) in explaining the origins and development of control strategies; and in Belgium, that implies a concern with the contrasting politics found in the different regional governments as a consequence of the Belgian constitutional/legal settlement. MAS implementation in two cities In both of the two case studies, local An Expanding Culture of Control? 71 politics was effective in implementing policies in ways that the federal government intended. In Antwerp, MAS is an important instrument, but only as the last part of an integral local policy chain. Liège was more reluctant to implement MAS, and only uses instruments that are already available in the criminal law enforcement system to regulate antisocial behaviour. Liège mostly invests in social welfare and prevention approaches instead of issuing fines. Conclusion and discussion In Belgium, there was a lack of confidence in crime-fighting models and criminal law enforcement approaches, such as the UK’s ASBOs and Criminal ASBOs (CRASBOs) (Bottoms 2006; Burney 2005; Card and Ward 2008) or Australian control orders (Zedner 2007) in the period covered by this research, with the socialists calling instead for an approach that would lead to a better quality of life in a preventative manner. As one of the causes of MAS was the flight to the extreme right in 1991 and the complete absence of justice in the regulation of minor crimes that citizens complained about, it is obvious that socialists wanted their electorate back, which they managed to do for 12 years in federal government. The two case studies (Antwerp and Liège) retained socialist mayors from 1976 until 2013, when a right-wing mayor took over the mayoral sash in Antwerp (but not in Liège). By ignoring minor offences and antisocial behaviour that disrupted people’s lives, fears of a second extreme right electoral turn became a reality. To conclude: local politics do matter. Our results elucidate the second factor in Garland’s analysis, the local factors determining control policy more than they do the importance of social forces associated with the onset of late modernity. Socialist mayors sitting in parliament from all five major cities in Belgium shaped the intentions, the assumptions and the objectives of MAS, together with their Socialist Party members at a federal level and at both regional levels. As this act is a nonmandatory instrument that is available for all mayors in their task of keeping peace and order, it is to be expected that politics will be an important factor. Although the act sets structural legal limitations not to punish behaviour that is already penalised in a federal or regional act, net widening can be a dangerous side-effect of this act, broadening the control perspective. Although our empirical observations explicitly showed no reason to believe that risk assessment and control measures were the intention of the original (socialist) moral entrepreneurs, local mayors can decide otherwise. As the Socialist Party had completely vanished in federal coalition and in Antwerp, right-wing and conservative parties demonstrated diverging policy agendas and were able to implement diverging strate- gies. Recently, Belgian austerity policy has led to huge budget cuts, specifically in social insurance and other programmes of social care, welfare and prevention. Conclusion and discussion The detailed reconstruction of the genesis of MAS and of, more generally, Belgian security policy from 1985 to 2010, broadened the scope of classic criminological research (Bruinsma and Huisman 2006) (which, when analysing national acts, usually focuses exclusively on the enforcement of criminal law measures that have been set out by ministers of the interior and of justice). The link with fragmented regional and local competences due to federalisation processes when studying a national act is rarely made: as Hamilton (2014) put it when discussing the fact that research has examined institutional arrangements, national culture and the interaction between the two, the impact of a jurisdiction’s political culture on penal policy has remained a largely underexamined topic. Prohibiting government officials in charge of police and justice from creating social approaches such as restorative justice and social prevention (state reform) sheds light on the intentions of governmental state actors. As we did not limit ourselves to the police senso stricto approach (the act installed by the federal government) but studied the political 72 E. Devroe et al. dynamics between federal, regional and local actors in the common Bpolicing goal of making cities clean, liveable and attractive again, the detailed reconstruction revealed some very unexpected results. dynamics between federal, regional and local actors in the common Bpolicing goal of making cities clean, liveable and attractive again, the detailed reconstruction revealed some very unexpected results. First, we found a coherent rationale among those who create and implement policy that is, to a certain degree, contrary to Garland's thesis, which emphasises volatility and ambivalence (see also O'Malley 1999); and the regulation of antisocial behaviour was strongly influenced by the ideological body of thought of Socialist Party officials (French and Dutch speaking) who shared a strong belief in social prevention and, as an ultimum remedium if prevention and warnings fail, a local tailor-made instrument in the hands of the city council to sanction frequent antisocial behaviour. We believe this socialist ideology to be the main element of resistance against purely punitive reactions in the Belgian political context. Conclusion and discussion Disconnecting local (sanctioning) policies from local preventative and welfare policies could turn MAS practices into a purely repressive and punitive arrangement. Second, Belgian socialist officials do not comply with regulations for state reform, which is only possible in the typical Belgian political-bargaining consensus model of governance. Our analysis showed that although the Minister of the Interior (an empty barrel) formerly had no say over municipalities and certainly not in the domain of social prevention (only in terms of repression), in fact, the minister found a way to govern municipalities from a distance by Bcontracting politics, so imposing on them a preventative and welfare-oriented ideology. This 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 repro- duction 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. An Expanding Culture of Control? 73 was not, as in the UK, about creating networks. In fact, the minister found a way to govern municipalities from a distance by using a community safety approach, so imposing on them to create crime prevention networks22 As prevention officers were mostly located in police stations, it was more about transferring processes of social justice and prevention to a tailor- made level: cities. This rather unusual approach, which is due to Belgium’s complex consti- tutional setting, touches on Lijphart’s (1999) distinction between consensus and majoritarian democracy as a key element in explaining the country’s moderate model of regulating antisocial behaviour. In conclusion, it can be observed that MAS offers municipalities a tool to steer local problems by providing administrative fines and is accompanied by tailor-made prevention programmes that are not oriented towards a dangerous other but are relevant to all municipal inhabitants. MAS cannot be called an expressive/symbolic dimension of criminology of the other (acting out) or as an illustration of a stronger repressive governmental grip. Our results are in line with the findings of Goddard (2012), who supports the development of a thesis that prevention and welfarist traditions survived the transition to late-modern risk-management developments, at least in policy intentions at the federal level, and in Liège. Our analysis has shown that the culture of the control framework contains elements that help explain and understand the emergence and application of MAS. It is clear, however, that not all aspects of regulating antisocial in Belgium fit within Garland’s limited scope of the sociology of punishment. Complementing Garland’s thesis with theoretical and empirical insights into public order issues in general and on regulating public space in particular would enlarge our understanding of control theories in late-modern society. How can we explain convergent or divergent regulating tools and practices between different metropolises embedded in the same nation state? Can we detect more explanatory variables than historical evolutions, linguistic characteristics and political ideologies? This last question illustrates the need for further research and confirms the culture of control thesis as a useful framework for analysis. However, Garland’s insights do not necessarily have an explanatory value in the empirical study of public order and antisocial behaviour at the local level. 22 The first governmental white paper on the integral security ideology dates from 2004. References Aberbach, J. D., & Rockman, B. A. (2002). Conducting and coding elite interviews’. Political Science and Politics, 35(4), 673–6. Aberbach, J. D., & Rockman, B. A. (2002). Conducting and coding elite interviews’. Political Science and Politics, 35(4), 673–6. Bauwens, T., Pleysier, S., De Pauw, E., & Van Den Broeck, T. (Eds.). (2016). 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https://openalex.org/W2591134468	http://ejurnal.its.ac.id/index.php/teknik/article/download/16521/3197	Indonesian	null	Penambahan Bulking Agent untuk Meningkatkan Kualitas Kompos Sampah Sayur dengan Variasi Metode Pengomposan	Jurnal Teknik/Jurnal Teknik ITS	2,016	cc-by-sa	5,783	Moh. Rohim, Arseto Y. Bagastyo Jurusan Teknik Lingkungan, Fakultas Teknik Sipil Dan Perencanaan, Institut Teknologi Sepuluh Nopember (ITS) Jl. Arief Rahman Hakim, Surabaya 60111 Indonesia e-mail: bagastyo@enviro.its.ac.id Moh. Rohim, Arseto Y. Bagastyo Jurusan Teknik Lingkungan, Fakultas Teknik Sipil Dan Perencanaan, Institut Teknologi Sepuluh Nopember (ITS) Jl. Arief Rahman Hakim, Surabaya 60111 Indonesia e-mail: bagastyo@enviro.its.ac.id Abstrak— Bahan utama pengomposan dalam penelitian ini adalah sayur dan sabut kelapa sebagai bulking agent. Kedua bahan tersebut digunakan karena ketersediannya yang melimpah di lokasi penelitian yaitu Pasar Puspa Agro, Sidoarjo. Variabel yang digunakan dalam penelitian ini adalah penambahan bulking agent dan metode pengomposan. Penambahan bulking agent yang digunakan yakni 40%, 50% dan 60% yang mana persentase tersebut diperoleh dari hasil perhitungan rasio C/N campuran dari kedua bahan yang masuk rentang 25-40. Sedangkan metode pengomposan yang dipakai adalah metode 1 (tidak dicacah, dilapis), metode 2 (dicacah, dicampur) dan metode 3 (dicacah, dilapis). Dimensi pengomposan yaitu 0,5x0,5x1 m dan pembalikan untuk metode 1 dan 3 yakni 3 hari sekali selama 2 minggu pertama, seminggu sekali sampai minggu keenam dan 2 minggu sekali sampai kompos matang. Sedangkan pada metode 2, pembalikan dilakukan 3 hari sekali sampai kompos matang. Parameter kualitas kompos yang digunakan mengacu pada SNI 19- 7030-2004 meliputi suhu, pH, kadar air, kadar C-organik, N total dan rasio C/N. Hasil dari penelitian ini adalah jika ditinjau dari penambahan bulking agent kualitas kompos yang paling baik yaitu dengan penambahan 60%. Apabila ditinjau dari metode pengomposan, kualitas kompos yang paling baik yaitu menggunakan metode 3 (dicacah, dilapis). biodegradable dan menghasilkansisa metabolisme berupa CO2, uap air dan panas [3]. Pasar Induk Puspa Agro telah menerapkan sistem pengomposan dalam mengolah sampah pasarnya. Akan tetapi, berdasarkan analisis laboratorium pada Januari tahun 2015 kompos masih bermutu kurang baik karena rasio C/N dalam kompos berada di bawah SNI spesifikasi kompos yaitu 7,53. Berdasarkan SNI 19-7030-2004 tentang Spesifikasi Kompos dari Sampah Organik dan Domestik, standar kualitas kompos untuk rasio C/N adalah 10-20, unsur karbon 9,8-32 % dan nitrogen minimum 0,4%. Dalam meningkatkan kualitas kompos maka diperlukan bahan tambahan dalam pengomposan. Dalam penelitian ini bahan yang ditambahkan adalah sabut kelapa. Sabut kelapa dipilih karena ketersediannya di Pasar Puspa Agro sangat melimpah. Selain itu sabut kelapa memiliki rasio C/N yang cukup tinggi yaitu 91 (Hasil analisis Laboratorium Kualitas Lingkungan, 2016). Sabut kelapa juga berperan sebagai bulking agent dalam proses pengomposan karena sifatnya yang porous sehingga aerasi dalam tumpukan dapat tetap berjalan [4]. Kompos yang bermutu baik akan dapat memperbaiki struktur tanah dan meningkatkan pertumbuhan tanaman [5]. F99 F99 JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) Moh. Rohim, Arseto Y. Bagastyo Jurusan Teknik Lingkungan, Fakultas Teknik Sipil Dan Perencanaan, Institut Teknologi Sepuluh Nopember (ITS) Jl. Arief Rahman Hakim, Surabaya 60111 Indonesia e-mail: bagastyo@enviro.its.ac.id Tujuan dari penelitian ini yaitu untuk menganalisis kualitas kompos yang dihasilkan ditinjau dari variasi penambahan bulking agent dan variasi metode pengomposan. Dari hasil peneltian yang didapatkan dapat menjadi acuan dalam pengomposan sampah sayur pada umumnya terutama pada TPST Pasar Puspa Agro, Sidoarjo. Kata Kunci— bulking agent, kompos, rasio C/N, sampah sayur, sistem windrow Kata Kunci— bulking agent, kompos, rasio C/N, sampah sayur, sistem windrow III. ANALISIS DAN PEMBAHASAN 4) Penumpukan bahan kompos Penumpukan pada metode 1 dan 3 dilakukan dengan cetakan dan disusun secara berlapis. Lapisan paling atas dan paling bawah sedapat mungkin adalah sabut kelapa. Susunan lapisan pada metode ini yakni sabut-sayur-sabut- sayur-sabut. Sedangkan pada metode 2 ditumpuk secara biasa menyerupai bentuk kerucut tanpa sistem lapisan, jadi bahan sayur dan sabut dicampur dari awal. Adapun gambaran tumpukan dapat dilihat pada Gambar 1. D. Metode Pengumpulan Data 1) Pembuatan cetakan Cetakan dibuat untuk pengomposan metode 2. Bahan dari kawat berlubang (lubang berbentuk kotak, dimensi 0,5 cm). Dimensi untuk masing-masing cetakan yaitu 0,5x0,5x1 m. Pada sisi sudut dari cetakan kompos ini dilapisi dengan bambu untuk memperkuat cetakan. Metode pengumpulan data dalam penelitian ini sebagaimana tercantum dalam Tabel 2; Table 2. Metode Pengumpulan Data No Parameter Metode Sumber 1 Kadar Air Pemanasan dengan oven [6] 2 pH Soil test - 3 Suhu Termometrik [7] 4 Kadar C organik Walkey and Black [8] 5 Kadar N total Kjeldahl dan spektrofotometri [6] 6 Rasio C/N Perbandingan - Table 2. Metode Pengumpulan Data 2) Pemilahan sampah Pemilahan sampah dilakukan untuk memisahkan bahan yang akan dikomposkan dengan bahan yang sulit untuk diuraikan misalnya plastik, karet dan kayu. Sampah yang digunakan sebagai bahan kompos hanyalah sampah sayur dan sabut kelapa saja. 3) Pencacahan bahan kompos Pencacahan dilakukan pada tumpukan metode 2 dan 3. Sampah dicacah dengan mesin pencacah hingga berukuran 1-5 cm. A. Kerangka Penelitian S alah satu sumber timbulan sampah kota yang cukup tinggi adalah pasar yaitu sebesar 7% [1]. Hal ini karena tidak semua bahan di pasar terutama sayuran siap untuk dijual melainkan harus dipilih dan dibersihkan terlebih dahulu. Pengolahan sampah pasar dapat dilakukan dengan cara pengomposan. Pengomposan cocok diterapkan karena komposisi sampah pasar ialah jenis organik berupa sisa sayuran dan makanan yang mencapai 95 % [2]. Prinsip pengomposan yaitu penguraian bahan organik (biodegradable) melalui proses biologis dengan bantuan organisme pengurai. Dalam prosesnya, mikroorganisme pengurai mengambil sumber makanan dari sampah atau bahan organik S Penelitian ini berawal dari adanya permasalahan dari kualitas kompos yang dihasilkan di TPST Pasar Puspa Agro Sidoarjo yang berada di bawah SNI. Penelitian ini dilakukan dalam skala pilot bertempat di TPST Pasar Puspa Agro, sementara untuk uji parameter C dan N dilakukan di laboratorium Teknik Lingkungan FTSP ITS, Surabaya. Penelitian pendahuluan dilakukan untuk mengetahui karakteristik dari masing-masing bahan pengomposan. Pelaksanaan penelitian ini membutuhkan waktu 2 bulan waktu pengomposan. Metode yang digunakan ada 3 yaitu metode 1 (tidak dicacah, dilapis), metode 2 (dicacah, dicampur) dan metode 3 (dicacah, dilapis). Jumlah tumpukan untuk masing- JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) F100 5) Pembalikan dan penyiraman Tujuan dari pembalikan adalah memberikan sirkulasi oksigen dalam tumpukan. Penyiraman dilakukan ketika tumpukan kompos terlihat kering atau melihat data kadar airnya telah berada di bawah 40%. 5) Pembalikan dan penyiraman Tujuan dari pembalikan adalah memberikan sirkulasi oksigen dalam tumpukan. Penyiraman dilakukan ketika tumpukan kompos terlihat kering atau melihat data kadar airnya telah berada di bawah 40%. masing metode adalah 3 tumpukan sesuai dengan variasi penambahan bulking agent (40%, 50%, 60%). Ketiga tumpukan diduplo sehingga masing-masing metode terdapat 6 tumpukan, jadi total terdapat 18 tumpukan ditambah 2 tummpukan kontrol. Pengumpulan data meliputi kadar air, pH, suhu, %C-organik, %N total dan rasio C/N. Selanjutnya data dianalisis dengan uji anova menggunakan software SPSS untuk mengetahui perbedaan rata-rata antar variasi dalam penelitian ini. 6) 6) Pemantauan parameter 6) Pemantauan parameter Pemantauan parameter meliputi kadar air (3 hari sekali), pH (3 hari sekali), suhu (setiap hari), C-organik (akhir pengomposan), N total (akhir pengomposan) dan rasio C/N. 7) Pemanenan dan pengayakan B. Penelitian Pendahuluan Pemanenan dilakukan dengan cara kompos diayak dengan mesin ayakan silinder. Pemanenan dilakukan ketika kompos telah menunjukkan ciri-ciri kematangan menurut SNI 19- 7030-2004. Penelitian pendahuluan dilaksanakan di laboratorium Teknik Lingkungan FTSP ITS yaitu melalui uji karakteristik bahan pengomposan. Parameter yang diuji terhadap bahan pengomposan yaitu kadar air, pH, %C-organik dan %N total. Hasil uji karakteristik ini menjadi pertimbangan untuk penambahan bulking agent yang perlu ditambahkan dalam pengomposan sampah sayur. Dari hasil penelitian penelitian didapatkan data seperti pada Tabel 1 berikut; Table 1. Hasil Uji Karakteristik Bahan Pengomposan Table 1. Table 1. Hasil Uji Karakteristik Bahan Pengomposan Bahan Kadar air (%) pH %C- organik %N total Sayur 93,00 3,25 34,50 2,52 Sabut kelapa 15,96 8,1 43,25 0,57 C. Tahapan Pembuatan Kompos C. Tahapan Pembuatan Kompos C. Tahapan Pembuatan Kompos Tahapan pembuatan kompos dalam penelitian ini merupakan langkah-langkah yang dilakukan dalam pengomposan mulai dari pembuatan cetakan hingga pemanenan kompos Gambar 1 Tumpukan untuk Ketiga Metode A. Bahan Pengomposan Bahan pengomposan yang dipakai dalam penelitian ini adalah sampah sayur dan sabut kelapa. Sampah sayur didapatkan dari Pasar Puspa Agro dan Pasar Sepanjang, sedangkan sabut kelapa didapatkan dari Puspa Agro saja. Adapun karakteristik sampah dari kedua pasar tersebut tidak sama pada saat pengambilan. Sampah sayur yang berasal dari Pasar Puspa Agro didominasi oleh sawi dan kubis sedangkan JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) F101 dilihat pada Gambar 2. Suhu tertinggi dicapai oleh P11 yaitu 42°C pada hari kedua pengomposan. P11 mencapai suhu tertinggi karena bahan kompos lebih didominasi oleh kubis. Hal ini karena kubis mengandung air lebih dari 90%, sehingga mudah mengalami pembusukan [10]. Oleh karena sifatnya yang mudah busuk, maka hasil dari respirasi mikroorganisme berupa energi dalam bentuk panas, CO2, dan air dengan cepat terbentuk. Pada hari ketiga dan seterusnya suhu mengalami penurunan dan menuju keadaan stabil. Hal ini menunjukkan bahan organik biodegradable terutama C-organik yang didekomposisi oleh bakteri semakin berkurang. dari Pasar sepanjang didominasi oleh kangkung dan daun singkong. Bahan awal pengomposan untuk masing-masing tumpukan dan berat awal pengomposan dapat dilihat pada Tabel 3. Table 3. Table 3. Bahan Pengomposan untuk Masing-masing Tumpukan Tumpukan (P) Bahan Pengomposan (Kg) Jenis Sayur dominan Asal Sayur Sabut kelapa P1 32,45 16,10 sawi, selada P. Puspa Agro P2 27,79 19,92 sawi P. Puspa Agro P3 21,72 23,89 sawi, selada P. Puspa Agro P11 37,19 15,93 kubis P. Puspa Agro P22 28,08 20,18 sawi, kubis P. Puspa Agro P33 22,10 24,21 kubis, seledri P. Puspa Agro P4 33,23 15,58 kubis, selada P. Puspa Agro P5 27,16 20,41 sawi, kubis P. Puspa Agro P6 21,50 23,49 sawi, seledri P. Puspa Agro P44 32,67 16,32 kubis, bayam P. Puspa Agro P55 22,43 19,82 kangkung P. Sepanjang P66 18,22 24,31 kangkung, klobot jagung P. Sepanjang P7 25,63 15,70 kangkung, bawang prei P. Sepanjang P8 21,34 20,15 kangkung, daun singkong P. Sepanjang P9 16,82 23,44 kangkung, bayam P. Sepanjang P77 34,21 15,90 kubis P. Puspa Agro P88 27,78 19,76 sawi, brokoli P. Puspa Agro P99 22,61 23,66 kubis, sawi P. Puspa Agro PK Tc 55,41 0 sawi, kubis P. Puspa Agro PK C 42,14 0 daun singkong, kangkung P. A. Bahan Pengomposan Sepanjang Bahan Pengomposan untuk Masing-masing Tumpukan p g Gambar 2 Perubahan suhu pada metode 1 Gambar 2 Perubahan suhu pada metode 1 Pada metode 2, pencacahan yang dilakukan pada sayur dan sabut kelapa akan meningkatkan luas permukaan bahan kompos, sehingga memudahkan mikroba dekomposer untuk menghancurkan bahan kompos [9]. Sabut kelapa yang dicacah dapat menahan panas dalam tumpukan. Strukurnya yang makin halus dapat menutupi celah-celah pori dalam tumpukan. Peningkatan dan penurunan suhu tumpukan pada metode 2 dapat dilihat pada Gambar 3. dapat dilihat pada Gambar 3. Gambar 3 Perubahan suhu pada metode 2 Pada Gambar 3, secara umum peningkatan suhu terjadi pada hari kedua hingga kelima. Waktu peningkatan suhu ini lebih panjang apabila dibandingkan dengan metode yang tidak dicacah. Hal ini karena bahan yang dicacah dapat dikonsumsi oleh mikroorganisme dengan lebih mudah, sehingga proses degradasi berlangsung dengan lancar. Oleh karena kemudahan dalam mendegradasi tersebut, suhu akan tetap tinggi seiring dengan proses degradasi yang menghasilkan panas. Pada metode 3 kondisi tumpukan yang tinggi membuat panas yang dihasilkan dari proses degradasi akan tertahan oleh sabut dan sulit keluar Tingginya suhu ini dapat menimbulkan Berat sayur dan sabut kelapa pada tabel di atas ditentukan berdasarkan perbandingan sesuai dengan variasi penambahan bulking agent. Perbedaan jenis sayuran dalam tumpukan kompos juga dapat mempengaruhi kondisi kompos. P1-P33 merupakan metode 1 (tidak dicacah, dilapis), dengan bulking agent P1 yaitu 40%, P2 50% dan P3 60%. Sementara P11-P33 adalah duplonya. P4-P66 merupakan metode 2 (dicacah, dicampur) dan P7-P99 yakni metode 3 (dicacah, dilapis). Persentase penambahan bulking agent urutannya sama seperti pada metode 1. PKTc merupakan perlakuan kontrol tanpa dicacah, sedangkan PKC adalah kontrol yang dicacah. p p Gambar 3 Perubahan suhu pada metode 2 P d G b 3 i k h j di p p Gambar 3 Perubahan suhu pada metode 2 B. Perubahan Suhu Gambar 3 Perubahan suhu pada metode 2 Tumpukan kompos mengalami perubahan suhu yang berbeda antar metodenya. Namun, secara umum pada awal proses pengomposan, suhu meningkat hingga hari ke 2-5, dan selanjutnya suhu turun terus hingga stabil. Pada metode 1, sayur dan sabut yang tidak dicacah menyebabkan struktur yang kasar. Ukuran partikel dan struktur bahan kompos mempengaruhi sistem aerasi. Semakin kasar struktur bahan kompos, maka makin besar volume pori udara dalam tumpukan [9]. Sehingga panas yang dihasilkan dari proses degradasi bahan organik biodegradable akan mudah terlepas ke udara dan mengakibatkan suhu dalam tumpukan menjadi turun. Fluktuatif suhu dalam tumpukan pada metode 1 dapat Pada Gambar 3, secara umum peningkatan suhu terjadi pada hari kedua hingga kelima. Waktu peningkatan suhu ini lebih panjang apabila dibandingkan dengan metode yang tidak dicacah. Hal ini karena bahan yang dicacah dapat dikonsumsi oleh mikroorganisme dengan lebih mudah, sehingga proses degradasi berlangsung dengan lancar. Oleh karena kemudahan dalam mendegradasi tersebut, suhu akan tetap tinggi seiring dengan proses degradasi yang menghasilkan panas. Pada metode 3 kondisi tumpukan yang tinggi membuat panas yang dihasilkan dari proses degradasi akan tertahan oleh sabut dan sulit keluar. Tingginya suhu ini dapat menimbulkan JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) F102 degradasi oleh bakteri sehingga kenaikan dan penurunan nilai pH tidak terjadi secara drastis. asap yang keluar dari dalam tumpukan, dan ini terlihat pada hari ketiga ketika akan dilakukan pembalikan. Fluktuasi suhu pada metode 3 dapat dilihat pada Gambar 4. Suhu terendah dalam metode ini yaitu P9, dikarenakan jumlah sayurnya yang sedikit dibanding sabut kelapa. Sehingga hasil proses degradasi dalam bentuk panas akan sedikit juga. Pada metode 2, adanya proses pencacahan menyebabkan penurunan pH berlangsung dalam jangka waktu yang lama yaitu hingga hari ke dua belas (Gambar 6). Gambar 6 Perubahan pH pada metode 2 g p j g Gambar 4 Perubahan suhu pada metode 3 C. Perubahan pH Gambar 6 Perubahan pH pada metode 2 Penurunan pH dalam jangka waktu yang lama ini terjadi karena sayur yang dicacah dapat dengan mudah didegradasi oleh bakteri. Sehingga proses pembentukan asam organik oleh bakteri berlangsung seiring dengan banyaknya bahan yang mudah didegradasi. Fluktuasi terjadi hingga hari kedua belas dan kelima belas waktu pengomposan, setelahnya pH kompos mendekati pH netral sampai proses pematangan. C. Perubahan pH C. B. Perubahan Suhu Perubahan pH Pengukuran pH dilakukan mulai pada hari ketiga pengomposan, hal ini dilakukan karena pada awal pengomposan sampah sayur dan sabut kelapa masih belum tercampur secara merata. Pada hari ketiga pengomposan, rata- rata nilai pH untuk semua tumpukan yaitu 6,2 – 6,9. Nilai pH yang rendah menunjukkan terjadinya pembentukan asam organik dari proses degradasi bahan organik [4]. Naiknya nilai pH disebabkan oleh penguraian protein menjadi ammonia (NH3) yang berpengaruh terhadap peningkatan pH kompos [11]. Pada metode 3, dengan adanya peletakan bahan kompos yang berlapis mengakibatkan sayur tidak tersebar seperti pada metode 2. Hal ini dapat membuat pembentukan asam organik oleh bakteri tidak semudah apabila dibandingkan dengan kondisi tercampur. Hal tersebut karena proses degradasi bisa terhambat akibat adanya sekat antar sayur. Apabila dilihat secara rata-rata, fluktuasi pH pada metode ini diawali dengan peningkatan hingga hari keenam dan kedua belas, kemudian terjadi penurunan hingga mendekati pH netral pada hari setelahnya hingga waktu pengomposan selesai. Fluktuasi pH untuk metode 3 dapat dilihat pada Gambar 7. Pada metode 1 pH tumpukan pada hari ketiga dan keenam pengomposan adalah 6,6 hingga 6,9. Kenaikan pH terjadi pada hari keenam pengomposan yang mana hal ini menunjukkan terbentuknya NH3 dari proses pengomposan. Dan ini terbukti pada pembalikan hari keenam hingga hari kedua belas, aroma tumpukan lebih menyengat seperti bau amonia. Fluktuasi pH pada metode 1 dapat dilihat pada Gambar 5. Gambar 7 Perubahan pH pada metode 3 D. Perubahan Kadar Air Gambar 5 Perubahan pH pada metode 1 D. Perubahan Kadar Air Kadar air pada awal pengomposan berbeda untuk masing- masing tumpukan, hal ini diakibatkan oleh perbandingan sayur dengan sabut kelapa, jenis sayur yang mendominasi pada tumpukan dan metode pengomposan. Karena kadar air sayur lebih besar dari pada sabut kelapa yaitu 93%. Selain itu sayur Fluktuasi pH tumpukan pada metode 1 terjadi hingga hari kedua belas, sedangkan hari setelahnya pH cenderung netral. Perlakuan sayur yang tidak dicacah sedikit menghambat proses JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) F103 Gambar 9 Perubahan kadar air pada metode 2 yang dicacah kandungan airnya lebih besar dari pada sayur yang tidak dicacah, karena pada sayur yang dicacah luas permukaannya semakin besar dan ukurannya kecil. Ukuran partikel yang semakin kecil, akan mempermudah bakteri melakukan degradasi dan akan menghasilkan cairan, sehingga kadar air akan meningkat [12]. Rujukan [13] menjelaskan kadar air 40-60 % adalah kelembaban optimum untuk metabolisme mikroba. Apabila kelembaban di bawah 40%, aktivitas mikroba akan mengalami penurunan. B. Perubahan Suhu Penurunan ini menyebabkan kurangnya air untuk melarutkan bahan organik biodegradable yang akan didegradasi oleh mikroorganisme sebagai sumber energinya [12]. Maka dari itu, perlunya dilakukan penyiraman dalam tumpukan apabila kadar air dalam tumpukan telah di bawah 40%. Semua tumpukan dalam metode 2 mengalami penurunan kadar air hingga hari kedua belas waktu pengomposan. Hal ini disebabkan oleh bentuk tumpukan yang hanya ditumpuk biasa dan adanya pencampuran dari awal antara sayur dan sabut kelapa. Oleh karena tercampur, maka lapisan bawah dari tumpukan bukan merupakan sabut. Sehingga air yang mengalir dari dalam tumpukan tidak dapat ditahan dan akan keluar sebagai lindi. Pada hari kedua belas, dilakukan proses penyiraman untuk menghindari tumpukan terlalu kering, sehingga setelah hari tersebut kadar air kembali naik. Pada metode 1, kadar air di awal pengomposan berkisar antara 45-60%. Kemudian pada hari keenam atau pada pembalikan kedua, kadar air pada semua tumpukan mengalami kenaikan hingga menjadi 55-62 %. Hal ini mengindikasikan telah terjadi penguraian bahan organik biodegradable oleh mikroba di mana hasil dari proses tersebut berupa air. Air yang berlebih tidak mampu ditahan oleh sabut kelapa, sehingga air tersebut mengalir keluar tumpukan sebagai lindi. Peningkatan dan penurunan kadar air ini dapat dilihat pada grafik dalam Gambar 8. Pada metode 2, penyusunan bahan secara berlapis antara sayur dan sabut kelapa, maka air yang mengalir ke arah bawah tumpukan dapat sedikit tertahan oleh adanya sabut kelapa pada lapisan paling bawah. Sehingga kadar air dalam tumpukan dapat terjaga. Namun, analisis tersebut tidak terjadi pada tumpukan P7, P8 dan P9 (dapat dilihat pada Gambar 10). Gambar 8. Gambar 8 Perubahan kadar air pada metode 1 Pada metode 2, sayur yang dicacah dapat meningkatkan kadar air dalam tumpukan pada fase awal. Karena semakin kecil ukuran bahan, maka air yang terkandung di dalamnya akan semakin banyak yang keluar. Perubahan kadar air dalam tumpukan pada metode 2 dapat dilihat pada Gambar 9. Gambar 8 Perubahan kadar air pada metode 1 Gambar 10 Perubahan kadar air pada metode 3 Gambar 8 Perubahan kadar air pada metode 1 Pada metode 2, sayur yang dicacah dapat meningkatkan kadar air dalam tumpukan pada fase awal. Karena semakin kecil ukuran bahan, maka air yang terkandung di dalamnya akan semakin banyak yang keluar. Perubahan kadar air dalam tumpukan pada metode 2 dapat dilihat pada Gambar 9. Gambar 10 Perubahan kadar air pada metode 3 Tumpukan P7, P8 dan P9 mengalami penurunan kadar air karena bahan dalam tumpukannya didominasi oleh kangkung. F. Mass Balance Kualitas Kompos Matang kandungan airnya lebih tinggi dari yang tidak dicacah, sehingga lindi yang dihasilkan lebih banyak. Table 4. Perubahan Rasio C/N dalam Pengomposan Tumpukan (P) Awal Akhir C- organik (%) N total (%) Rasio C/N C- organik (%) N total (%) Rasio C/N P1 38,00 1,74 21,84 19,17 3,47 5,53 P2 38,87 1,55 25,16 21,03 3,38 6,21 P3 39,75 1,35 29,44 21,17 3,14 6,74 P11 38,00 1,74 21,84 18,35 3,58 5,12 P22 38,87 1,55 25,16 19,76 3,55 5,56 P33 39,75 1,35 29,44 24,62 3,35 7,34 P4 38,00 1,74 21,84 15,72 1,61 9,76 P5 38,87 1,55 25,16 16,78 1,49 11,29 P6 39,75 1,35 29,44 18,02 1,36 13,21 P44 38,00 1,74 21,84 15,87 1,70 9,35 P55 38,87 1,55 25,16 16,21 1,35 11,97 P66 39,75 1,35 29,44 17,65 1,34 13,21 P7 38,00 1,74 21,84 16,75 1,64 10,22 P8 38,87 1,55 25,16 18,88 1,59 11,88 P9 39,75 1,35 29,44 19,24 1,54 12,50 P77 38,00 1,74 21,84 16,96 1,79 9,47 P88 38,87 1,55 25,16 18,23 1,62 11,24 P99 39,75 1,35 29,44 18,69 1,59 11,79 PK Tc 34,50 2,52 13,69 4,12 4,61 0,89 PK C 34,50 2,52 13,69 3,53 3,48 1,01 Mass Balance Tumpukan (P) Berat kering (Kg) Persentase reduksi total (%) awal akhir P1 15,80 10,55 33,22 P2 18,69 13,18 29,45 P3 21,60 16,85 21,96 P11 15,99 10,39 35,03 P22 18,92 13,39 29,26 P33 21,89 16,88 22,91 P4 15,42 3,30 78,59 P5 19,05 4,60 75,85 P6 21,25 5,15 75,78 P44 16,00 4,98 68,88 P55 18,23 6,06 66,73 P66 21,71 10,47 51,77 P7 14,99 7,59 49,37 P8 18,43 9,40 48,98 P9 20,88 10,94 47,61 P77 15,76 6,37 59,55 P88 18,55 8,88 52,15 P99 21,47 10,36 51,73 PK Tc 3,88 2,02 47,98 PK C 2,95 1,26 57,17 G. Kualitas Kompos Matang Pemanenan kompos dilakukan pada waktu umur pengomposan 60 hari. Hal ini dilakukan karena ciri-ciri kematangan kompos telah terlihat yaitu warna coklat kehitaman bau seperti tanah suhu dan pH juga sudah stabil Table 4. B. Perubahan Suhu Sedangkan pada tumpukan P77, P88 dan P99 lebih didominasi oleh sayuran kubis dan sawi. Kadar air kangkung lebih rendah dari pada kubis, hal ini dibuktikan dalam penelitian [14]. Dengan kadar air yang sedikit, seiring berjalannya waktu, maka lama lama kadar air yang tedapat dalam tumpukan akan turun. Sedangkan pada tumpukan P77, P88 dan P99 yang bahannya memiliki kandungan air lebih tinggi, air yang keluar dan hasil dari proses degradasi (yang berupa air juga) tidak bisa secara langsung keluar tumpukan, akan tetapi tertahan dengan adanya lapisan sabut kelapa. Selain sifatnya yang JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) F104 mampu menahan air, sabut kelapa yang dicacah dapat mengurangi jumlah pori dalam tumpukan sehingga kemungkinan air untuk keluar dalam tumpukan akan berkurang. Rasio C/N pada metode 1 tidak ada yang mencapai rentang SNI (10-20). Sedangkan pada metode 2, hanya perbandingan 60:40 yang belum mencapainya. Pada metode 3 hampir semuanya masuk dalam kriteria SNI, hanya P77 yang tidak masuk rentang. Metode 3 (dicacah, dilapis) dalam hal ini menjadi yang terbaik apabila dilihat dari rasio C/N. E. Perubahan Rasio C/N Proses degradasi dalam pengomposan membutuhkan C- organik untuk pemenuhan energi dan pertumbuhan, dan nitrogen untuk pemenuhan protein sebagai zat pembangun sel bakteri [15]. Hasil pengukuran C organik dan nitrogen total dalam penelitian ini menujukkan penurunan selama proses pengomposan. Perubahan rasio C/N bahan pengomposan antara awal dan akhir dapat dilihat pada Tabel 4. Penurunan kandungan C-organik dalam tumpukan terjadi karena karbon digunakan oleh bakteri sebagai sumber energi untuk pertumbuhannya. F. Mass Balance Perubahan berat total yang menurun merupakan indikator kehilangan massa bahan organik biodegradable sebagai hasil respirasi [4]. Berat bahan yang hilang adalah gas-gas hasil penguraian oleh mikroba yang terbuang ke udara, misalnya amonia sehingga menyebabkan berat bahan akhir menjadi berkurang [14]. Selain itu hasil samping dari proses degradasi berupa CO2 dan air juga keluar dari tumpukan akibat penguapan atau menjadi lindi. Metode yang mempunyai penurunan berat bahan yang besar adalah metode 2 (dicacah, dicampur) yaitu 78% (berdasarkan berat kering). Hal ini karena metode ini dicacah dan dicampur ditumpuk biasa tanpa dicetak. Sehingga mikroba lebih mudah mendegradasinya karena tidak ada sekat atau lapisan sabut kelapa seperti pada metode 1 dan 3. Perubahan berat bahan pengomposan dalam penelitian ini dapat dilihat pada diagram mass balance pada Tabel 5. Sedangkan metode 3 (dicacah, dilapis) penurunan berat bahannya sebesar 49%, hal ini lebih tinggi dari pada metode 1 (tidak diicacah, dilapis) yakni 33%. Hal ini disebabkan karena meskipun sama-sama disusun secara berlapis, bahan yang dicacah dapat meningkatkan reduksi sampah. Pada metode 1 kandungan C-organik lebih tinggi dari metode 2 dan 3, hal ini karena bahan yang tidak dicacah membuat mikroba kesulitan dalam mendegradasinya. Sehingga di akhir pengomposan C-organik yang tersisa juga lebih tinggi dari metode dengan pencacahan. Sedangkan pada pada metode 2 dan 3, bahan yang dicacah dapat membuat mikroba lebih mudah dalam mendegradasinya. Sehingga penurunan C- organik oleh mikroba lebih besar dari metode 1. Sedangkan kandungan N total yang besar ada pada metode 1, hal ini disebabkan sayur yang tidak dicacah akan membuat tingkat degradasi oleh mikroba lebih sulit. Sedangkan penurunan nitrogen total pada metode 2 dan 3 diakibatkan oleh hilangnya nitrat yang larut bersama lindi. Pada bahan yang dicacah, kandungan airnya lebih tinggi dari yang tidak dicacah, sehingga lindi yang dihasilkan lebih banyak. Table 5. Mass Balance Tumpukan (P) Berat kering (Kg) Persentase reduksi total (%) awal akhir P1 15,80 10,55 33,22 P2 18,69 13,18 29,45 P3 21,60 16,85 21,96 P11 15,99 10,39 35,03 P22 18,92 13,39 29,26 P33 21,89 16,88 22,91 P4 15,42 3,30 78,59 P5 19,05 4,60 75,85 P6 21,25 5,15 75,78 P44 16,00 4,98 68,88 P55 18,23 6,06 66,73 P66 21,71 10,47 51,77 P7 14,99 7,59 49,37 P8 18,43 9,40 48,98 P9 20,88 10,94 47,61 P77 15,76 6,37 59,55 P88 18,55 8,88 52,15 P99 21,47 10,36 51,73 PK Tc 3,88 2,02 47,98 PK C 2,95 1,26 57,17 G. F. Mass Balance Perubahan Rasio C/N dalam Pengomposan Tumpukan (P) Awal Akhir C- organik (%) N total (%) Rasio C/N C- organik (%) N total (%) Rasio C/N P1 38,00 1,74 21,84 19,17 3,47 5,53 P2 38,87 1,55 25,16 21,03 3,38 6,21 P3 39,75 1,35 29,44 21,17 3,14 6,74 P11 38,00 1,74 21,84 18,35 3,58 5,12 P22 38,87 1,55 25,16 19,76 3,55 5,56 P33 39,75 1,35 29,44 24,62 3,35 7,34 P4 38,00 1,74 21,84 15,72 1,61 9,76 P5 38,87 1,55 25,16 16,78 1,49 11,29 P6 39,75 1,35 29,44 18,02 1,36 13,21 P44 38,00 1,74 21,84 15,87 1,70 9,35 P55 38,87 1,55 25,16 16,21 1,35 11,97 P66 39,75 1,35 29,44 17,65 1,34 13,21 P7 38,00 1,74 21,84 16,75 1,64 10,22 P8 38,87 1,55 25,16 18,88 1,59 11,88 P9 39,75 1,35 29,44 19,24 1,54 12,50 P77 38,00 1,74 21,84 16,96 1,79 9,47 P88 38,87 1,55 25,16 18,23 1,62 11,24 P99 39,75 1,35 29,44 18,69 1,59 11,79 PK Tc 34,50 2,52 13,69 4,12 4,61 0,89 PK C 34,50 2,52 13,69 3,53 3,48 1,01 Table 4. Perubahan Rasio C/N dalam Pengomposan Pemanenan kompos dilakukan pada waktu umur pengomposan 60 hari. Hal ini dilakukan karena ciri-ciri kematangan kompos telah terlihat yaitu warna coklat kehitaman, bau seperti tanah, suhu dan pH juga sudah stabil. JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) F105 bawah 0,05, maka dapat dikatakan bahwa parameter C- organik, N total dan rasio C/N menunjukkan perbedaan nilai rata-rat yang bermakna terhadap metode pengomposan. Untuk melihat metode pengomposan mana saja yang berbeda rata- ratanya, maka dilakukan uji lanjut yaitu uji Bonferroni. Uji Bonferroni ini dilakukan karena hasil uji kehomogenan varian mempunyai nilai signifikansi di atas 0,05. Berdasarkan uji Bonferroni (Tabel 9), didapatkan bahwa metode pengomposan yang menunjukkan perbedaan rata-rata parameter (ada tanda ) adalah metode 1 dengan 2, dan metode 1 dengan 3. Sedangkan metode 2 dengan 3 tidak menunjukkan perbedaan rata-rata yang bermakna. Adapun kualitas kompos yang dihasilkan dalam penelitian ini dapat dilihat pada Tabel 6. dapat dilihat pada Tabel 6. F. Mass Balance 3 1.78333 .07926 .000 1.5698 1.9968 Met. 2 Met. 1 -1.93667* .07926 .000 -2.1502 - 1.7232 Table 7 Apabila ditinjau dari variasi penambahan bulking agent, kualitas kompos yang dihasilkan dengan penambahan 60% lebih baik dari persentase yang lain. Namun jika ditinjau dari metode pengomposannya, baik penambahan 40%, 50% maupun 60% yang paling baik adalah pada metode 3 (dicacah, dilapis). Di antara penambahan tersebut, penambahan bulking agent 50% adalah yang paling baik dikarenakan rasio C/Nnya yang mendekati rasio C/N tanah (10-12). F. Mass Balance Table 6 Kualitas Kompos Matang Tumpukan (P) Karbon (%) Nitrogen (%) Rasio C/N Kadar Air (%) suhu (°C) pH SNI 19- 7030-2004 9,8 - 32 0,4 10 -20 maks 50 maks 30 6,8 - 7,5 P1 19,17 3,47 5,53 27,87 26 7,2 P2 21,03 3,38 6,21 38,36 26 7,2 P3 21,17 3,14 6,74 29,57 26 7,2 P11 18,35 3,58 5,12 29,82 27 7,2 P22 19,76 3,55 5,56 25,29 26 7,2 P33 24,62 3,35 7,34 41,18 26 7,2 P4 15,72 1,61 9,76 45,77 26 7,2 P5 16,78 1,49 11,29 54,06 26 7,2 P6 18,02 1,36 13,21 51,79 26 7,2 P44 15,87 1,70 9,35 40,89 26 7,2 P55 16,21 1,35 11,97 52,73 26 7,2 P66 17,65 1,34 13,21 46,57 26 7,2 P7 16,75 1,64 10,22 46,05 26 7,2 P8 18,88 1,59 11,88 54,23 26 7,2 P9 19,24 1,54 12,50 43,84 26 7,2 P77 16,96 1,79 9,47 49,38 26 7,2 P88 18,23 1,62 11,24 40,74 26 7,2 P99 18,69 1,59 11,79 37,10 26 7,2 PK Tc 4,12 4,61 0,89 12,80 27 7,2 PK C 3,53 3,48 1,01 29,95 27 7,2 yang bermakna. Table 7 Uji Anova Parameter Terhadap Penambahan Bulking Agent Sum of Squares df Mean Square F Sig. C_organik Between Groups 22.886 2 11.443 2.852 .089 Within Groups 60.193 15 4.013 Total 83.078 17 N_total Between Groups .181 2 .090 .097 .908 Within Groups 14.011 15 .934 Total 14.192 17 Rasio_CN Between Groups 22.281 2 11.140 1.395 .278 Within Groups 119.777 15 7.985 Total 142.058 17 Table 8 Uji Anova Parameter Terhadap Metode Pengomposan Sum of Squares df Mean Square F Sig. C_organik Between Groups 48.705 2 24.353 10.627 .001 Within Groups 34.373 15 2.292 Total 83.078 17 N_total Between Groups 13.909 2 6.954 368.98 1 .000 Within Groups .283 15 .019 Total 14.192 17 Rasio_CN Between Groups 113.990 2 56.995 30.460 .000 Within Groups 28.068 15 1.871 Total 142.058 17 Table 9 Uji Bonferroni Parameter Terhadap Metode Pengomposan Dependent Variable (I) Metode (J) Metode Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound C_organik Met. 1 Met. 2 3.97500* .87398 .001 1.6207 6.3293 Met. 3 2.55833* .87398 .031 .2040 4.9126 Met. 2 Met. 1 -3.97500* .87398 .001 -6.3293 - 1.6207 Met. 3 -1.41667 .87398 .378 -3.7710 .9376 Met. 3 Met. 1 -2.55833* .87398 .031 -4.9126 -.2040 Met. 2 1.41667 .87398 .378 -.9376 3.7710 N_total Met. 1 Met. 2 1.93667* .07926 .000 1.7232 2.1502 Met. H. Hasil Analisis Data dengan Uji Anova Analisis data dengan anova dilakukan untuk menguji adakah perbedaan rata-rata dalam kandungan C-organik, N total dan rasio C/N terhadap jumlah bulking agent yang ditambahkan dan metode pengomposan. Angka significance level (taraf signifikansi) yang dipakai dalam uji ini yaitu 0,05. Apabila nilai signifikansi di atas 0,05, maka dapat dikatakan tidak ada perbedaan rata-rata pada nilai parameter (C-organik, N total dan rasio C/N) terhadap variabel bebasnya. Namun apabila nilainya di bawah 0,05, maka ada perbedaan rata-rata parameter terhadap variabel bebasnya. Sehingga apabila kejadiannya seperti itu, perlu diuji lanjut untuk mengetahui variabel mana saja yang berbeda. Uji Bonferroni Parameter Terhadap Metode Pengomposan Dependent Variable (I) Metode (J) Metode Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound C_organik Met. 1 Met. 2 3.97500* .87398 .001 1.6207 6.3293 Met. 3 2.55833* .87398 .031 .2040 4.9126 Met. 2 Met. 1 -3.97500* .87398 .001 -6.3293 - 1.6207 Met. 3 -1.41667 .87398 .378 -3.7710 .9376 Met. 3 Met. 1 -2.55833* .87398 .031 -4.9126 -.2040 Met. 2 1.41667 .87398 .378 -.9376 3.7710 N_total Met. 1 Met. 2 1.93667* .07926 .000 1.7232 2.1502 Met. 3 1.78333* .07926 .000 1.5698 1.9968 Met. 2 Met. 1 -1.93667* .07926 .000 -2.1502 - 1.7232 Dari hasil uji anova antara parameter dengan jumlah penambahan bulking agent pada Tabel 7, nilai signifikansi semuanya di atas 0,05, maka dapat dikatakan tidak ada perbedaan yang bermakna dari rata-rata parameter terhadap jumlah penambahan bulking agent. Apabila dilihat dari hasil uji parameter dengan metode pengomposan pada Tabel 8, didapatkan nilai signifikansi di JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) JURNAL TEKNIK ITS Vol. 5, No. 2, (2016) ISSN: 2337-3539 (2301-9271 Print) F106 Works Association/Water Environment Federation, Washington DC, USA (2005). Works Association/Water Environment Federation, Washington DC, USA (2005). Dependent Variable (I) Metode (J) Metode Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound Met. 3 -.15333 .07926 .216 -.3668 .0602 Met. 3 Met. 1 -1.78333* .07926 .000 -1.9968 - 1.5698 Met. 2 .15333 .07926 .216 -.0602 .3668 Rasio_CN Met. 1 Met. 2 -5.54833* .78976 .000 -7.6757 - 3.4209 Met. 3 -5.10000* .78976 .000 -7.2274 - 2.9726 Met. 2 Met. 1 5.54833* .78976 .000 3.4209 7.6757 Met. 3 .44833 .78976 1.000 -1.6791 2.5757 Met. 3 Met. 1 5.10000* .78976 .000 2.9726 7.2274 Met. 2 -.44833 .78976 1.000 -2.5757 1.6791 [7] R. Rynk, M.v.D. Kamp, G.B. Wilson, M.E. Singley, T.L. Richard, J.J. Kolega, F.R. Gouin, L.L Jr, D. H. Hasil Analisis Data dengan Uji Anova Kay, D.W. Murphy, H.A.J. Hoitink, W.F. Brinton, On-Farm Composting Handbook. Ithaca, NY: Northeast Regional Agricultural Engineering Service (1992). [8] Soil Survey Standard Test Method Organic Carbon, Department of Sustainable Natural Resources, New South Wales, Australia. [9] R.D.M. Simanungkalit, D.A. Suriadikarta, R. Saraswati, D. Setyorini, W. Hartatik, Pupuk Organik dan Pupuk Hayati. Bogor: Balai Besar Penelitian dan Pengembangan Sumberdaya Lahan Pertanian (2006). [10] B.N. Widarti, W.K. Wardhini, E. Sarwono, “Pengaruh rasio C/N bahan baku pada pembuatan kompos dari kubis dan kulit pisang,” Jurnal Integrasi Proses, Vol. 5, No. 2 (2015) 75-80. [11] A.A.N. Supadma, D.M. Arthagama, “Uji formulasi kualitas pupuk kompos yang bersumber dari sampah organik dengan penambahan limbah ternak ayam, sapi, babi dan tanaman pahitan,” Bumi Lestari, Vol. 8, No. 2 (2008) 113-121. [12] E.S. Pandebesie, D. Rayuanti, “Pengaruh penambahan sekam pada proses pengomposan sampah domestik,” Jurnal Lingkungan Tropis, Vol. 6, No. 1 (2013) 31-40. [13] Isroi, Pengomposan Limbah Kakao. Jember: Pelatihan TOT Budidaya Kopi dan Kakao (2007) 5. IV. KESIMPULAN DAN SARAN [14] A.H. Permana, R.S. Hirasmawan, “Pembuatan kompos dari limbah padat organik yang tidak terpakai (limbah sayuran kangkung, kol dan kulit pisang),” (2009) 1-6. Kesimpulan dari penelitian adalah apabila ditinjau dari variasi penambahan bulking agent, kualitas kompos yang paling baik yaitu dengan penambahan 60%. Hasil yang diperoleh adalah suhu 26°C, pH 7,2, kadar air 46,57%, C- organik 17,65%, N total 1,34% dan rasio C/N 13,21. Sedangkan ditinjau dari metode pengomposan, kualitas kompos yang paling baik yaitu menggunakan metode 3 (dicacah, dilapis). Hasil yang diperoleh yakni suhu 26°C, pH 7,2, kadar air 40,74%, C-organik 18,23%, N total 1,62% dan rasio C/N 11,24. Namun, ditinjau dari reduksi totalnya, metode 2 (dicacah, dicampur) yang paling besar dengan nilai mencapai 78,59%. [15] A. Ismayana, N.S. Indrasti, Suprihatin, A. Maddu, A. Fredy, “Faktor rasio C/N awal dan laju aerasi pada proses co-composting bagasse dan blotong,” Jurnal Teknologi Industri Pertanian, Vol. 22, No. 3 (2012) 173-179. Saran yang dapat diberikan dalam penelitian ini adalah perlunya diukur parameter lain seperti kalium, phospor dan kemampuan ikat air untuk membandingkan lebih jauh lagi kualitas kompos yang dihasilkan dari variasi penelitian. Selain itu bahan pengomposan untuk semua tumpukan diusahakan seragam agar perbandingan kualitasnya bisa valid antar variasi. Pengukuran parameter C dan N sebaiknya dilakukan dengan periode 5 hari-an atau per minggu agar dapat melihat laju penurunannya. DAFTAR PUSTAKA [1] Dinas Kebersihan dan Pertamanan Kabupaten Sidoarjo, “Timbulan dan Komponen Sampah Kabupaten Sidoarjo”, (2013). [2] R. Sudradjat, Mengelola Sampah Kota. Bogor: Penebar Swadaya (2006) 7. [3] B. Subali, Ellianawati, “Pengaruh waktu pengomposan terhadap rasio unsur C/N dan jumlah kadar air dalam kompos,” presented at the Prosiding Pertemuan Ilmiah XXIV HFI Jateng dan DIY, Semarang, (2010). [4] J. Nugroho, N.S. Bintoro, T. Nurkayanti, “Pengaruh variaisi jumlah dan jenis bulking agent pada pengomposan limbah organik sayuran dengan komposter mini,” presented at the Prosiding Nasional Perteta, Purwokerto, (2010). [5] L. Murbando, Membuat Kompos. Jakarta: Penebar Swadaya (2008) 11. [6] Apha, Awwa, Wpcf, Standard Methods for The Examination of Water and Wastewater. American Public Health Association/American Water
https://openalex.org/W2548279154	https://europepmc.org/articles/pmc5118616?pdf=render	English	null	Basement membrane ultrastructure and component localization data from uterine tissues during early mouse pregnancy	Data in brief	2,016	cc-by	3,722	Basement membrane ultrastructure and componen localization data from uterine tissues during early mouse pregnancy Celestial R. Jones-Paris a,b, Sayan Paria b, Taloa Berg b, Juan Saus c,d, Gautam Bhave e,f, Bibhash C. Paria g,n,1, Billy G. Hudson a,c,e,f,h,i,j,k,n,1 y a Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States b Aspirnaut, Vanderbilt University Medical Center, Nashville, Tennessee, United States c Valencia University Medical School, Valencia, Spain d FibroStatin, SL, Valencia, Spain e Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States f Center for Matrix Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States g Division of Neonatology, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, United States h Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States i Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States j Vanderbilt Ingram Cancer Center, Nashville, Tennessee, United States k Vanderbilt Institute of Chemical Biology, Nashville, Tennessee, United States a Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States b Aspirnaut, Vanderbilt University Medical Center, Nashville, Tennessee, United States c Valencia University Medical School, Valencia, Spain d FibroStatin, SL, Valencia, Spain e Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States f Center for Matrix Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States g Division of Neonatology, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, United States h Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States i Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States j Vanderbilt Ingram Cancer Center, Nashville, Tennessee, United States k Vanderbilt Institute of Chemical Biology, Nashville, Tennessee, United States f Center for Matrix Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States g Division of Neonatology, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, United States k Vanderbilt Institute of Chemical Biology, Nashville, Tennessee, United States Contents lists available at ScienceDirect Contents lists available at ScienceDirect Contents lists available at ScienceDirect DOI of original article: http://dx.doi.org/10.1016/j.matbio.2016.09.005 n Corresponding authors. E-mail addresses: bc.paria@Vanderbilt.Edu (B.C. Paria), billy.hudson@Vanderbilt.Edu (B.G. Hudson). 1 These authors contributed equally to this work. Data in Brief 9 (2016) 931–939 Data in Brief 9 (2016) 931–939 http://dx.doi.org/10.1016/j.dib.2016.10.033 2352-3409/& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). a r t i c l e i n f o Article history: Received 6 September 2016 Received in revised form 7 October 2016 Accepted 31 October 2016 Available online 5 November 2016 Keyword: Basement membrane Basement membranes (BMs) are specialized extracellular scaffolds that provide architecture and modulate cell behaviors in tissues, such as fat, muscle, endothelium, endometrium, and decidua. Properties of BMs are maintained in homeostasis for most adult tissues. However, BM ultrastructure, composition, and localization are rapidly altered in select uterine tissues that are reprogrammed during pregnancy to enable early maternal-embryo interactions. Here, our data exhibit both static and dynamic BMs that were tracked in mouse uterine tissues during pre-, peri-, and postimplantation periods of pregnancy. The data exhibit spatial-temporal patterns of BM property regulation DOI of original article: http://dx.doi.org/10.1016/j.matbio.2016.09.005 n Corresponding authors. E-mail addresses: bc.paria@Vanderbilt.Edu (B.C. Paria), billy.hudson@Vanderbilt.Edu (B.G. Hudson 1 These authors contributed equally to this work. 932 C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 that coincide with the progression of adapted physiology. Further interpretation and discussion of these data in this article are descri- bed in the associated research article titled, “Embryo implantation triggers dynamic spatiotemporal expression of the basement mem- brane toolkit during uterine reprogramming” (C.R. Jones-Paris, S. Paria, T. Berg, J. Saus, G. Bhave, B.C. Paria, B.G. Hudson, 2016) [1]. & 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (h // /l /b / /) that coincide with the progression of adapted physiology. Further interpretation and discussion of these data in this article are descri- bed in the associated research article titled, “Embryo implantation triggers dynamic spatiotemporal expression of the basement mem- brane toolkit during uterine reprogramming” (C.R. Jones-Paris, S. Paria, T. Berg, J. Saus, G. Bhave, B.C. Paria, B.G. Hudson, 2016) [1]. & 2016 The Authors. Published by Elsevier Inc. Value of the data These data of H&E stainings and schematics are useful for researchers interested in tissue and cellular-level histological changes that occur in mouse uterine tissues during early pregnancy. The presented electron micrographs provide a context of cells and basement membrane for interpretation of cell–matrix and cell–cell orientation and interactions. The presented electron micrographs provide a context of cells and basement membrane for interpretation of cell–matrix and cell–cell orientation and interactions. The immunoﬂuorescence data include entire cross-sections through uterine tissues that allow comparative analysis of dynamic and homeostatic localization of basement membrane components during pregnancy. The immunoﬂuorescence data include entire cross-sections through uterine tissues that allow comparative analysis of dynamic and homeostatic localization of basement membrane components during pregnancy. g p g y The positive and negative control data provide evidence of immunoﬂuorescence detection speci- ﬁcity and are informative to researchers that apply the same antibodies in future experiments. The positive and negative control data provide evidence of immunoﬂuorescence detection speci- ﬁcity and are informative to researchers that apply the same antibodies in future experiments. Biology Matrix Biology during Uterine Physiology for Pregnancy Biology Matrix Biology during Uterine Physiology for Pregnancy Image (microscopy) Brightﬁeld microscope (Olympus Bx51 with DP72 camera), Transmission electron microscope (Philips/FEI T-12); High-resolution immunoﬂuorescence automated microscope (Leica Ariol SL-50 or Apiro Versa 200) Raw, processed Physiological responses to pregnancy; pseudopregnancy reaction; Collected uterine tissues that were processed for microscopy-based analysis Histological staining; electron micrographs; immunoﬂuorescence Nashville, Tennessee, United States Speciﬁcations Table Speciﬁcations Table a r t i c l e i n f o This is an open access article under the CC BY license (h // i /li /b /4 0/) Speciﬁcations Table Subject area Biology More speciﬁc subject area Matrix Biology during Uterine Physiology for Pregnancy Type of data Image (microscopy) How data was acquired Brightﬁeld microscope (Olympus Bx51 with DP72 camera), Transmission electron microscope (Philips/FEI T-12); High-resolution immunoﬂuorescence automated microscope (Leica Ariol SL-50 or Apiro Versa 200) Data format Raw, processed Experimental factors Physiological responses to pregnancy; pseudopregnancy reaction; Collected uterine tissues that were processed for microscopy-based analysis Experimental features Histological staining; electron micrographs; immunoﬂuorescence Data source location Nashville, Tennessee, United States Data accessibility Data is with this article. 1. Data The data in this article include hematoxylin and eosin (H&E) histological visualization of uterine tissue changes that naturally occur during early mouse pregnancy (Fig. 1). A schematic (Fig. 2) (included in part in [1]) is presented for anatomical spatial orientation of dynamic basement mem- branes (BMs) that is beneﬁcial in cross-examination of BM ultrastructure (Fig. 3) and localization of individual components: peroxidasin (Fig. 4); GPBP (Fig. 5); GPBP-1, a variant of GPBP that can be trafﬁcked to extracellular spaces (Fig. 6); and collagen IV and laminin (Fig. 7). 933 C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 2.1. Animals 2.1. Animals Healthy adult CD1 mice (Charles River Laboratories, Crl:CD1(ICR), Wilmington, MA, USA) were bred and the presence of a vaginal plug the following morning was established as day 1 of pregnancy [2]. Uterine horns were harvested on days 1 through 4 of pregnancy. Chicago Blue 6B dye (Sigma, St. Louis, MO) was used to distinguish implantation sites on day 5 of pregnancy with methods previously described [3]. Implantation sites from days 6 through 8 of pregnancy were collected by anatomical identiﬁcation. The deciduomata, a decidual reaction triggered by non-embryonic experimental sti- mulus, was induced by intraluminal injection sesame oil into the uterine horn on day 4 of pregnancy then tissues were collected 48 h post injection as described [4]. Uterine samples were collected in the mornings on designated days of pregnancy, snap frozen, and stored at 80 °C. All animal procedures were conducted in accordance with guidelines and standards of the Society for the Study of Repro- duction (SSR) and approved by Vanderbilt University's Institutional Animal Care and Use Committee (IACUC). 2. Materials and methods 2.1. Animals 2.1. Animals 2.2. Histochemical staining and Immunoﬂuorescence 2.2. Histochemical staining and Immunoﬂuorescence Uterine samples were embedded in Optimal Cutting Temperature (OCT) compound (Sakura Tissue-Tek, Torrance, CA), cryosectioned (Leica Biosystems, Buffalo Grove, IL), and collected onto slides (Fisher Scientiﬁc, Waltham, MA). Samples were processed and stained with hematoxylin and eosin (H&E) using routine methods at the Vanderbilt University Medical Center Translational Pathology Shared Resource (TPSR, Nashville, TN). Brieﬂy, samples were carefully acclimated to room temperature, washed with phosphate-buffered saline (PBS) for 3 min to remove OCT, ﬁxed with 10% neutral buffered formalin for 3 min, and rinsed with running water. Staining was carried out by incubating in hematoxylin and then eosin by dipping 15 times in each dye, rinsing with running water between dyes. Sections were then dehydrated in 3 exchanges of ethanol and 2 exchanges of xylene and mounted in non-aqueous media with a coverslip. p Cryosections processed for immunoﬂuorescence were air-dried and ﬁxed in 20 °C acetone (Sigma, St. Louis, MO) then were washed several times with phosphate-buffered saline (PBS) (Corning, Corning, NY) and PBS with 0.2% Tween (Sigma, St. Louis, MO). Nonspeciﬁc binding was blocked by Fig. 1. Histological examples of uterine tissue organization during early pregnancy. Brightﬁeld micrographs of H&E-stained mouse uterus during early pregnancy. Scale bar¼500 μm. Blue inset squares in A’–E’ are indicative of the ﬁeld of view in A”–E”. Asterisk (n) indicates the location of the embryo. Fig. 1. Histological examples of uterine tissue organization during early pregnancy. Brightﬁeld micrographs of H&E-stained mouse uterus during early pregnancy. Scale bar¼500 μm. Blue inset squares in A’–E’ are indicative of the ﬁeld of view in A”–E”. Asterisk (n) indicates the location of the embryo. C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 934 incubation of samples with 10% normal goat or horse serum (Invitrogen, Grand Island, NY). All anti- bodies were diluted in PBS/0.1% Tween with 5% goat serum. The following primary antibodies were applied to the sections: rat anti-collagen IV NC1 (1:500 dilution, JK2, were from Y. Sado, Shigei Medical Research Institute, Okayama, Japan [5,6]), rabbit anti-laminin (1:50 dilution for tissues not treated with dissociation buffer and 1:500 dilution for those that were pre-treated with dissociation buffer, ab11575, Abcam, Cambridge, MA), rabbit anti-peroxidasin (1:250 dilution, were from G. 2.2. Histochemical staining and Immunoﬂuorescence Bhave, Vanderbilt Uni- versity, Nashville, TN [7]), Alexa546 conjugated mouse anti-GPBP (1:50 dilution, mAb N26 to the N-terminal serine-rich domain conserved across all GPBP isoforms in several species [8], from Fibros- tatin, SL, Valencia, Spain), and Alexa546 conjugated mouse anti-GPBP-1 (1:50 dilution, mAb e11-2 to the 26-amino acid residue of exon 11 not present in GPBP-2, also called CERT [9], from Fibrostatin, SL, Valencia, Spain). A previously described dissociation buffer was used on sections co-stained with col- lagen IV antibody (JK2) [1,5] followed by several washes with PBS and PBS/0.2% Tween. Detection and co-detection with GPBP antibodies (mAbs N26 and e11-2) were performed without dissociation treatment. Sections were washed with PBS/0.2% Tween before applying the following secondary anti- bodies: Alexa555 goat anti-rat (1:200 dilution, ab150166, Abcam) and Alexa488 goat anti-rabbit (1:200 dilution, ab150081, Abcam). Nuclei were stained with Hoechst dye. Following several washes, sections were mounted in Prolong Gold (Life Technologies, Grand Island, NY). After mounting solution cured, sections were imaged with 20x objective on a automated microscope (Ariol SL-50 or Apiro Versa 200 Leica Biosystems, Buffalo Grove, IL) at the Vanderbilt Univeristy Medical Center Digital Histology Shared Resource (Nashville, TN). Consistent Linear adjustments were applied to brightness and contrast of images with settings established relative to representative controls. Fig. 2. Diagrams of uterine tissue anatomy and basement membrane arrangement during early pregnancy. A) Schematic of BM localization during tissue-level adapted physiology of uterus during early mouse pregnancy. (B) Schematic of BM localization during cell-level adapted physiology of uterus during early mouse pregnancy. PMT, plasma membrane transformation. Pre-, peri-, and postimplantation stage changes exempliﬁed and basement membranes are represented by pink patterns in (A) and (B). Fig. 2. Diagrams of uterine tissue anatomy and basement membrane arrangement during early pregnancy. A) Schematic of BM localization during tissue-level adapted physiology of uterus during early mouse pregnancy. (B) Schematic of BM localization during cell-level adapted physiology of uterus during early mouse pregnancy. PMT, plasma membrane transformation. Pre-, peri-, and postimplantation stage changes exempliﬁed and basement membranes are represented by pink patterns in (A) and (B). C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 935 Early pregnancy basement membrane ultrastructure in endometrium and decidua. Transmission electron m magniﬁcation micrographs of mouse uterus day 4–8 of pregnancy with pericellular lamina densa (basem represented in Fig. 1, uncropped. Arrows point toward regions of lamina densa. Fig. 3. 2.2. Histochemical staining and Immunoﬂuorescence Early pregnancy basement membrane ultrastructure in endometrium and decidua. Transmission electron microscopy 15000x magniﬁcation micrographs of mouse uterus day 4–8 of pregnancy with pericellular lamina densa (basement mem- brane) represented in Fig. 1, uncropped. Arrows point toward regions of lamina densa. C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 936 Fig. 4. Collagen IV and peroxidasin localization during early pregnancy. A) Full cross-section of mouse uterus double immu- noﬂuorescence of collagen IV and peroxidasin during early pregnancy with collagen IV in red, peroxidasin in green, and nuclei in blue. Co-localization of collagen IV and peroxidasin is indicated by orange/yellow color. Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls preimplantation (B) and postimplantation (C). Fig. 4. Collagen IV and peroxidasin localization during early pregnancy. A) Full cross-section of mouse uterus double immu- noﬂuorescence of collagen IV and peroxidasin during early pregnancy with collagen IV in red, peroxidasin in green, and nuclei in blue. Co-localization of collagen IV and peroxidasin is indicated by orange/yellow color. Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls preimplantation (B) and postimplantation (C). Fig. 5. Localization of GPBP during early pregnancy. A) Full cross-section of mouse uterus immunoﬂuorescence of GPBP during early pregnancy with GPBP in red and nuclei in blue. Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls preimplantation (B) and postimplantation (C). Fig. 5. Localization of GPBP during early pregnancy. A) Full cross-section of mouse uterus immunoﬂuorescence of GPBP during early pregnancy with GPBP in red and nuclei in blue. Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls preimplantation (B) and postimplantation (C). C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 937 Fig. 6. Isoform GPBP-1 and laminin localization during early pregnancy. A) Diagram depicting areas of GPBP isoforms that mAb N26 and e11-2 speciﬁcally bind. The N26 antibody recognizes all isoforms of GPBP while the e11-2 antibody detects the 26- amino acid region that is encoded by exon 11 (shown as the red block), which is not present in GPBP-2 also called GPBPΔ26 or CERT. B) Full cross-section of mouse uterus double immunoﬂuorescence of GPBP-1 and laminin during early pregnancy with GPBP-1 in red, laminin in green, and nuclei in blue. Colocalization of GPBP-1 and laminin is indicated by orange/yellow color. 2.3. Transmission Electron Microscopy (TEM) Fresh uterine tissues were ﬁxed in glutaraldehyde with sodium cacodylate. Then, samples were further processed in the Vanderbilt University Medical Center Cell Imaging Shared Resource. Brieﬂy, tissues underwent several exchanges of ﬁxative and dehydrating solvents. Samples were then inﬁl- trated gradually with epoxy resin (ER). Then samples were cured [10]. Thin sections were cut using an ultramicrotome (Leica ultracut, Buffalo Grove, IL) and then mounted onto copper grids. The sections were stained with uranyl acetate and Reynold lead citrate before they were imaged at 30,000x on an electron microscope (Philips T-12 equipped with an AMT CCD camera system, FEI, Hillsboro, OR). Further details of immunoﬂuorescence and electron microscopy methods are available in the accompanying research article [1]. 2.2. Histochemical staining and Immunoﬂuorescence Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls pre- implantation (C) and postimplantation (D). C.R. Jones Paris et al. / Data in Brief 9 (2016) 931 939 937 Fig. 6. Isoform GPBP-1 and laminin localization during early pregnancy. A) Diagram depicting areas of GPBP isoforms that mAb N26 and e11-2 speciﬁcally bind. The N26 antibody recognizes all isoforms of GPBP while the e11-2 antibody detects the 26- amino acid region that is encoded by exon 11 (shown as the red block), which is not present in GPBP-2 also called GPBPΔ26 or CERT. B) Full cross-section of mouse uterus double immunoﬂuorescence of GPBP-1 and laminin during early pregnancy with GPBP-1 in red, laminin in green, and nuclei in blue. Colocalization of GPBP-1 and laminin is indicated by orange/yellow color. Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls pre- implantation (C) and postimplantation (D). C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 938 Fig. 7. Collagen IV and laminin localization during early pregnancy. A) Full cross-section of mouse uterus double immuno- ﬂuorescence of collagen IV and laminin during early pregnancy with collagen IV in red, laminin in green, and nuclei in blue. Colocalization of collagen IV and laminin is indicated by orange/yellow color. Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls preimplantation (B) and postimplantation (C). Fig. 7. Collagen IV and laminin localization during early pregnancy. A) Full cross-section of mouse uterus double immuno- ﬂuorescence of collagen IV and laminin during early pregnancy with collagen IV in red, laminin in green, and nuclei in blue. Colocalization of collagen IV and laminin is indicated by orange/yellow color. Asterisk (n) indicates the location of the embryo. Scale bar¼500 μm. Representative immunoﬂuorescence controls preimplantation (B) and postimplantation (C). Conﬂict of interest The authors declare no conﬂicts of interest. 939 C.R. Jones-Paris et al. / Data in Brief 9 (2016) 931–939 Acknowledgements We acknowledge the efforts of Fernando Revert, Ph.D. for producing and characterizing antibodies (mAb N26 and mAb e11-2) at Fibrostatin, SL. This work was supported by NIH, United States Grants F31 CA165787-03 (to C.R.J.), R25 DK096999 (to B.G.H. for S.P. and T.B.), K08 DK097306 (to G.B.), R01 HD044741 (to B.C.P.), and R01 DK018381 (to B.G.H). This study was also supported by RT2014-2415-1 / Prometeo II -2014-048 (to J.S). References [1] C.R. Jones-Paris, S. Paria, T. Berg, J. Saus, G. Bhave, B.C. Paria, B.G. Hudson, Embryo implantation triggers dynamic spatiotemporal expression of the basement membrane toolkit during uterine reprogramming, Matrix Biol. (2016) (Sep 10; PMID: 27619726). [1] C.R. Jones-Paris, S. Paria, T. Berg, J. Saus, G. Bhave, B.C. Paria, B.G. Hudson, Embryo implantation triggers dynamic spatiotemporal expression of the basement membrane toolkit during uterine reprogramming, Matrix Biol. 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https://openalex.org/W2923218948	https://www.econstor.eu/bitstream/10419/245216/1/10.1080-23322039.2019.1595291.pdf	English	null	Determinants of livelihood strategies of fishing households in the volta Basin, Ghana	Cogent economics & finance	2,019	cc-by	9,454	Article Cogent Economics & Finance Provided in Cooperation with: Taylor & Francis Group Provided in Cooperation with: Taylor & Francis Group Suggested Citation: Amevenku, F. K. Y.; Asravor, R. K.; Kuwornu, John K. M. (2019) : Determinants of livelihood strategies of fishing households in the volta Basin, Ghana, Cogent Economics & Finance, ISSN 2332-2039, Taylor & Francis, Abingdon, Vol. 7, Iss. 1, pp. 1-15, https://doi.org/10.1080/23322039.2019.1595291 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Documents in EconStor may be saved and copied for your personal and scholarly purposes. 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Cogent Economics & Finance ISSN: (Print) 2332-2039 (Online) Journal homepage: https://www.tandfonline.com/loi/oaef20 Determinants of livelihood strategies of fishing households in the volta Basin, Ghana . K. Y. Amevenku, R. K. Asravor & J. K. M. Kuwornu \| Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=oaef20 F. K. Y. Amevenku, R. K. Asravor & J. K. M. Kuwornu \| To cite this article: F. K. Y. Amevenku, R. K. Asravor & J. K. M. Kuwornu \| (2019) Determinants of livelihood strategies of fishing households in the volta Basin, Ghana, Cogent Economics & Finance, 7:1, 1595291, DOI: 10.1080/23322039.2019.1595291 To link to this article: https://doi.org/10.1080/23322039.2019.1595291 © 2019 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. Published online: 06 Apr 2019. Submit your article to this journal Article views: 2133 View related articles View Crossmark data Citing articles: 4 View citing articles Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 ECONOMETRICS \| RESEARCH ARTICLE F. K. Y. Amevenku1, R. K. Asravor2* and J. K. M. Kuwornu3 F. K. Y. Amevenku1, R. K. Asravor2* and J. K. M. Kuwornu3 Received: 13 November 2018 Accepted: 07 March 2019 First Published: 01 April 2019 Corresponding author: R. K. Asravor, Ghana Technology University, Ghana E-mail: rkasravor@yahoo.com Reviewing editor: Xibin Zhang, Econometrics and Business Statistics, Monash University, Melbourne, Australia Additional information is available at the end of the article Received: 13 November 2018 Accepted: 07 March 2019 First Published: 01 April 2019 Abstract: The main objective of this study is to identify livelihood strategies of fishing households in response to prevalent vulnerabilities they face within the Volta Basin. Questionnaires were administered to fishery households in the Basin using the multistage sampling technique. A non-hierarchical k-means cluster analysis partitioned the households into four livelihood strategies on which the multinomial logit regression was performed. These four identified strategies are fishery only, fishery and farming, fishery and non-farm, and fishery, non-farming and farming. The results from the multinomial logit regression revealed that marital status of head of household, number of months of food shortage experienced by a household per year, access to credit, access to extension services, distance to regular markets and district capital as well as experience in fishery were the major determinants of livelihood strategies. Implications for policy include the need for public extension services and training to invigorate fishery households’ income. As majority of the fishery households combined fishing and non-fishing strategies, livelihood inter- vention programmes should prioritize improvement of the non-fishing activities and lead to opening other opportunities for rural development. This will take pressure off the fish stock by facilitating the regeneration of fish stock. © 2019 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. 1. Introduction Diverse livelihood portfolios are often viewed as an essential part of household economies in developing countries, especially rural economies. The relationship between fishing and liveli- hood diversification is important because fishing is an important component of rural liveli- hoods of households in the coastal areas. The Big Numbers Project (BNP) (2008) estimate that between 93 and 97 million of rural households in developing countries are either directly or indirectly involved in fishing or are into the processing and marketing of small-scale fisheries. Fishing is a highly gender-segregated profession, with men catching fresh fish, and women processing fish (Britwum, 2009). Despite the importance of fishery to the rural economy, steady increases in the number of rivers being dammed have been reported to affect the aquatic ecosystems that provide important environmental and socioeconomic benefits (Liu et al., 2013). Analyzing the role of natural resource-based occupational changes along the spectra of socioeconomic and infrastructure development in coastal communities in East African, Cinner and Bodin (2010) suggest that increases in household-level specialization in most occupational sectors, including fishing and farming. Cinner and Bodin (2010), further reported that at the community-level, rural coastal economies have diversified due to infra- structural and developmental changes. According to Prado, Seixas, and Berkes (2015), many coastal communities in developing countries have been going through complex dynamics of change relating to the degradation of ecosystems and changing government policies. Many of these policies severe consequences on the livelihood strategies of coastal residents. Prior to the construction of the dam on the Volta River in Ghana, Volta River supported sub- stantial local fishery especially in the Lower Volta Area. The Volta basin, as an ecosystem, func- tioned primarily for food production, supporting fishery, flood protection, water infiltration and groundwater recharge. An economic assessment by Lawson in 1963 showed that an estimated 1500 to 2000 women were involved in clam picking along the river between Tefle and Torgome. These women were earning an average income of approximately £100,000.00 (1963 British Pounds) annually (Lawson, 1968). However, the damming of the Volta River has led to the alterations of the livelihood strategy and income of coastal households. This has negatively affected inhabitants of the riparian communities in Ghana and their livelihoods. Shepherd, Kessy, Higgens, Scott, and Luvanda (2011) stated that the capacity to diversify livelihoods and income sources under such situations has become crucial for the survival of households. Subjects: Agricultural Development; Agricultural Economics; Fisheries Science Keywords: volta Basin; cluster analysis; k-means; livelihood strategy; diversification Keywords: volta Basin; cluster analysis; k-means; livelihood strategy; diversification PUBLIC INTEREST STATEMENT The major dams in Ghana have contributed in many ways to the social and economic develop- ment of inhabitants, but the resultant changes in the environment have led to losses of livelihoods. After five decades of existence of the first of these dams, this article describes the types and factors that have influenced livelihood strategies adopted by households in response to changes made to the original Volta River. The results show that many of the households combined fishing and non-fishing approaches for survival. The decision on what approach to choose was determined by variables such as marital status of head of household, number of months of food shortage experienced by a household per year, access to credit, access to extension services, distances to regular markets and district capital and number of years spent fishing. An appreciation of these variables could assist to guide future interventions and policies to construct dams on rivers. F. K. Y. Amevenku is a Senior Research Scientist at the Council for Scientific and Industrial Water Research Institute, Fisheries and Aquaculture Division, Ghana. He holds a PhD in Agricultural Economist and Policy. His research interest is in the Socioeconomics of Fisheries and Aquaculture, Economics of the Environment and Project Analysis. R. K. Asravor is a research consultant with special interested in agricultural, environmental and health economics. He holds a PhD in Agricultural Economist and a Master of Philosophy degree in economics. He is a research assistant in applied econometrics for PhD stu- dents at the University of Ghana, Department of Agricultural Economics and Agribusiness, College of Basic and Applied. g pp J. K. M. Kuwornu is an Associate Professor of Agribusiness Management in the Department of Food, Agriculture and Bioresources, School of Environment, Resources and Development, Asian Institute of Technology, Pathum Thani 12120, Thailand. © 2019 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. Page 1 of 15 Page 1 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 1. Introduction The postconstruc- tion habitat modifications and resource decline started after the coming to being of the Akosombo dam but worsened upon the formation of the Kpong dam. This manifesting changes in the ecosystems of the Lower Volta Area as diminished income of households and lead to losses of livelihood opportunities. The construction of these two dams have led to activities such as fuelwood harvesting, charcoal burning (Tonah, 2008), palm wine tapping, local gin distilling, mat weaving, sand winning (Obour, Owusu, Agyeman, Ahenkan, & Madrid, 2016) and stone quarrying in the Volta basin. These activities are extractive in nature and depended on locally available resources. The intensity of the exploitation of these extractive resources has raised questions of sustainability and resilience of livelihood strategies as they expose future generations to significant ecological risks and scarcities (Tonah, 2008; Tsikata, 2005). In the context of the Volta basin in Ghana, the over- dependence on fishing as a source of livelihood was fast depleting the natural resource and deepening poverty (Asante, 2006). Fabio et al. (2003a, 2003b) diagnostic study on the poverty profiling and the diversification strategies of households in the Volta Basin is the only studies on the entire international Volta Basin of Ghana. Page 2 of 15 Page 2 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 Within the interplay of the ecological changes occasioned by the creation of these two dams and its attendant livelihood loses, the pertinent questions worth asking are; ●Which sustainable livelihood strategies have evolved among fishery households? ●What principal factors influence the choice of those strategies? The broad objective of this paper, therefore, is to examine the livelihood strategies of fishery households within the Volta Basin in Ghana. The specific objectives are: The broad objective of this paper, therefore, is to examine the livelihood strategies of fishery households within the Volta Basin in Ghana. The specific objectives are: ●To identify the major livelihood strategies adopted by fishery households; ●To identify the major livelihood strategies adopted by fishery households; ●To examine the determinants of the choices of fishery households livelihood strategies. To examine the determinants of the choices of fishery households livelihood strategies. The remainder of the paper proceeds as follows: the next section discusses theoretical and empirical literature on livelihood diversification. This is followed by a narration of the sampling technique, study areas, data collection and analytical methods adopted. 1. Introduction Section three is a presentation of the detail results gathered from the analyzed data. The discussions of the results with reference to the literature presented are also made in this section. The last section of the study presents the conclusions and recommendations of the study. 2. Brief literature review While empirical studies on diversification may be widely applicable in the economic and socio- cultural contexts, results from empirical studies differed between the different regions of the world. In spite of this, it is generally agreed in the empirical literature that the purpose of diversification is to develop portfolios of income generating activities with low covariate risk (Asravor, 2017; Ellis, 2000). The literature also shows that numerous factors affect the diversifica- tion strategies of household in agriculture and aquaculture but in a general context, these factors can be categorized into “pull” (positive) or “push” (negative) factors (Brugère, Holvoet, & Allison, 2008; Ellis, 2000). According to Asravor (2017), the pull factors are favorable factors or opportu- nity-led and leads to diversification of livelihood strategies, whereas push factors are survival-led or harsh conditions that force farm households to diversify their income activities off their main income generating activity. Empirical studies devoted to the diversification of fishery households are limited as many studies have focused on crop and livestock farmers (Asravor, 2017; Eneyew, 2012). Martin, Lorenzen, and Bunnefeld (2013) examined the relationship between fishing, livelihood diversifi- cation and poverty in the lower Mekong basin, in Laos. From their analysis of the household survey data, Martin et al. (2013) found that participation in fishing is common and positively associated with higher occupational diversity and more agricultural activities. Additionally, Martin et al. (2013) reported that alternative livelihoods within the rural setting are unlikely to cause fishers to leave fishery, but instead strengthen the livelihood portfolio as a supplementary activity. Fishing forms a greater proportion of income, employment and food security for the poor in the coastal areas. Cinner, McClanahan, and Wamukota (2010) posited that household livelihoods in tropical coastal communities are not dependent on a single livelihood strategy but often a multiplicity of occupa- tional sectors, such as agriculture, fisheries, and informal economic activities. These non-fishing and agricultural economic activities include small shops, transportation, and mechanic work among others. To understand the diversification of smallholder farmers’ in Ghana, Asravor (2017) employed the Margalef index of diversiﬁcation. The results revealed that households had diversiﬁed on their farm and in informal economic activities. It also showed that social capital (dependency ratio, marital status), and human capital (extension services) significantly affected the type of diversification strategy practiced. Page 3 of 15 Page 3 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 3.1. Study area The study was undertaken in selected coastal administrative districts in Ghana. These selected districts are located in the segments labeled LV1, LV2 and LV3 included North, Central and South Tongu, Ada East, Asuogyaman, and Lower Manya (Figure 1). In addition, the Pru district in the Brong Ahafo region, which constitute Stratum VII was also sampled for this study (Figure 1). The characteristic of the study area is presented in Table 1. 3.2. Sampling technique The multistage sampling technique was employed to sample the fishing households. The Volta Basin encompasses four administrative regions, that is, the Greater Accra, Volta, Eastern regions and Brong Ahafo region. The first stage of the multistage sampling involved the selections of districts in these regions. The districts were purposively selected because they were known to have many communities and households involve in fishery, hence are affected by any activities along the Volta Basins. After purposively selecting these districts, the second stage involved the use of the simple random sampling technique to select the various com- munities that can be found in these selected districts. To sample these communities, District Assemblies were first contacted for the list of all the communities that fall within each of these districts. At the community level, all the households within each community were numbered. The “sample” command in R was then employed to select households that were administered with the questionnaire. Before the administration of the questionnaire to each household, opinions and community leaders were each informed about the survey and permission were sought. On average 2 h was spent administering the questionnaire to each household. In all, the 802 households were selected for the study. 6°00'N 6°30'N Agotaga 0°00' Kpong Old Ningo 0°30'E 0 5 Akplabanya Ada Big Ada Agave 1°00'E 10 Wuti Atiteti Anyanui Anloga Agodome Volivo Akosombo Amedeka Kpong Senchi Asutsuare Adomi Gyakiti Ajena Bator Sokpoe Bakpo Tefle Adidome Sogakope LEGEND River / Stream Road Town Bridge Sampling Point 15 20km Scale Abotia C O T E D' I V OI R E 10°00'N 11°00'N B U R K I N A F A S O 0 20 80 SCALE 40 60 T O G O 5°30'N LOWER VOLTA Mepe (LV1) (LV2) (LV3) 9° 8° 7° 6° 5° 1° 0° 1° 0 100 Km. I II III IV V VI VII VIII N THE VOLTA LAKE AND ITS MAJOR TRIBUTRIES IN GHANA e 1. Study Areas: LV1, LV2, nd the principal strata of olta Lake. ce: CSIR Water Research tute, Accra Page 4 of 15 THE VOLTA LAKE AND ITS MAJOR TRIBUTRIES IN GHANA Figure 1. Study Areas: LV1, LV2, LV3 and the principal strata of the Volta Lake. Figure 1. Study Areas: LV1, LV2, LV3 and the principal strata of the Volta Lake. Figure 1. Study Areas: LV1, LV2, LV3 and the principal strata of the Volta Lake. 3.2. Sampling technique 6°00'N 6°30'N Agotaga 0°00' Kpong Old Ningo 0°30'E 0 5 Akplabanya Ada Big Ada Agave 1°00'E 10 Wuti Atiteti Anyanui Anloga Agodome Volivo Akosombo Amedeka Kpong Senchi Asutsuare Adomi Gyakiti Ajena Bator Sokpoe Bakpo Tefle Adidome Sogakope LEGEND River / Stream Road Town Bridge Sampling Point 15 20km Scale Abotia C O T E D' I V OI R E 10°00'N 11°00'N B U R K I N A F A S O 0 20 80 SCALE 40 60 T O G O 5°30'N LOWER VOLTA Mepe (LV1) (LV2) (LV3) 9° 8° 7° 6° 5° 1° 0° 1° 0 100 Km. 3.2. Sampling technique I II III IV V VI VII VIII N THE VOLTA LAKE AND ITS MAJOR TRIBUTRIES IN GHANA LV1, LV2, trata of search 6°00'N 6°30'N Agotaga 0°00' Kpong Old Ningo 0°30'E 0 5 Akplabanya Ada Big Ada Agave 1°00'E 10 Wuti Atiteti Anyanui Anloga Agodome Volivo Akosombo Amedeka Kpong Senchi Asutsuare Adomi Gyakiti Ajena Bator Sokpoe Bakpo Tefle Adidome Sogakope LEGEND River / Stream Road Town Bridge Sampling Point 15 20km Scale Abotia C O T E D' I V OI R E 10°00'N 11°00'N B U R K I N A F A S O 0 20 80 SCALE 40 60 T O G O 5°30'N LOWER VOLTA Mepe (LV1) (LV2) (LV3) Source: CSIR Water Research Institute, Accra Page 4 of 15 Page 4 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 Segment Characteristics LV1 (Lower Volta Area I) Formation of sandbar at the estuary limiting inflow of seawater into the river at high tide; Reduction of floodplain agriculture; Explosion of aquatic weeds that blocked fishing grounds; Infestation of creeks, streams and main fishing channels by aquatic weeds; LV2 Lower Volta Area II Collapse of floodplain farming; Cessation of influx of seawater at high tide leading to collapse of shell fish fishery; Trapped between the 2 Volta dams; Receives limited inflow of seawater; LV3 Lower Volta Area III Experiences reduced water flow (speed) in the main channel; Collapsed flood plain agriculture; Weed-choked creeks, streams and main channel limiting extent of fishing; Constitutes the most riverine segment of the Volta Lake; Stratum VII Under the influence of large inflows thus, providing large volumes of water and fish; Constitutes the hub of fishery on the Volta Lake; Benefited from a number of national and international interventions aimed at improving livelihoods of households Sources: Institute of Aquatic Biology [IAB] (1995); Petr (1974); Fisheries Department (1995) and Henle and Eckert (1970); FAO (2008). 3.4.1. Diversiﬁcation measure The income-based approach is the main basis for the analysis of identifying household livelihood strategies (Barrett, Bezuneh, Clay, & Reardon, 2005; Brown, Stephens, Ouma, Murithi, & Barrett, 2006). Specifically, the k-means Cluster Analysis procedure was employed in this study. The k-means approach used the Euclidean measure to determine dissimilarities, that is, the distance between the final cluster points. It further assigned each observation (xi) to only one cluster (Marzban & Sandgathe, 2005). Thus, more succinctly, given a set of observation xi; x2; . . . :; xn ð Þ with each observation being a d-dimensional of a real vector, the purpose of k-means clustering is to partition the sampled observations (n) into k (≤n) sets f ¼ fi; f2; . . . :; fk ð Þ in order to minimize the variance or within-cluster sum of squares (WCSS). Thus, the within-cluster is written as: argfmin ∑ k i¼1 ∑ x2fi k x μi k2¼ argfmin ∑ k i¼1 fij jVar fi (1) (1) where the mean of points in fi is given by μi and is equivalent to the minimization of the pairwise squared deviations of points in the same cluster: where the mean of points in fi is given by μi and is equivalent to the minimization of the pairwise squared deviations of points in the same cluster: argfmin ∑ k i¼1 1 2 fij j ∑ x;y2fi x y k k2 (2) argfmin ∑ k i¼1 1 2 fij j ∑ x;y2fi x y k k2 (2) Using the identity of equation (3) we deduce the equivalence. From the law of total variance, where the total variance is assumed to be a constant equation (3) and is equivalent to maximizing the sum of squared deviations between points in different clusters (between-cluster sum of squares). (3) ∑x2fi x μi k k2 ¼ ∑xy2f x μi ð Þ μi y ð Þ ∑x2fi x μi k k2 ¼ ∑xy2f x μi ð Þ μi y ð Þ (3) The choice of k-means over the wards and hierarchical clustering approaches was because the k-means approach helped the researcher identify strategies which are “similar” and are “dissim- ilar” to the other strategies belonging to other clusters. Thus, the k-mean approach enabled the researcher to segregate groups with similar traits (livelihood strategies) and assign them into clusters (strategy). Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 3.3. Data collection A total of 802 household heads constituted the respondents from which data was obtained through the administration of a structured pretested set of questionnaires from July 2015 to December 2015 in the study area. However, after data cleaning, during which nonresponses and unrelated feedbacks were eliminated, 715 respondents became the sample on which the analysis was based.1 3.4. Estimated model In this study, analytical technique and model utilized are presented in this section. The analytical technique employed to measure the diversification strategies of the fishery households was the k-means while the multinomial logit model was used to examine the determinants of the fishery household diversification strategies. 3.4.1. Diversiﬁcation measure The k-means was used in this study because it is conceptually simple and is computationally fast. According to Kaushik and Mathur (2014), there is no clear evidence that any other clustering algorithm performs better in k-means general as it has the advantage of cluster- ing large data sets with its performance increasing as the number of clusters increases. 3.2. Sampling technique Segment Characteristics LV1 (Lower Volta Area I) Formation of sandbar at the estuary limiting inflow of seawater into the river at high tide; Reduction of floodplain agriculture; Explosion of aquatic weeds that blocked fishing grounds; Infestation of creeks, streams and main fishing channels by aquatic weeds; LV2 Lower Volta Area II Collapse of floodplain farming; Cessation of influx of seawater at high tide leading to collapse of shell fish fishery; Trapped between the 2 Volta dams; Receives limited inflow of seawater; LV3 Lower Volta Area III Experiences reduced water flow (speed) in the main channel; Collapsed flood plain agriculture; Weed-choked creeks, streams and main channel limiting extent of fishing; Constitutes the most riverine segment of the Volta Lake; Stratum VII Under the influence of large inflows thus, providing large volumes of water and fish; Constitutes the hub of fishery on the Volta Lake; Benefited from a number of national and international interventions aimed at improving livelihoods of households Sources: Institute of Aquatic Biology [IAB] (1995); Petr (1974); Fisheries Department (1995) and Henle and Eckert (1970); FAO (2008). Page 5 of 15 3.4.2. Determinant of diversification 3.4.2. Determinant of diversification To identify the determinants of rural household’s decision to engage in various livelihood strate- gies, the researchers assumed that a rational household would choose among mutually exclusive livelihood strategy alternatives that offered them the maximum utility (Eneyew, 2012; Eneyew & Bekele, 2012). From the random utility argument, fishery households’ decision to choose a livelihood strategy is categorized as a function of a set of livelihood strategies. Given the four- Page 6 of 15 Page 6 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 alternative choices (fishery only; fishery and farming; fishery and non-farming; and fishery, farming and non-farming), we applied the multinomial logit model to estimate the livelihood choice with discrete dependent variable. alternative choices (fishery only; fishery and farming; fishery and non-farming; and fishery, farming and non-farming), we applied the multinomial logit model to estimate the livelihood choice with discrete dependent variable. Given the random utility model (RUM), we assume that the decision-maker (fishers) choose from a set of mutually exclusive alternatives j ¼ 1; 2; 3; . . . ; J and obtains a certain level of utility Uij from each alternative. Since we do not observe the decision makers’ utility, but observe some attributes of the alternatives faced by households, then the utility is decomposed into determinis- tic ðVijÞ and random (εijÞ) part: Uij ¼ Vij þ εij (4) A fisher selects livelihood strategy U ¼ 1 if Uik > Uk (5) Uij ¼ Vij þ εij (4) A fisher selects livelihood strategy U ¼ 1 if (5) Uik > Uk Where Uik denotes a random utility associated with the livelihood strategy j ¼ k, and Vik is an index function denoting the fishers’ average utility linked with this alternative. The second term εik is a random error which is specific to a producer’s utility preference (McFadden, 1974). The livelihood strategies modeled is stated as: Lij ¼ βjXij þ εij (6) (6) Lij ¼ βjXij þ εij where Lij is a vector of the livelihood choices j ¼ 1 for fishery only; 2 for fishery and farming; 3 for fishery and non-farming; and 4 for fishery, farming and non-farming of ith fisher, βj is a vector of channel-specific parameters. εij is the error term assumed to be normally distributed (mean 0 and variance 1) and Xij is a vector of fishers’ characteristics. 3.4.2. Determinant of diversification where Lij is a vector of the livelihood choices j ¼ 1 for fishery only; 2 for fishery and farming; 3 for fishery and non-farming; and 4 for fishery, farming and non-farming of ith fisher, βj is a vector of channel-specific parameters. εij is the error term assumed to be normally distributed (mean 0 and variance 1) and Xij is a vector of fishers’ characteristics. If we make Y to be the unordered categorical dependent variable that takes on a value of zero or one for each of the J choices, then the general multinomial logit model is given as: Pr Yi ¼ j ð Þ ¼ exp β0 jXi ∑j j¼0exp β0 jXi for J ¼ 1; 2; 3 Pr Yi ¼ j ð Þ ¼ exp β0 jXi ∑j j¼0exp β0 jXi for J ¼ 1; 2; 3 (7) (7) Where: Pr Yi ¼ j ð Þ is the probability of choosing either fishing only, fishing and farming, fishing and non- farming, and fishing, farming and non-farming; j is the number of livelihood activities engaged in; Xi is a vector of explanatory factors conditioning the choice of the jth alternatives; β is a vector of the estimated parameters. The estimated equations provide a set of probabilities for the j þ 1 choice restricted for a decision- maker with characteristics. In order to remove an indeterminacy in the model, a convenient normal- ization that solves the problem is β0 ¼ 0. Therefore, one can define the general form of the probability that individual ith choose the alternative jth in the following way: Pr Yi ¼ j=Xi ð Þ ¼ exp β0 jXi 1 þ ∑j j¼0exp β0 jXi for all J > 0 (8) (8) The MNL coefficients are difficult to interpret and associating the βj with the j outcome is tempting and misleading. To interpret the effects of explanatory variables on the probabilities, marginal effects are usually used and derived as (Greene, 2003): The MNL coefficients are difficult to interpret and associating the βj with the j outcome is tempting and misleading. To interpret the effects of explanatory variables on the probabilities, marginal effects are usually used and derived as (Greene, 2003): θj ¼ @Pj @Xj ¼ Pj βj ∑ j j¼1 Pjβj " # ¼ pj βj β h i (9) θj ¼ @Pj @Xj ¼ Pj βj ∑ j j¼1 Pjβj " # ¼ pj βj β h i (9) Page 7 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 The marginal effects measure the expected change in the probability of a specific outcome being made with respect to a unit change in an explanatory variable (Greene, 2003). The marginal effects measure the expected change in the probability of a specific outcome being made with respect to a unit change in an explanatory variable (Greene, 2003). The choice of the multinomial logit model over the ordered logit and the mixed logit or the probit or logit model is because the predictor variables are individual speciﬁc, and the choices facing the household are unordered. STATA version 13 was the statistical package used to analyze the data gathered from the survey. Sources: Authors computation (2016). 3.5. Definitions of variables Table 2 presents the deﬁnition of variables used in the estimation of the multinomial logit model. In terms of the socioeconomic characteristics of the households, the statistics shows that approxi- mately 79.2% of the household heads were married. The household with the minimum members had the household size of three (3) persons whereas the highest household size was nine (9) persons. The number of dependents within the sampled households ranges from 0 to 8. This is an indication that there were households without any dependent. The human capitals or assets of the sampled study showed that the average fishing experience of the fishing households in the study area was 21 years while the minimum experience was 9 years and the maximum fishing experience was 33 years. Many of the household heads do not have any formal education (69.4%). The summary of the explanatory variable on agricultural extension services shows that very few of the fisher households had access to agricultural extension service. Table 2. Socio-demographic and economic characteristics the fishing households Explanatory variables Descriptions Social capitals/assets: Marital status 1 = Married, 0 = otherwise 0.792 Household size Number of persons in the households 6.0 ± 3.31 Dependency ratio Level of dependency on household workforce 1.0 ± 0.90 Human capitals/assets: Fishing experience (HH) Years of fishing 21.0 ± 12.43 Formal education (HH) 1 = Formal education, 0 = otherwise 0.694 Extension services 1 = Access to extension service, 0 = otherwise 0.021 Financial capitals/assets: Receipt of remittances 1 = Receives remittance, 0 = otherwise 0.07 Access to credit 1 = Access to credit, 0 = otherwise 0.84 Natural capitals/assets Fishing seasons 1 = distinct number of fishing seasons, 0 = otherwise 0.161 Food shortage Number of months of food shortage 3.84 ± 1.13 Physical capitals/assets Distance to regular fishing site Kilometres 2.77 ± 1.94 Distance to regular market Kilometres 6.54 ± 3.78 Value of fishing assets Value in Ghana Cedis 5,444.00 ± 4,767.99 Distance to district capital Kilometres 9.13 ± 4.31 Sources: Authors computation (2016). Page 8 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 The analysis in terms of financial capital or asset showed that most fisher households had access to credit (84%) for their fishing activities. Very few expressed receiving remittances (7%) from household members or relatives leaving in the big cities. 3.5. Definitions of variables The natural capitals/assets indicated a fishing regime (season) with no distinct number of fishing seasons. An indication that fisher households have an all-year-round fishing activity. On average, three months of food shortage was experience within a year by respondents. The distance to the regular fishing site, regular market and district capital averaged 2.77, 6.54 and 9.13 km respectively. In addition, physical capital or asset showed that the value of fishing asset owned by the household averaged GHC 5,444.00 but range between 5,444.00 ± 4,767.99. The continuous variables of all the binary variables reported in Table 2 were also collected during the data collection period. Table 32 shows some of the analysis. The empty spaces imply that households did not provide the researcher with the needed information. Table 3 shows that on average the entire Volta Basin recorded 3 days of extension visit, an average of GHc100 remittance and GHc120 worth of credits. Table 3 shows that majority of the responding house- holds did not provide details of the amount of money received in the form of remittance or credit. For majority of households visited, these, were not applicable or they could not recall the amount received. 4.2. Choice of livelihood strategy in the volta basin Table 5. presents the result of the choice of livelihood strategies in the Volta Basin. The result indicated that 14 of the explanatory variables were significant at 1%, 5% and 10% significant levels. Overall, the diagnostics statistics shows that the model was statistically significant at 1% while the pseudo R-square show that 17% of the variation in the dependent variables was explained by the changes is the independent variables. The base strategy for the determinant of livelihood diversification strategies was fishery, non- farm and farming. Detail results presented in Table 4 show that compared to households in the base category, household heads who were married decreased their participation in fishery only strategy by 19%, whereas married household heads increased their participation in fishery and non-farming strategies by 25%. The coefficient of household size was positive for fishery and farming. This implies that com- pared to households in the base category, an increase in household size is more likely to increase the probability of engaging in fishery and farming strategies by 0.8% at the 5% level of signifi- cance. The study also reports that compared to the base category having access to credit significantly reduce the participation of households in fishery strategy by 31%. Table 5 also shows that compared to the base category, an increase in the dependency ratio significantly leads to a 3% reduction in the adoption of fishery and farming strategies. Receipt of remittances from member of the households living outside the community was found to adverse impact in the adoption of fishery plus farming strategies by approximately 7%. Compared to the base category, an increase in the number of fishing seasons positively affected the participation in fishery only but negatively affected the participation in fishery and non-farming livelihood strategy. Thus, the finding shows that a month increase in the fishing season leads to a 24% increase in the fishery only livelihood strategy and a 24% decrease in fishery plus non- farming livelihood strategy of households in the Volta basin. The distance to the regular market center and also the distance to the regular fishing site were significant and directly influence the participation in fishery plus farming strategy and fishery plus non-farming strategy by 0.08% and 0.36%, respectively. The availability of extension services had a direct relationship on diversification into fishery plus farming (96%) and fishery plus non-farming plus farming activities (46%). 4.2. Choice of livelihood strategy in the volta basin The finding also shows that compared to the base category, an increase in the value of fishery assets will lead to a decrease in the participation of fishery only as a livelihood diversification strategy but increase the participation in fishery and non-farming livelihood diversification strategy. 4.1. Livelihood strategies Table 4 presents the livelihood strategies identified through non-hierarchal (k-means) cluster analysis for the Volta Basin, Ghana. The results show that most of the household in the Volta Basin are engaged in fishery and non-farming (56.81%) The least livelihood diversification strategy identified in this study was fishery and farming (8.20). The farming practices of households are the cultivation of crop and the rearing of livestock and Table 3. Continuous variables of the Dummy responses Volta Basin LV1 LV2 LV3 Stratum7 Number of extension visit (days) 3 2 1 Average remittances received (GHc) 100 100 Amount received as credit (GHc) 120 120 2These were dropped by Stata during the course of the analysis hence we settled on using the dummy responses 2These were dropped by Stata during the course of the analysis, hence we settled on using the dummy responses. Table 4. Livelihood Strategies identified through Non-hierarchal (k-means) cluster analysis for the Volta Basin, Ghana Cluster 1 2 3 4 Name of strategy Fishery only Fishery and Farming Fishery and Non-farming Fishery, Farming and Non-farming Number of HH 198 56 388 41 % of HH 28.99% 8.20% 56.81% 6.00% Source: Authors compilation (2016). Page 9 of 15 Page 9 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 poultry birds. The common crops cultivated by these households are maize and cassava while the common livestock reared were sheep, goats and pigs. The main poultry birds kept were chicken while a few kept ducks and guinea fowls. These farming practices were undertaken on smallholder bases, and most farm produce are kept for consumption purposes. There were fewer cases where farm produce was sold in the market for cash. The non-farm livelihood activities engage in by the households were generally petty trading. Women were mostly involved in the sale of food stuffs and toiletries while men were mainly mechanics and carpenters. 5. Discussion The households studied can be said to be diversified in their livelihood activities. This corresponds with the assertion by Smith, Nguyen Khoa, and Lorenzen (2005) and Martin et al. (2013) that Page 10 of 15 Page 10 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 Note: The marginal effects are defined by the dy/dx values and , , * are, respectively, significant at <1%, 5% and 10% probability level. 3 The base strategy is Fishery, non-farm and farming. 4Though 715 observations were used for the MLR, some observation were dropped hence 683 observations were used for the final estimation. e 11 of 15 Table 5. 5. Discussion Estimates of choice of livelihood strategy by fishery households of the Volta basin of Ghana Strategy3 Fishery only Fishery and farming Fishery and non-farming Explanatory variables Coefficients Marginal effects Coeffients Marginal effects Coefficient Marginal effects Marital status 1.0592 (0.5227) −0.1859 (0.0826) 0.0616 (0.1838) −0.0322 (0.0454 1.0592 (0.229)* 0.2471 (0.0686)* Formal education (HH) −0.1126 (0.0991) −0.0350 (0.25838) −0.0653 (0.2537) −0.0014 (0.0065) −0.3781 (0.6021) −0.1348 (0.0998) Household size 0.4929* (0.1834) −0.2172 (0.31880) 0.1216 (0.0357)* 0.0084 (0.0027)*) 0.3252 (0.4030) 0.0115 (0.10377) Dependency ratio −0.2289 (0.2519) −0.1976 (0.2829) 0.3493 (0.1614) −0.0269 (0.0124)* −0.0658 (0.0452) −0.0826 (0.1218) Access to credit 15.7478 (4.6684)* −0.3067 (0.0105)* 0.0136 (0.0924) −0.3807 (0.2664) 0.0119* (0.0066) 0.0756 (0.1093) Number of months of food shortage 0.2137 (0.0947) −0.0476 (0.0159) −0.063 (0.1007) −0.3058 (0.2553) −0.0149 (0.0153) −0.0811 (0.1116) Receipt of remittances −0.1977 (0.2887) −0.0060 (0.0063) 1.3602 (0.6712)* −0.0668 (0.0214) −0.3502 (0.2685) −0.0146 (0.0334) Number of fishing seasons 1.1763 (0.3919) 0.2369 (0.0870) 0.4355 (0.5911) 0.0063 (0.0087) 1.1763 (0.3952) −0.2383 (0.0967) Distance to regular fishing site 0.1840 (0.0616) −0.0348 (0.0122) −0.5003 (0.3885) −0.0364 (0.0874) 0.1840 (0.0620) 0.0036 (0.0013) Distance to regular market 0.0028 (0.0069) 0.0889 (0.10166) 0.1127 (0.0424) 0.0084 (0.0016)*** 0.0011 (0.0028) 0.0022 (0.0035) Value of fishing assets −0.00003 (1.45E-05)* −7.10e-06 (3.28E-06)* 0.0209 (0.0362) 0.0233 (0.06972) 0.00003 (1.2E-05) 4.16e-06 (1.27E-06) Fishing experience (HH) −0.031 (0.0123) −0.0058 (0.0023) −0.0457 (0.0340) −0.0242 (0.097) 0.0310 (0.0107) 0.00003 (9.38E-06) Distance to district capital 0.1717* (0.0867) 0.0267 (0.01827) 0.0349 (0.0161)* 0.0024 (0.0012)* 0.0058 (0.0354) −0.1337 (0.0863) Access to extension services 0.0534 (0.0902) 0.0047 (0.0027) 17.9287 (3.9322)* 0.9617 (0.2136)* −0.0232 (0.0384) −0.10777 (0.0860) Wald Chi2 3749.54* 3749.54* 248.63* Pseudo R2 0.1702 0.1702 0.1702 Prob>Chi2 = 0.0000 Number of observations4 = 683 Note: The marginal effects are defined by the dy/dx values and , , are, respectively, significant at <1%, 5% and 10% probability level. 3 The base strategy is Fishery, non-farm and farming. 4Though 715 observations were used for the MLR, some observation were dropped hence 683 observations were used for the final estimation. Page 11 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 specialization in fisheries rarely happens in developing countries and majority of fishery house- holds diversified their livelihood strategy. specialization in fisheries rarely happens in developing countries and majority of fishery house- holds diversified their livelihood strategy. Marital status is an important determinant of the diversification behavior of household heads. 5. Discussion Marriage is usually accompanied by numerous responsibilities, especially child care and the pay- ment of bills. Household heads will, therefore, diversify their income, hence the decrease in the reliance of fishery only to the diversification into fishery plus non-farming strategy. As indicated by Asravor (2017) farming is seen as a very risky business, hence many farm households diversify their farming activities away from farming by entering into non-farm. Similarly, it could be that the households studied were avoiding the riskiness of relying on fishery alone by diversifying their activities. Those with formal education were better enlightened to efficiently manage their activities. In developing countries, such as Ghana, increasing household size is associated with more labor for agriculture activities. Households with more family labor are more diversified than those with very little labor (Asravor, 2017). In the fishing communities, diversification in both fishery and farming strategies are paramount to larger households since it provides these households with alternative sources of income and consumption of balanced diets (protein and carbohydrate diet). The higher dependency ratio is associated with a lower level of participation in fishery livelihood strategy. The plausible explanation was that an increase in dependency ratio increased the number of household members within the bracket (less than 18 and greater than 65 years) were not able to either farm or fish or do both. Additionally, fishers who had access to credit reduced their reliance on fishery only strategy. Access to financial credit has been documented to lead to diversification (Jansen, Damon, Pender, Wielemaker, & Schipper, 2003) among rural households. With access to credit, households have basic capital to diversify into either farming or non-farming business or both. Jansen et al. (2003) argue that households with large access to credit promote nonfarm activities rather than increased investments in inputs for their basic or main fishery livelihood activity. Extension services significantly influenced the diversification strategies of households. This finding was confirmed by Asravor (2017). Extension services gave the fisher folks the knowledge, training and skills needed to either diversify or specialized their livelihood activities. In most cases, fisher households with access to agricultural extension service have a higher tendency to diversify their farming activities than those with very little access to extension services. 5. Discussion The empirical findings of this study broadly confirmed the empirical literature, which posits that despite the risk involved in fishing, households located in coastal areas of developing countries preferred fishing (Cinner, Daw, & McClanahan, 2009). The importance of fisheries to the small-scale families is that being a safety net and its contributions to pro-poor growth. From the random utility maximization perspective, we argue that fishery households’ decision to choose a particular livelihood strategy is a function of the set of livelihood choice available. The choice made by the household is, therefore, based on the maximum satisfaction that the head of household derives (in terms of the income generated) from the livelihood activity(ies) subject to a set of constraints that he or she faces. This largely confirms the assertion of Eneyew and Bekele (2012) and Eneyew (2012). 6. Conclusions and recommendations This study undertook an appraisal of the livelihood strategies of households in the Volta Basin of Ghana. The study concludes that although fishing is risky and laborious venture, it remains the major occupa- tions preferred by households in the Volta Basin of Ghana. Our study concludes that the riskiness of fishing had led many fishing households in the Volta Basin to diversified their livelihood activities. in their livelihood strategies, although fishery activities remained central in the composition of all strategies. The four main livelihood strategies employed by the fishery households are fishery only, fishery plus farming, fishery plus non-farming and fishery plus farming plus non-farming. The study concludes that most Page 12 of 15 Page 12 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 households in the Volta Basin engage in an additional livelihood activity apart fishery. Fishery plus non- farming was the dominant strategy employed whereas the least strategy used was fishery, farming and non-farming. On the aggregated, the decision to adopt a particular strategy was mostly influenced by the demographic characteristics of the households. In spite of this, the human capital, financial capital, and natural capital also play important roles in the livelihood diversification strategy of households. In details, the conclusion is that the marriage household heads, household heads with larger family size and a higher number of dependents were more diversified. Furthermore, livelihood diversification strategies were significantly influenced by access to credit, food insecurity, remittances, fishing season, distances to fishery activity grounds and markets, value of fishery assets, experience of head of house- hold, distance to district capital, and extension contact. In the sub aggregated Lower Volta south of the Akosombo dam, again marital and formal educational statuses of the head of household, household size, dependency ratio, access to credit, distance to fishery activity grounds, distance to markets; experience of head of household and extension contact. Based on these conclusions, we recommend that livelihood intervention programmes should prioritize the improvement of non-fishing activities as it led to the opening of other opportunities for rural development and take pressure off the fish stock to facilitate regeneration. To be successful, however, support in the form of credit and training must go together if alternative income sources of the house- holds are to be relied upon. Author details F. K. Y. Amevenku1 E-mail: fykamevenku@gmail.com ORCID ID: http://orcid.org/0000-0003-1652-9948 R. K. Asravor2 E-mail: rkasravor@yahoo.com ORCID ID: http://orcid.org/0000-0003-1652-9948 J. K. M. Kuwornu3 E-mail: jkuwornu@gmail.com 1 Council for Scientific and Industrial Research – Water Research Institute, Accra. 2 Department of Agricultural Economics & Agribusiness, School of Agriculture, College of Basic & Applied Sciences, University of Ghana, Legon. 3 Agribusiness Management/Agricultural Systems and Engineering, School of Environment, Resources and Development, Asian Institute of Technology, Pathum Thani 12120, Thailand. References Asante, F. A. (2006). Socioeconomics of fisheries depen- dent communities in the Volta Basin of Ghana. International Journal of Ecology and Environmental Sciences, 32(1), 127–132. Asravor, R. K. (2018). Livelihood diversification strategies to climate change among smallholder farmers in Northern Ghana. Journal of International Development, 30(8), 1318-1338. 1 Council for Scientific and Industrial Research – Water Barrett, C. B., Bezuneh, M., Clay, C. D., & Reardon, T. (2005). 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Binary outcomes as used in this paper are widespread and perceived to be advantageous due to its simplicity in the interpretation. The challenge of using it is the loss of information which leads to the loss of power to detect relationships, and the probable inflation of type I error. To overcome these challenges, attempts were made to use some of the richer disaggregated data collected during the study (see Section 3.5). These variables were automatically dropped by the software whiles running the multinomial logit model. 1. During the analysis using the MNL, Stata automatically dropped some variables. Thus, the analysis was based on 683 respondents. Citation information (1974). The measurement of urban travel demand. Journal of Public Economics, 3, 303–328. doi:10.1016/0047-2727(74)90003-6 Eneyew, A. (2012). Determinants of livelihood diversifica- tion in pastoral societies of Southern Ethiopia. Journal of Agriculture and Biodiversity Research, 1(3), 43–52. Obour, P. 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Conservation Biology, 23, 124–140. doi:10.1111/j.1523- 1739.2008.01041.x Lawson, G. W. (1963). Volta Basin research project. Nature, 199, 858-859. Liu, J., Zang, C., Tian, S., Liu, J., Yang, H., Jia, S., … Zhang, M. (2013). Water conservancy projects in China: Achievements, challenges and way forward. Global Environmental Change, 23(3), 633–643. doi:10.1016/j.gloenvcha.2013.02.002 Cinner, J., McClanahan, T., & Wamukota, A. (2010). Differences in livelihoods, socioeconomic character- istics, and environmental perceptions between fish- ers and non-fishers living near and far from marine parks on the Kenyan coast. Marine Policy, 34, 22–28. doi:10.1016/j.marpol.2009.04.003 Martin, S. M., Lorenzen, K., & Bunnefeld, N. (2013). Fishing farmers: Fishing, livelihood diversification and pov- erty in rural Laos. Human Ecology, 41, 737–747. doi:10.1007/s10745-013-9567-y Cinner, J. E., & Bodin, O. (2010). Livelihood diversification in tropical coastal communities: A network-based approach to analyzing ‘livelihood landscapes’. PloS one, 8, 1–13. Marzban, C., & Sandgathe, S. (2005). Cluster analysis for verification of precipitation fields. Weather and Forecasting, 21, 824–836. doi:10.1175/WAF948.1 Ellis, F. (2000). Rural LIVELIHOODS and diversity in devel- oping countries. Oxford: Oxford University Press. McFadden, D. Citation information Amoah (Ed.), New developments of health, agricultural resources and socio-economic activities in the Volta Basin, Ghana VBRP (pp. 136–154). Institute of Aquatic Biology (IAB). (1995). Biology of selected commercially important fishes of Stratum VII of the Volta Lake, IAB-IDAF Project GHA/93/008. Accra: Institute of Aquatic Biology of the Council for Scientific and Industrial Research. Tsikata, D. (2005). Downstream rural communities and the Volta River Project: Long term responses to environmental changes and state neglect. Ghana Journal of Social Sciences, 3(1), 1–10. Jansen, H., Damon, A., Pender, J., Wielemaker, W., & Schipper, R. (2003). Policies for sustainable develop- ment in the hillsides of Honduras: A quantitative Page 14 of 15 Page 14 of 15 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 Amevenku et al., Cogent Economics & Finance (2019), 7: 1595291 https://doi.org/10.1080/23322039.2019.1595291 © 2019 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. You are free to: Share — copy and redistribute the material in any medium or format. Adapt — remix, transform, and build upon the material for any purpose, even commercially. The licensor cannot revoke these freedoms as long as you follow the license terms. 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No additional restrictions You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits. Citation information Cogent Economics & Finance (ISSN: 2332-2039) is published by Cogent OA, part of Taylor & Francis Group. Publishing with Cogent OA ensures: • Immediate, universal access to your article on publication • High visibility and discoverability via the Cogent OA website as well as Taylor & Francis Online • Download and citation statistics for your article • Rapid online publication • Input from, and dialog with, expert editors and editorial boards • Retention of full copyright of your article • Guaranteed legacy preservation of your article • Discounts and waivers for authors in developing regions Submit your manuscript to a Cogent OA journal at www.CogentOA.com Page 15 of 15 Cogent Economics & Finance (ISSN: 2332-2039) is published by Cogent OA, part of Taylor & Francis Group. Publishing with Cogent OA ensures: • Immediate, universal access to your article on publication • High visibility and discoverability via the Cogent OA website as well as Taylor & Francis Online • Download and citation statistics for your article • Rapid online publication • Input from, and dialog with, expert editors and editorial boards • Retention of full copyright of your article • Guaranteed legacy preservation of your article • Discounts and waivers for authors in developing regions Submit your manuscript to a Cogent OA journal at www.CogentOA.com cis Online Page 15 of 15
https://openalex.org/W4230228097	https://www.qeios.com/read/I56X5C/pdf	English	null	Proximal 16p11.2 microduplication syndrome	Definitions	2,020	cc-by	109	Qeios · Definition, February 10, 2020 Open Peer Review on Qeios INSERM INSERM Qeios ID: I56X5C · https://doi.org/10.32388/I56X5C Source INSERM. (1999). Orphanet: an online rare disease and orphan drug data base. Proximal 16p11.2 microduplication syndrome. ORPHA:370079 Proximal 16p11.2 microduplication syndrome is a rare chromosomal anomaly syndrome resulting from a partial duplication of the short arm of chromosome 16 characterized by developmental delay and intellectual disability of a highly variable degree, autism spectrum, obsessive-compulsive, attention deficit hyperactivity disorder, speech articulation abnormalities, muscular hypotonia, tremor, hyper- or hyporeflexia, seizures, microcephaly, neuroimaging abnormalities, decreased body mass index and schizophrenia or bipolar disorder later on in life. Qeios ID: I56X5C · https://doi.org/10.32388/I56X5C 1/1
https://openalex.org/W2190002939	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1004528&type=printable	English	null	Modelling Odor Decoding in the Antennal Lobe by Combining Sequential Firing Rate Models with Bayesian Inference	PLOS computational biology/PLoS computational biology	2,015	cc-by	20,823	OPEN ACCESS Citation: Cuevas Rivera D, Bitzer S, Kiebel SJ (2015) Modelling Odor Decoding in the Antennal Lobe by Combining Sequential Firing Rate Models with Bayesian Inference. PLoS Comput Biol 11(10): e1004528. doi:10.1371/journal.pcbi.1004528 Editor: Konrad P. Kording, Northwestern University, UNITED STATES Editor: Konrad P. Kording, Northwestern University, UNITED STATES Received: April 1, 2015 Accepted: August 28, 2015 Published: October 9, 2015 Received: April 1, 2015 Accepted: August 28, 2015 Published: October 9, 2015 Copyright: © 2015 Cuevas Rivera 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. Modelling Odor Decoding in the Antennal Lobe by Combining Sequential Firing Rate Models with Bayesian Inference Dario Cuevas Rivera1,2, Sebastian Bitzer1,3, Stefan J. Kiebel1,2,3 , , 1 Department of Psychology, Technische Universität, Dresden, Germany, 2 Biomagnetic Centre, Department of Neurology, University Hospital Jena, Jena, Germany, 3 Department of Neurology, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany 1 Department of Psychology, Technische Universität, Dresden, Germany, 2 Biomagnetic Centre, Department of Neurology, University Hospital Jena, Jena, Germany, 3 Department of Neurology, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany dario.cuevas_rivera@tu-dresden.de Abstract The olfactory information that is received by the insect brain is encoded in the form of spatio- temporal patterns in the projection neurons of the antennal lobe. These dense and overlap- ping patterns are transformed into a sparse code in Kenyon cells in the mushroom body. Although it is clear that this sparse code is the basis for rapid categorization of odors, it is yet unclear how the sparse code in Kenyon cells is computed and what information it represents. Here we show that this computation can be modeled by sequential firing rate patterns using Lotka-Volterra equations and Bayesian online inference. This new model can be understood as an ‘intelligent coincidence detector’, which robustly and dynamically encodes the presence of specific odor features. We found that the model is able to qualita- tively reproduce experimentally observed activity in both the projection neurons and the Kenyon cells. In particular, the model explains mechanistically how sparse activity in the Kenyon cells arises from the dense code in the projection neurons. The odor classification performance of the model proved to be robust against noise and time jitter in the observed input sequences. As in recent experimental results, we found that recognition of an odor happened very early during stimulus presentation in the model. Critically, by using the model, we found surprising but simple computational explanations for several experimental phenomena. Introduction Understanding how a brain encodes and decodes olfactory input has been an active field of study for decades [1,2]. The relatively simple circuitry in the insect brain for odor processing offers a good opportunity to understand the basic principles of sensory processing in brains. Some findings have been key in understanding how the insect brain makes sense of the olfac- tory information that it acquires from the outside world: (i) There are three stages of stimulus processing: in the antennae, the receptor neurons bond with odorants creating a time-invariant spatial pattern of activations in these neurons, which is sent to the antennal lobe [3]. In the antennal lobe, the projection neurons (PNs) react with odor-specific spatiotemporal patterns [4], whose duration far surpasses that of the stimulus itself [5]. In the mushroom body (MB), the target of the PNs, a small number of highly-specific Kenyon cells (KC) respond with short- lived activation periods, often only with a single spike. (ii) Odor-specific trajectories can be measured in the PN firing rate phase space, and the separation between the trajectories for dif- ferent odors is greatest during a period of slow dynamics which lasts for about 1.5s after odor onset. (iii) The spatiotemporal patterns that arise in the PN population encode the identity of the odor [6], but can be difficult to differentiate for any two odors [7]. It is only at the KC level that the trajectories are easily identifiable, due to the sparseness of KC responses [2]. In response to an odor, only a few of KCs fire spikes (population sparseness) and the firing rates are limited to usually one or two spikes during the presentation of the odor (lifetime sparseness). The causes of this KC sparseness and its precise role in odor decoding are still unknown. It has been suggested that the KCs act as coincidence detectors [5,8], i.e., a KC becomes active only when a number of its input PNs are active. Another proposal offers an explanation for the lifetime sparseness of the response based on spike frequency adaptation [9], albeit without providing an explicit functional role for the sparseness. During the period of slow dynamics in the response of the PNs to a stimulus, the firing rates of single PNs rise and fall sequentially in an odor-specific order, creating a sequence of “active” PNs through time [10]. Modelling Insect Olfaction with Bayesian Inference underlie a fast and accurate recognition which is highly robust against neuronal or sensory noise. This model replicates several key experimental findings and explains how the insect brain achieves both speed and robustness of odor recognition as observed in experiments. Author Summary Data Availability Statement: The authors confirm that all data underlying the findings are fully available without restriction. Source code for the implementation can be found at [https://github.com/ dcuevasr/Olfaction/] Odor recognition in the insect brain is amazingly fast but still not fully understood. It is known that recognition is performed in three stages. In the first stage, the sensors respond to an odor by displaying a reproducible neuronal pattern. This code is turned, in the sec- ond and third stages, into a sparse code, that is, only relatively few neurons activate over hundreds of milliseconds. It is generally assumed that the insect brain uses this temporal code to recognize an odor. We propose a new model of how this temporal code emerges using sequential activation of groups of neurons. We show that these sequential activations Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. 1 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Results We developed a model of how the insect brain encodes the qualities of an odor and subse- quently decodes this information to identify the odor. To do this, we assumed that the underly- ing encoding mechanism in the brain is based on sequential neuronal dynamics [15], as in the Laurent-Rabinovich model introduced in [11]. The proposed model consists of two compo- nents. The first is a generative model in accordance with key experimental findings that will be briefly reviewed below. The second is a Bayesian inference scheme based on this genera- tive model. As we will show, this Bayesian model can recognize synthetic PN input using sequential KC activity. Note that the Bayesian inference scheme is the key device to derive a fast and robust decoding model while maintaining, at a firing rate level, neurobiological plausi- bility by relying on the Bayesian brain hypothesis [12,16–19]. Based on experimental findings that the representations for different odors appear to be well differentiated at the KC level (as opposed to the PN level) [20], we refer to successful odor recognition when the model can successfully reproduce the spatiotemporal KC representation expected for a specific odor (see Methods for details). In the following, we first briefly review the experimental findings which we used to derive the generative model for the activity of the KC and PN population, followed by the model description and results. Modelling Insect Olfaction with Bayesian Inference activations? (ii) What is the mechanism behind the hypothesized coincidence detection of the KCs? In this work, we present a new model that addresses these two questions. The model com- bines a nonlinear generative model with approximate Bayesian inference for nonlinear dynam- ical systems [12–14]. Specifically, as a generative model, we use a modified version of the Laurent-Rabinovich model introduced in [11], which is based on sequential neuronal dynamics to describe the dynamics of PNs. In the proposed generative model, the PN activity is the input to the model. We propose to use Bayesian inference to infer the states of the hidden variables, i.e. the firing rates of the KCs. The model dynamics exhibit the observed behavior of both PNs and KCs in the insect brain and mechanistically explains how sparse code in the KCs emerges from the dense coding of the PNs. In addition, the Bayesian inference approach enables the rec- ognition of odors from PN activation dynamics, which may be understood as an ‘intelligent coincidence detector’ implemented by the KCs. In sum, we present a model which (1) repli- cates experimentally observed dynamics at two hierarchical levels of the odor recognition sys- tem of insects, (2) provides simple explanations for several key experimental findings and (3) implements fast and robust odor recognition based on firing rate input. Introduction Such a sequence-generating device could be the basis of an odor recog- nition mechanism at the KC or downstream levels. There are two previous modelling approaches addressing this. The first approach used Lotka-Volterra equations to model PN activations [11]. The solution of these equations visits the vicinity of a set of equilibrium points, giving rise to a trajectory in the phase space akin to those observed in experiments. This model is an abstract system that behaves in a similar way to that observed in the PN population, albeit without providing a mechanism for decoding stimulus responses at the KC level. The second approach presented a computational mechanism through which lifetime sparseness can be achieved in the KC population as information is passed on from the PNs [9]; however, the sequential nature of the activity at both the PN and KC level was not part of this model. Although these two previous computational models addressed fundamental questions, it is still unclear how the PNs and KCs interact mechanistically to enable odor recognition. In par- ticular, the following two key questions remain to be answered: (i) How does the insect brain achieve its speed and robustness of odor recognition by transforming highly overlapping spa- tiotemporal PN patterns into a potentially unique and easily recognizable sequence of KC PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 2 / 31 Modelling Insect Olfaction with Bayesian Inference observed, different from those in the first phase. The activity then slowly fades back to baseline activity over the course of a few seconds. Sequential firing patterns of PNs. Single PNs can respond to a stimulus in a multiphasic way [4]: they can be at a baseline firing rate of around 3Hz [5], in an excited state or inhibited under baseline. Correspondingly, the PN population can be divided into three groups at any given time during the response: inhibited, excited and at baseline. The ensuing response can be seen as a sequence of spatial patterns of activation, each being a combination of excited, inhib- ited and baseline PNs. The duration of each of these patterns is not fixed, even for the same insect-odor combination, but the order in which they appear (i.e. the order of the sequence) is reproducible across trials [7,10]. The representation of an odor in the PNs of the antennal lobe can be understood as a sequence whose elements (the spatial patterns) appear in the same order for different trials of the same odor, even if the exact timing changes. These elements are largely overlapping: a PN that is excited (or inhibited) during one of these elements will proba- bly also be excited (or inhibited) during other elements of the sequence for that odor or for other odors. The evolution of these sequences of spatial patterns can be understood as trajectories in the phase space of the firing rates of the PNs. While all trajectories are odor-specific throughout their duration, the maximum separation between trajectories for different odors appears dur- ing the first phase of the response [7]. Sparse KC code. The dense and largely overlapping spatial patterns in the PNs are trans- formed into sparse, highly odor-specific sequences of spikes produced by a small number of odor-specific KCs, while all other KCs remain silent. This means that the KC population reacts to a stimulus in clusters comprising a small number of KCs that increase their firing rate, as suggested for example in [3] and can be seen in the raster plots in [5]. Furthermore, it has been found that, while the number of odors that a KC responds to is much lower than that of the PNs, some KCs activate in response to more than one odor [5]. Previous experimental findings Slow evolution of PN firing pattern. In response to a stimulus, the PNs exhibit temporal spiking patterns in two frequency ranges that are independent of the frequencies of variation in the stimulus [4]: in fast, synchronized oscillations of 20–30Hz [4] and in a slower evolution, on a timescale of seconds. The slow evolution of the response to an odor is divided into three phases [7]: the onset sequential phase, which occurs first, is characterized by sequen- tial dynamics, in which the whole PN population responds with spatial patterns of activation which evolve in time. When the stimulus is presented for more than ca. 1.5s, the system enters the steady state phase, where it exhibits steady state dynamics, with stagnant PN activity; when the stimulus lasts less than 1.5s, this second phase is skipped. The steady state phase lasts until stimulus offset, which triggers the offset sequential phase, where again sequential dynamics are PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 3 / 31 A novel model of odor recognition In this paper, we build on the core idea of the Laurent-Rabinovich model that odor recognition is based on neuronal sequences and extend the model in three ways. Firstly, experimental results show that KCs respond to more than one odor and are activated in small groups [5,20]. Motivated by these results, we replace the rather simple neuronal sequences of Eq 1 used in [11] by sequences of small neuronal clusters and use these equations to describe sequences in KCs as opposed to the PNs. As we will show below, this extension massively increases the number of possible odors that can be recognized by the model and is critical in explaining how a KC can represent multiple odors. In addition, this cluster extension makes the decoding at the KC level highly robust against failures of single KCs because at each point in time during odor recognition multiple KCs sparsely share the decoding. Secondly, we combine the KC-cluster sequences with a model for PN activity. This will enable us to model the hierarchical decoding of the dense PN code by sparse KC activity. Thirdly, we combine the resulting PC/KN model with Bayesian inference. This will make the model a recognition model, i.e. the model can receive and decode PN input. Critically, we will show that this recognition model can identify specific odors (out of a selection of odors) very rapidly, and is robust against several noise sources, for instance unexpectedly activated/ inactivated PNs. In sum, these three extensions enable us to answer our two questions: (i) How does the insect brain achieve its speed and robustness of odor recognition by transforming highly-over- lapping spatiotemporal PN patterns into a potentially unique and easily recognizable sequence of KC activations? (ii) What is the mechanism underlying the hypothesized coincidence detec- tion of the KCs? In the following we will describe each of the three extensions in detail. Sequences of KC clusters. Because KCs respond to more than one odor and are activated in small groups [5,20], we modified the conditions on the connectivity matrix presented in [11,21,22] to allow clusters of KCs, instead of single neurons, to represent an element in an odor-specific sequence (see Fig 1). With this neuronal cluster approach, a single KC can be part of the representations of multiple odors and activate more than once in response to a sin- gle odor. Modelling Insect Olfaction with Bayesian Inference model. This means that the model cannot recognize one specific odor among many possible alternatives but rather follows the dynamics of a specific, pre-set odor. Secondly, as the input is only used to fix the model parameters, there is no way of finding out how robust the model is against sensory or neuronal noise. For example, neuronal noise may mean that for a given odor a PN does not fire although it should, or if a PN does fire although it should not. Clearly, a model of odor recognition should be robust against such neuronal noise. Thirdly, the Laurent- Rabinovich model is meant to model PN activity but does not describe the sparse KC activity. This means that one of the most prominent questions, i.e. how dense PN activity is trans- formed into the sparse KC code, cannot be addressed by the model proposed in [11]. Previous modeling work for sequential activity Previous efforts for modelling the sequential dynamics during the onset sequential phase of an odor response in the antennal lobe used the Lotka-Volterra equations [11,21]. This model used the Lotka-Volterra equations to obtain reproducible sequential dynamics in the PN population. The equations are the following: _xi ¼ xiðsiðIÞ þ X i6¼j rijðIÞxjÞ þ Zi 1 1 where xi is the ﬁring rate of the i-th neuron, σi is a parameter, η is noise and ρij is the connectiv- ity matrix among the neurons; I is the input to the system, i.e. the odor being perceived. Because of the continuum of possible input odors, the parameters ρ and σ are continuous func- tions of the input I. Under the conditions over ρij and σi given in [22] the system has a set of equilibrium points Qi = (0,0,. . .,σi,. . .,0), where the non-zero entry is at the i-th position, and its solution presents a stable heteroclinic sequence (SHS), which is the union of these equilib- rium points and trajectories that join them in a speciﬁc sequence. An odor is represented by a sequence of equilibrium points visited by the solution. When presented with a stimulus, which sets a value for σi and the connectivity matrix ρij, the system responds by visiting a sequence of equilibrium points in which the order of the points is constant across trials. Although this model (which we call the Laurent-Rabinovich model from now on) captures the most prominent feature of the PN data, i.e., neuronal sequential activations, the model has several limitations. Firstly, there is no mechanism for how the sensory input is received by the PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 4 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Each KC could connect with a neuron that is not part of the next cluster; these connections are labeled KCn, KCn+1, etc. and arise because KCs can belong to other clusters as well. (B) Conditions for the connectivity matrix in the proposed model and in the [11] model. In the proposed model, all components of the connectivity matrix ρ are negative (inhibition), whereas the [11] model uses both inhibition and excitation. Ck is the k-th cluster, i.e. i Ck for all neurons in cluster k. (C) Example of connections between KCs and PNs. KCs work as coincidence detectors, being activated only when both the PNs they are connected to are activated. Shown, a population of 6 KCs divided in two clusters. Four PNs are connected to the KCs so that each KC sees half of the PN population. When PNs 1, 2 and 3 are active, cluster 1 is activated. Cluster 2 is activated with PNs 2, 3 and 4. doi:10.1371/journal.pcbi.1004528.g001 inhibition, which is almost all-to-all, as direct connections between KCs, but it can equivalently be implemented as a pool of inhibition that is connected to all neurons in the population. Note that there is empirical evidence for the latter connection scheme [23] supporting our model assumption that the effect of KC activity on other KCs is mostly inhibitory. A full description of these connections can be found in the section ‘Model parameters used for simulations’ below. To illustrate the difference between our approach and the previous work in [11], in Fig 2A we show data of KC population activity as would be expected from the [11] model (Eq 1). We use a projection rate of 1:20 into the PN population (i.e. one KC connects to 20 PNs, equivalent to connecting each PN to around 60 KCs), to create the closest resemblance to the experimen- tally observed patterns in the PNs. This should be compared against Fig 2B, where we show KC population activity generated by our model. It can be seen that the resulting PN activity pat- terns are denser than in the Laurent-Rabinovich model and qualitatively more similar to exper- imentally observed PN activity. See also below. In addition, in our model, KC activity is sparse but not restricted to exactly one neuron active at a time. Modelling Insect Olfaction with Bayesian Inference Fig 1. Connectivity among KCs and PNs. (A) Clusters of KCs and their connectivity. The connections shown here are the low-inhibition connections (blue) and high-inhibition (red) between specific KCs and the medium inhibition between members of a cluster (green). The high-inhibition all-to-all connections are not depicted. Each neuron is connected (as an example) to three other neurons outside of its cluster. A line with a single dot at the end means that the connection is one-way only. The neurons in one cluster connect to each other and to elements outside the cluster. Those neurons that receive input from more than one KC in a cluster form the cluster to be activated next. Each KC could connect with a neuron that is not part of the next cluster; these connections are labeled KCn, KCn+1, etc. and arise because KCs can belong to other clusters as well. (B) Conditions for the connectivity matrix in the proposed model and in the [11] model. In the proposed model, all components of the connectivity matrix ρ are negative (inhibition), whereas the [11] model uses both inhibition and excitation. Ck is the k-th cluster, i.e. i Ck for all neurons in cluster k. (C) Example of connections between KCs and PNs. KCs work as coincidence detectors, being activated only when both the PNs they are connected to are activated. Shown, a population of 6 KCs divided in two clusters. Four PNs are connected to the KCs so that each KC sees half of the PN population. When PNs 1, 2 and 3 are active, cluster 1 is activated. Cluster 2 is activated with PNs 2, 3 and 4. Fig 1. Connectivity among KCs and PNs. (A) Clusters of KCs and their connectivity. The connections shown here are the low-inhibition connections (blue) and high-inhibition (red) between specific KCs and the medium inhibition between members of a cluster (green). The high-inhibition all-to-all connections are not depicted. Each neuron is connected (as an example) to three other neurons outside of its cluster. A line with a single dot at the end means that the connection is one-way only. The neurons in one cluster connect to each other and to elements outside the cluster. Those neurons that receive input from more than one KC in a cluster form the cluster to be activated next. A novel model of odor recognition This enables us to encode several sequences (odors) within a single connectivity matrix, while a single KC can respond to more than one odor. This was not possible in the Lau- rent-Rabinovich model. Inhibitory connections between the KCs, which translate into negative constants ρij, create the sequential dynamics between neuronal clusters. We used two general types of inhibitory connections with high and low levels of inhibition between different KCs. Connections with low inhibition encode the neuronal sequences associated with odors. They only exist between two neurons if they appear in the same sequence. Connections with high inhibition exist between two neurons that do not appear together in any sequence. We implement this high PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 5 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference Fig 2. PN output of our model compared to the Laurent-Rabinovich model. Firing rates of 100 KCs and 30 PNs in mushroom body and the antennal lobe, respectively, for the proposed model and an implementation of the Laurent-Rabinovich model. Each panel represents the activity of the KC and PN populations at times t1, t2, t3 and t4, in which the clusters of the sequence are at their maximum value (see Fig 5), and each square in a panel represents a neuron (10x10 for KCs, 6x5 for PNs). (A) An SHS model, as presented in [11], used for the KC population, plus a random 1 KC to 20 PNs projection. (B) Data generated by the proposed model, with clusters of three KCs each. Fig 2. PN output of our model compared to the Laurent-Rabinovich model. Firing rates of 100 KCs and 30 PNs in mushroom body and the antennal lobe, respectively, for the proposed model and an implementation of the Laurent-Rabinovich model. Each panel represents the activity of the KC and PN populations at times t1, t2, t3 and t4, in which the clusters of the sequence are at their maximum value (see Fig 5), and each square in a panel represents a neuron (10x10 for KCs, 6x5 for PNs). (A) An SHS model, as presented in [11], used for the KC population, plus a random 1 KC to 20 PNs projection. (B) Data generated by the proposed model, with clusters of three KCs each. Fig 2. PN output of our model compared to the Laurent-Rabinovich model. Firing rates of 100 KCs and Fig 2. PN output of our model compared to the Laurent-Rabinovich model. Firing rates of 100 KCs and 30 PNs in mushroom body and the antennal lobe, respectively, for the proposed model and an implementation of the Laurent-Rabinovich model. Each panel represents the activity of the KC and PN populations at times t1, t2, t3 and t4, in which the clusters of the sequence are at their maximum value (see Fig 5), and each square in a panel represents a neuron (10x10 for KCs, 6x5 for PNs). (A) An SHS model, as presented in [11], used for the KC population, plus a random 1 KC to 20 PNs projection. (B) Data generated by the proposed model, with clusters of three KCs each. Fig 2. By using clusters the model can pro- duce more varied PN activity, closer to that reported, for example, in [24] for mitral cells and the more nuanced firing rates curves of PNs shown in [6,7,25]. A combined model of principal neurons and Kenyon cells. To combine the PN and KC levels in the model, we used two experimental findings (reviewed above, also see Fig 3 for an PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 6 / 31 Modelling Insect Olfaction with Bayesian Inference Fig 3. Illustration of experimental findings. (A) Raster plots for the full response (three phases) to a stimulus of PNs (left) and KCs (right) using a modified version of the generative model (see Methods for details; also see the S1 Text). This modified version was only used for the generation of this figure and no inversion was performed on it. Although the model consisted of 30 PNs and 100 KCs, most KCs were inactive during the simulations. Only the 27 active KCs are shown. The simulation was run for ten trials and the responses of different neurons are separated by thick black lines. For the PNs, periods of inhibition and excitation can be observed. During the steady state phase the population no longer evolved. KC responses are very sparse, with only a few spikes per KC throughout a trial. (B) Instantaneous firing rate for a sample PN. (C) The periods of excitation and inhibition (with respect to a baseline) during the dynamic phase for the PN in B. The baseline shown is calculated as half the minimum PN activity that the generative model can produce before it is zero. d i 10 1371/j l bi 1004528 003 Fig 3. Illustration of experimental findings. (A) Ras Fig 3. Illustration of experimental findings. (A) Raster plots for the full response (three phases) to a stimulus of PNs (left) and KCs (right) using a modified version of the generative model (see Methods for details; also see the S1 Text). This modified version was only used for the generation of this figure and no inversion was performed on it. Although the model consisted of 30 PNs and 100 KCs, most KCs were inactive during the simulations. Only the 27 active KCs are shown. The simulation was run for ten trials and the responses of different neurons are separated by thick black lines. For the PNs, periods of inhibition and excitation can be observed. During the steady state phase the population no longer evolved. KC responses are very sparse, with only a few spikes per KC throughout a trial. (B) Instantaneous firing rate for a sample PN. (C) The periods of excitation and inhibition (with respect to a baseline) during the dynamic phase for the PN in B. The baseline shown is calculated as half the minimum PN activity that the generative model can produce before it is zero. PN output of our model compared to the Laurent-Rabinovich model. Firing rates of 100 KCs and 30 PNs in mushroom body and the antennal lobe, respectively, for the proposed model and an implementation of the Laurent-Rabinovich model. Each panel represents the activity of the KC and PN populations at times t1, t2, t3 and t4, in which the clusters of the sequence are at their maximum value (see Fig 5), and each square in a panel represents a neuron (10x10 for KCs, 6x5 for PNs). (A) An SHS model, as presented in [11], used for the KC population, plus a random 1 KC to 20 PNs projection. (B) Data generated by the proposed model, with clusters of three KCs each. doi:10.1371/journal.pcbi.1004528.g002 doi:10.1371/journal.pcbi.1004528.g002 illustration): (1) A single PN can respond to a stimulus in a multiphasic way during the dynamic phase (see Fig 3C for an illustration). (2) The PN population responds to a stimulus with a sequence of dense spatial patterns, whose order is reproducible across trials, even if the exact timing varies (see Fig 3A). To accomplish this, we used a linear, one-to-many projection from individual KCs to the PNs to create the activity of the PN population during data genera- tion (a connection direction that is later inverted by Bayesian inference; see below for details). Experimental evidence for feedback from the KCs to the PNs has been found, for example, in drosophila [26]. The PNs are connected randomly with the KCs under the constraint that each KC connects with a fixed number of PNs via an observation equation. This equation is: Y ¼ YX þ G 2 2 Y ¼ YX þ G 2 where Y is the N×1 PN ﬁring rates vector, Θ is the N×M observation matrix, X is the M×1 vec- tor from the Lotka-Volterra system, representing KC activity, and Γ is Gaussian noise. The observation matrix Θ connects each KC with 20 random PNs. For details, see Methods. Eqs 1 and 2 as described in this and the previous sections are used to form what is called a generative model (see Methods for details), which is the basis of the dynamics of the KCs and PNs in our model. The connections contained in the generative model are used as a starting PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 7 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference Fig 4. Diagram of the proposed model. The generative model was used in two ways: first, to generate the PN data that was used as input to the Bayesian inference; Second, to describe the internally expected dynamics of the PN and KC populations. Top left: Specifically, the generative model uses the Lotka- Volterra equations to generate activity of the KC population. Bottom left: This activity was projected into the PN population via Eq 2 giving synthetic PN activity that was used as input to the Bayesian inference. Bottom right: Through the unscented Kalman filter, the PN activity observed was balanced with the expected dynamics of the KCs (prescribed by the generative model). Top right: This enabled the model to infer KC activity that is consistent with the PN input data. Fig 4. Diagram of the proposed model. The generative model was used in two ways: first, to generate the PN data that was used as input to the Bayesian inference; Second, to describe the internally expected dynamics of the PN and KC populations. Top left: Specifically, the generative model uses the Lotka- Volterra equations to generate activity of the KC population. Bottom left: This activity was projected into the PN population via Eq 2 giving synthetic PN activity that was used as input to the Bayesian inference. Bottom right: Through the unscented Kalman filter, the PN activity observed was balanced with the expected dynamics of the KCs (prescribed by the generative model). Top right: This enabled the model to infer KC activity that is consistent with the PN input data doi:10.1371/journal.pcbi.1004528.g004 Importantly, the Bayesian inference approach allows us to use a single, fixed connectivity matrix which encodes all previously stored sequences. The dynamic input to the system, i.e. the PN population activity, is used by the Bayesian inference to determine which of the stored sequences is the most consistent with the currently observed PN activity. This adds neurobio- logical plausibility as one would not expect (as for example in Eq 1, [11]) that the process of odor recognition would change the connectivity between neurons on a short time-scale. The online recognition is also useful for modelling ecologically valid scenarios where the actual input deviates from the expected (stored) sequence. doi:10.1371/journal.pcbi.1004528.g003 point by the Bayesian inference (see below) to build the full set of interactions between the neu- rons in our model. Additionally, we used this generative model to generate the synthetic data that were used as input to the model in the following sections. Additionally, we used this generative model to generate the synthetic data that were used as input to the model in the following sections. Bayesian inference. As third extension, we combine a generative model (built with Eqs 1 and 2; see Methods) with a Bayesian online inference scheme. This enables the model to read PN activity and infer the state of the KCs that is consistent with the PN activity, effectively making the PNs output to the KCs, as physiologically expected, inverting the projection from the KCs to the PNs of the generative model. This approach is useful for three reasons: Firstly, this extension makes the model an online recognition model. By online we mean that the Bayesian inference used here reads at each time step the current PN input and changes the KC states. This emulates precisely the online decoding of an insect brain. In particular, as we will show, it enables the model to recognize stimuli online and rapidly, i.e. before a full odor stimu- lus has been played to the model. Secondly, it provides the model with robustness to noise from several sources, e.g. neuronal noise at both the PN and KC levels. This robustness is important because it explains how a model based on potentially noisy neurons can recognize a stimulus accurately and reliably. Thirdly, it adds cluster-specific KC connections necessary for robustness and intelligent coincidence detection (see below) and connections from the PNs to the KCs. These PN-KC connections fit experimental observations [27]. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 8 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference generative model for Bayesian inference. We used populations of 100 KCs and 30 PNs with a KC:PN connectivity ratio of 1:20. The sizes of the populations were chosen in order to have a much larger number of KCs than PNs while keeping the population sizes small for computa- tional efficiency. We used four clusters per sequence and three KCs per cluster. As a proof of concept, we typically embedded two odors, for computational expediency, into these KC and PN populations. This means that per simulation at most 24 KCs were used for representing the two odors, although the exact number changes from simulation to simulation because the KCs which represent an odor are selected randomly from the population and a KC may be involved, as found experimentally, in representing both odors. In the generative model, there were four types of connections (see Fig 1), all of which are one-directional. For a cluster size of N KCs, these are: (i) Low-inhibition connections from the neurons in a cluster to those of the next cluster in a sequence, with weight 1/(10N). (ii) Medium-inhibition intra-cluster connections, among the neurons within a cluster, with a weight of 1/(3N). (iii) High-inhibition connections from neurons in a cluster to those in the previous cluster in a sequence, with strength 2 / N. (iv) Highest-inhibition connections between neurons not in any of the three past categories (e.g. a neuron and those that do not share any odor representation with it), with weight 2. Notice that the fourth type of connection is not normalized by the size of the clusters. The connections from the KCs to the PNs are selected randomly and the weights are all set to one. Unless otherwise stated, data generated with the generative model were obtained with addi- tive Gaussian noise in the PNs (Eq 2) with a signal-to-noise ratio (SNR) of 10, defined here as the activity of one PN when only one of its KCs is active, divided by the covariance of the noise. We used no noise in the KC dynamics (Eq 1) for data generation. In Fig 5A we present synthetic data generated by the generative model during the onset sequential phase for twenty representative PNs which connect to the same KC. It can be seen that the activity of individual PNs can be classified according to the succession of inhibition and excitation epochs through time, as found in experimental data [27]. The activity of the KC population is shown in Fig 5B. This sparse activity is representative of what has been found experimentally: only a few KCs are active at a time in odor-dependent clusters that activate together, while the rest of the population (i.e., those KCs not responsive to this particular stim- ulus) remains completely inactive. Note that before choosing these model parameters, we investigated cluster sizes from two to as many as ten KCs per cluster, and sequence lengths of up to 20 clusters per sequences. We found that these variations had no impact on the qualitative results shown below, i.e. the pro- posed model is robust over variations in cluster size and sequence length. In addition, the model also seems to scale with the number of KCs in total: We successfully simulated models from 50 to 1000 KCs with a proportional number of PNs. We do not report these results because they were qualitatively similar to the results presented here. Since the PN population has been found to change its activity pattern completely every 50– 300ms [7,29], we selected four clusters per sequence, which amounts to around 300ms per clus- ter in response to an odor. Lacking experimental results for the number of KCs that activate together, we settled on three neurons per cluster. As we will show, the model can, depending on the size of a deviation from the expected input, either explain the deviations away as sensory noise, or rapidly switch to another internally stored odor sequence, which may explain the cur- rent input better. Through Bayesian inference, the KCs’ role in the proposed model is consistent with the tra- ditional view of insect olfaction, in which the KC population reacts to the activity in the anten- nal lobe with a pattern of on/off activity, unequivocally identifying the odor perceived [28]. In addition to the on/off patterning discussed in [28], we were able to model the temporal dynam- ics of the response to an odor in both the antennal lobe and the mushroom body. This temporal patterning can be considered an extension to the ideas presented in [28]. Furthermore, evi- dence of these patterns has been found for the PNs [4,7] and preliminary evidence for the KCs [5,6]. In summary, the resulting model aims at decoding PN activity using dynamic expectations about KC activity; see Fig 4. Model parameters used for simulations. In the simulations below, we used the same parameters both for generating the synthetic data used as input to our model and for the 9 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference Fig 5. Example activity of the generative model’s PN-KC network. Y axis represents instantaneous firing rate, X axis is time. (A) Activity of 20 sample PNs that all connect to the same KC, showing epochs of excitation and inhibition. The shaded area is the onset sequential phase; the rest is the steady state phase. (B): Activity of the KCs during the onset sequential phase where each colored line represents a cluster of three KCs. In total, 15 KCs are shown (five clusters), with maximum firing rates around times t1, t2, t3 and t4. As in Fig 3, the baseline shown is calculated as half the activity level of a PN when only one KC of those connected to it is active. Fig 5. Example activity of the generative model’s PN-KC network. Y axis represents instantaneous firing Fig 5. Example activity of the generative model’s PN-KC network. Y axis represents instantaneous firing rate, X axis is time. (A) Activity of 20 sample PNs that all connect to the same KC, showing epochs of excitation and inhibition. The shaded area is the onset sequential phase; the rest is the steady state phase. (B): Activity of the KCs during the onset sequential phase where each colored line represents a cluster of three KCs. In total, 15 KCs are shown (five clusters), with maximum firing rates around times t1, t2, t3 and t4. As in Fig 3, the baseline shown is calculated as half the activity level of a PN when only one KC of those connected to it is active. Fig 5. Example activity of the generative model’s PN-KC network. Y axis represents instantaneous firing rate, X axis is time. (A) Activity of 20 sample PNs that all connect to the same KC, showing epochs of excitation and inhibition. The shaded area is the onset sequential phase; the rest is the steady state phase. (B): Activity of the KCs during the onset sequential phase where each colored line represents a cluster of three KCs. In total, 15 KCs are shown (five clusters), with maximum firing rates around times t1, t2, t3 and t4. As in Fig 3, the baseline shown is calculated as half the activity level of a PN when only one KC of those connected to it is active. doi:10.1371/journal.pcbi.1004528.g005 fast odor recognition, in accordance with experiments. Simulations and replication of experimental findings In this section, we will show the usefulness of the model in explaining how odor recognition based on the PN-KC hierarchy can be both rapid and robust. In addition, we will present a mechanistic explanation for several key experimental phenomena. There are five sections, where each addresses a specific experimental aspect. Firstly, we show how the model performs 10 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference Early odor recognition. In experiments, it has been found that the PN trajectories in the phase space of PN firing rates are maximally separated from each other during the onset sequential phase, facilitating odor discrimination [7]. In both insects and rodents the reaction time for odor recognition is much shorter (< 200ms) than this 1.5s-long first phase [6,30–32]. What is the function of the full 1.5s dynamic response to an odor? The answer might be a speed-accuracy tradeoff [30], which has been observed experimentally in rodents [30,33]; in this case, the 1.5s neuronal response may allow the brain to perform integration of information over time, improving accuracy in hard discrimination tasks, where the two odorants to be dis- criminated are presented in solutions with varying concentration ratios between them. In these tasks, the representations in the PNs of the two odors are very similar. Here we show that in our model, consistent with experimental results, the reaction time increases with harder tasks while maintaining a given accuracy level. We also find with our model that the reaction time is very short for most tasks. To show this, we embedded two odor representations (sequences) in a system, with the sys- tem’s parameters set as described in section ‘Model parameters used for simulations’ and used this to generate PN data to provide input to the model. We performed simulations with increasing degrees of task difficulty. To control the task difficulty, we changed the similarity between the two embedded odor representations, as is done experimentally [31]. To assess model performance in the recognition task, we compared the reaction times (see Methods). Choice comparisons were uninformative because the model performed mostly at ceiling for all tasks. The easiest task for the system was when the two sequences were similar only at the KC level. We implemented this by defining the first clusters for the two odor sequences such that they shared two out of three KCs. When playing one of the two odors to the model, we found that the model identified it correctly and rapidly (<60ms). This was expected because a sequence difference in a single KC expresses itself as a massive difference in up to 20 (out of 30) PNs in the projection pattern of the two sequences. To make the recognition task more challenging we used similar KC clusters with two shared KCs between odors. Secondly, we show that the experimen- tally established steady state phase of the odor response can be explained by our model as a prolonged activation of a single neuronal sequence element. Thirdly, we demonstrate the robustness of the model against several types of neurobiologically expected noise. Fourthly, we show how the KCs in our model behave as intelligent coincidence detectors of both PN and KC activity. Fifthly, we show that the model KC trajectories reduced to three dimensions look pre- cisely like their experimentally observed counterparts. This finding in particular indicates that our model captures a fundamental aspect of KC activity measured during insect odor recognition. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 11 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 However, differently from the easy task, for the differing KC we employed PN representations that were more similar than in the easy task. The similarity is expressed as the number of PNs that are connected to both of the differing KCs in the two clusters, where each KC connects with 20 PNs. For example, an easy task of this type is when there are only three PNs out of 20 (see Fig 6A) that connect to both KCs and the hardest when there are 20 (see Fig 6B). When 20 (out of 20) PNs are shared between the two KCs, the task is actually impossible to solve because the two odors are represented by exactly the same PNs during the first cluster. With increasing difficulty, we found that the system required, on average, more time to identify the correct sequence (i.e. the correct odor); see Fig 6C. For the easiest task, the mean reaction time was 60ms, which increased for harder tasks, where more PNs were shared between the two different KCs. For the hardest task, the model could not solve the task during the first cluster (50% accuracy) but had to wait for the onset of the second cluster (ca. 170ms) to then solve the task with a mean reaction time of 200ms. Note that these simulations present a worst-case scenario to the model because the differ- ence between the two odors was right at the beginning of the odor sequence. If these differences are found rather towards the end, the high similarity would not impede or slow down recogni- tion at all: We found that, if the two embedded representations are different enough at the beginning (i.e. their initial cluster is different), being identical later on has no effect on the per- formance of the system, as the correct representation has already been found and the system sticks with this sequence. This may be a general strategy for odor recognition: represent odors such that they are most dissimilar at the beginning of the response. As we have found in our PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 12 / 31 Modelling Insect Olfaction with Bayesian Inference Fig 6. Performance of the model in difficult tasks. The connectivity matrix has two sequences which were similar to each other. (A-B) Each single line represents one trial (out of 100 trials) simulated from the model. The Y axis represents the recognition variable, which is calculated from the difference between the Euclidean distances from the observed KC activity to the correct one and from the observed KC activity to the incorrect one (see Eq 6 in Methods). If the KCs are displaying the correct representation (encoding the displayed odor), the corresponding line is near the top. If the KCs are displaying the incorrect representation (i.e. the other sequence stored in the system), the corresponding line is near the bottom. Lines near the middle are as close to the correct representation as to the incorrect one. The activity of the clusters in the expected (correct) sequence is in the plot background as a time reference to show that many single trials quickly jump to the correct representation after the second cluster in the sequence starts: The shaded areas (2 colors) represent two clusters in the sequence (as in Fig 5B). (A) Results for a task in which the PN data for the two stored sequences are very similar during the first cluster: Only three out of twenty PNs are different for the two sequences during the first cluster. Due to noise, the KC activity represents sometimes the incorrect odor for a brief period of time. (B) Results for a task in which the PN data for the two stored sequences are identical during the first cluster but dissimilar for the rest of the odor. During exposure to the first segment of the odor, the representations on the KCs are correct around 50% of the time, as expected, consistent with random chance. When the second cluster in the sequence begins (at ca. 170ms), the KC activity quickly jumps to the correct representation. (C) Average reaction time of 100 trials plotted against difficulty of the task (as defined with the number of PNs that belong to the representations of both odors; see Methods). The PN representations of the two stored sequences are similar during the first 170ms; afterwards, they diverge and become easily identifiable. The more similar the PN representations of two stored odors are during this initial period, the longer it takes the KCs to identify it. The maximum reaction time, around 200ms, corresponding to the case in B, is obtained when all 20 PNs are the same. The case in A corresponds to 17 identical PNs. Fig 6. Performance of the model in difficult tasks. The connectivity matrix has two sequences which were similar to each other. (A-B) Each single line represents one trial (out of 100 trials) simulated from the model. The Y axis represents the recognition variable, which is calculated from the difference between the Euclidean distances from the observed KC activity to the correct one and from the observed KC activity to the incorrect one (see Eq 6 in Methods). If the KCs are displaying the correct representation (encoding the displayed odor), the corresponding line is near the top. If the KCs are displaying the incorrect representation (i.e. the other sequence stored in the system), the corresponding line is near the bottom. Lines near the middle are as close to the correct representation as to the incorrect one. The activity of the clusters in the expected (correct) sequence is in the plot background as a time reference to show that many single trials quickly jump to the correct representation after the second cluster in the sequence starts: The shaded areas (2 colors) represent two clusters in the sequence (as in Fig 5B). (A) Results for a task in which the PN data for the two stored sequences are very similar during the first cluster: Only three out of twenty PNs are different for the two sequences during the first cluster. Due to noise, the KC activity represents sometimes the incorrect odor for a brief period of time. (B) Results for a task in which the PN data for the two stored sequences are identical during the first cluster but dissimilar for the rest of the odor. During exposure to the first segment of the odor, the representations on the KCs are correct around 50% of the time, as expected, consistent with random chance. When the second cluster in the sequence begins (at ca. 170ms), the KC activity quickly jumps to the correct representation. (C) Average reaction time of 100 trials plotted against difficulty of the task (as defined with the number of PNs that belong to the representations of both odors; see Methods). The PN representations of the two stored sequences are similar during the first 170ms; afterwards, they diverge and become easily identifiable. The more similar the PN representations of two stored odors are during this initial period, the longer it takes the KCs to identify it. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference To illustrate how this steady state phase emerges in our model, we generated PN data corre- sponding to the full stimulus response with the three phases (Fig 7A, see Methods for details) and used it as input to the Bayesian inference. It can be seen that this PN input is remarkably similar to that observed experimentally. The model KC activity, in response to this PN input, is clearly divided into the three phases, as found experimentally when recording data from PNs (Fig 7B). In particular, the KC responses of the model show the steady state phase of the response with a lower firing rate than the sequential phases. Remarkably, the transitions between phases (from sequential to steady state and back to sequential) are reproduced by the model, even though we did not explicitly encode such a mechanism in the dynamics of the KCs in our model that would account for this behavior. The explanation for this finding is that during the steady state phase, the last element in the sequence lingers on as long as the stimulus is presented. Our model explains this phase as just another element of the sequence that lasts longer than the preceding elements. Thus, it contains as much information about the odor identity as any other spatial pattern of the response. How- ever, the information encoded in the temporal evolution is missing, which would explain the experimental finding that the steady state phase contains information about the odor stimulus but not as much as during the onset sequential phase. This model finding suggests a highly effi- cient representation of odors in insects because any odor duration can be represented by stretching the last element of the odor encoding sequence in time. Our model suggests that the insect odor system may be primarily geared towards fast recognition of any occurring odor and probably uses the last element of the sequence as a device to represent the constant odor input. This model behavior is due to the Bayesian inference technique: The temporal stretching of the last cluster of the sequence is the model’s successful attempt to fit its internal expectations (sequential dynamics) and the external input (constant). These results suggest that KC function in the insect antennal lobe may be largely aimed at representing neuronal sequences. The pro- posed model suggests a simple mechanism for how the qualitatively different three phases [7] can be encoded parsimoniously. The explanation for this finding is that during the steady state phase, the last element in the sequence lingers on as long as the stimulus is presented. Our model explains this phase as just another element of the sequence that lasts longer than the preceding elements. Thus, it contains as much information about the odor identity as any other spatial pattern of the response. How- ever, the information encoded in the temporal evolution is missing, which would explain the experimental finding that the steady state phase contains information about the odor stimulus but not as much as during the onset sequential phase. This model finding suggests a highly effi- cient representation of odors in insects because any odor duration can be represented by h h l l f h d d d l h h Robustness against noise and time-jitter. As odor recognition in an insect has to be robust against adverse conditions in nature, a model should emulate this robustness (i.e. per- form well even under adverse conditions). We tested this by making odor recognition difficult for the model by exposing it to noise and interferences. To do this, we used three different types of noise and interferences: (i) In nature, odors may be perceived with a low signal-to- noise (SNR) ratio, e.g. because the concentration of odor is low. We will test whether the pro- posed model can deal with such low SNRs. (ii) The representation of an odor in the PNs has been found to be in the form of a trial-variant sequence of spatial patterns, whose exact timings have been found to change for different trials of the same insect-odor combination [7]. There- fore, the insect brain’s recognition can be assumed to be robust against time jitter in the repre- sentation of an odor. We will test whether the model has the same robustness for time jitter. (iii) In experiments, insects were presented with an odorant for some time; before and after this stimulus, the insect is typically presented with clean, odorless air [6]. This brings the PNs and KCs back to baseline activity, in which they are not having any discernible response. In nature, such ‘bringing back to baseline’ may not happen. The maximum reaction time, around 200ms, corresponding to the case in B, is obtained when all 20 PNs are the same. The case in A corresponds to 17 identical PNs. doi:10.1371/journal.pcbi.1004528.g006 simulations, minute differences among odor representations, as expressed in PN activation pat- terns, are sufficient to recognize the correct odor rapidly, with a high probability. Such an encoding strategy may explain experimental results reporting the lack of evidence supporting a speed-accuracy tradeoff [31,34]: two odors that, on average, have highly similar representations in the brain might be different enough at the beginning to allow for rapid discrimination. Steady state phase. The second phase in the PN activity in response to a stimulus is a steady state phase, in which the temporal evolution has been found to halt until odor offset [7]. During this phase, the PN activity shows sustained firing at a lower average firing rate than during the first dynamical phase [7]. See Fig 3B for an illustrative example. The KC activity fol- lows this trend, having lower sustained activity during this phase [5]. This phase is typically observed when the insect has been presented with a stimulus for longer than ca. 1–2 seconds and the PN response has been found to contain information about the identity of an odor. However, during this phase, it is harder to distinguish between the representations of different odors than during the first dynamical phase of the response [7]. The function of the steady state phase is currently unknown, but based on the proposed model we suggest an explanation that is both mechanistic and functional. As the model is based on neuronal sequences (Eq 1, also see Methods), we explicitly avoided model compo- nents like fixed point attractors which may serve as a relatively direct explanation of a steady state phase. This means that the proposed model should have difficulties to represent a steady state phase. However, when we created PN data with the typical sequence of onset sequential phase followed by the steady state phase, we found that this steady state phase can be repre- sented easily be the model: The steady state phase is simply the consequence of the odor- encoding KC cluster sequence having run out of sequence elements to represent this specific odor over time. 13 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 doi:10.1371/journal.pcbi.1004528.g007 The brain must be able to recognize an odor that is presented right after another; in terms of neuronal activity, it means that recog- nition should be robust against the activity of KCs being different from baseline at odor onset. We will test whether the model can handle these deviations from a baseline state when recog- nizing odors. To test the model against neuronal noise, we ran all our simulations with white noise in the PN data. We used several levels of signal to noise ratio (SNR): 4.6, 2.6, 2.1, 1.9 and 1.8, which PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 14 / 31 Modelling Insect Olfaction with Bayesian Inference Fig 8. Performance of the model under adverse conditions. (A) Simulations to test robustness against noise in the PNs. Percentage of correct trials (i.e. trials in which the model displayed the correct KC representation) for low SNRs. For these simulations, we added white noise of different variances to all PN activity. The model is said to display the correct response if the Euclidean distance in the firing-rate-space between the inferred and expected (i.e. used to create the data) KC responses is smaller than a threshold (see main text). A decrease in performance can be observed only for SNRs smaller than 2.6. (B) Simulations to test robustness against KC states not at baseline at the onset of an odor. Histogram of reaction times (see Methods) for trials with initial KC states set to a baseline (light gray) or a random state (blue). These reaction times were calculated for two completely dissimilar sequences with high SNR. Therefore recognition is rapid. The reaction times for trials with random initial conditions are slightly more varied and the mean reaction time is 19ms, as compared to 17ms when the initial KC activity is at baseline. d i 10 1371/j l bi 1004528 008 Fig 8. Performance of the model under adverse conditions. (A) Simulations to test robustness against noise in the PNs. Percentage of correct trials (i.e. trials in which the model displayed the correct KC representation) for low SNRs. For these simulations, we added white noise of different variances to all PN activity. The model is said to display the correct response if the Euclidean distance in the firing-rate-space between the inferred and expected (i.e. used to create the data) KC responses is smaller than a threshold (see main text). A decrease in performance can be observed only for SNRs smaller than 2.6. (B) Simulations to test robustness against KC states not at baseline at the onset of an odor. Histogram of reaction times (see Methods) for trials with initial KC states set to a baseline (light gray) or a random state (blue). These reaction times were calculated for two completely dissimilar sequences with high SNR. Therefore recognition is rapid. The reaction times for trials with random initial conditions are slightly more varied and the mean reaction time is 19ms, as compared to 17ms when the initial KC activity is at baseline. Modelling Insect Olfaction with Bayesian Inference Fig 7. PN and KC activity for the full response to a stimulus. (A) Sample of four PNs’ activity throughout the full response. Left: experimental data as reported in [7]; Right: data generated by our model. The qualitative behavior of the PNs observed in experiments can be matched by our model by choosing an appropriate observation matrix. (B) Bayesian inversion of the model: Inferred KC activity as a response to the PN data generated from the full response shown in A. Each color represents a cluster of three different KCs. can be seen that the inferred KC activity displays the three phases of the odor response, although the generative model given by Eq 1 includes only the sequential dynamics. doi:10.1371/journal.pcbi.1004528.g007 Fig 7. PN and KC activity for the full response to a stimulus. (A) Sample of four PNs’ activity throughout the full response. Left: experimental data as reported in [7]; Right: data generated by our model. The qualitative behavior of the PNs observed in experiments can be matched by our model by choosing an appropriate observation matrix. (B) Bayesian inversion of the model: Inferred KC activity as a response to the PN data generated from the full response shown in A. Each color represents a cluster of three different KCs. It can be seen that the inferred KC activity displays the three phases of the odor response, although the generative model given by Eq 1 includes only the sequential dynamics. 15 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference Fig 9. Time-jittered KC activity from the model. Using one connectivity matrix, we generated PN data with no jitter and used that data to create time- jittered PN activity. The KC activity output by our model is shown: each row represents a sequence displayed by our model; each color represents a cluster in the sequence. The first sequence (top) has no time-jitter in the input. The following sequences are time-jittered, i.e. the time each cluster is active is different. It can be readily seen that the Bayesian inference has no trouble handling time-jitter that cannot be generated directly by the generative model. Fig 9. Time-jittered KC activity from the model. Using one connectivity matrix, we generated PN data with no jitter and used that data to create time- jittered PN activity. The KC activity output by our model is shown: each row represents a sequence displayed by our model; each color represents a cluster in the sequence. The first sequence (top) has no time-jitter in the input. The following sequences are time-jittered, i.e. the time each cluster is active is different. It can be readily seen that the Bayesian inference has no trouble handling time-jitter that cannot be generated directly by the generative model. doi:10.1371/journal.pcbi.1004528.g009 baseline between odors. The finding that there is a slight increase of ‘failed’ recognition trials (from <0.1% to 6%) when the system is not in its ‘ready’ baseline state, is interesting in light of similar findings in cognitive neuroscience where perceptual performance has been found to depend on the internal state of participants [35,36]. To test the robustness against time-jitter in the odor presentation by PNs, we created time- jittered PN data by processing the responses generated by the Lotka-Volterra equations to change the time each cluster is active during the sequence. In Fig 9 we show time-jittered exam- ple sequences in the KCs obtained with our model. We found no discernible effect of the time- jitter on the obtained responses from our model, i.e. mean reaction times are statistically not different and performance is at ceiling. That is, the model KCs display the expected (time-jit- tered) response to the stimulus, closely following the time jitter of the data. This is quite remarkable because the generative model prescribes a specific duration of each cluster response. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 doi:10.1371/journal.pcbi.1004528.g008 cover the spectrum between low noise, with ceiling recognition performance, and high noise causing highly degraded performance. For each of these noise levels, we computed the number of correct trials (out of 100) by using a threshold on the Euclidean distance between expected and inferred KC activity, as was done for the reaction time (see Methods). This conservative way of computing a correct response may be interpreted as counting a response as correct only if there is certainty on the response. This explains why we found for low SNRs performance levels of below 50% (which would be chance level in a forced-response paradigm). We found that the combination of the Lotka-Volterra equations with Bayesian inference is robust against this kind of noise for low SNRs higher than 2.6 and exhibits a graded deterioration of perfor- mance for smaller SNRs; see Fig 8A. To test the robustness against a wide range of non-baseline KC activity at odor onset, we randomized the initial states of the KCs in our model with a uniform probability distribution between zero (inactive) and one (the maximum normalized firing rate). We found that this random initial activity slightly impedes the accuracy of the response at the KC level when mea- sured with the conservative threshold approach above (<6% of cases, against <0.1% for base- line initial states). The mean reaction time for the non-baseline KC activity is slightly delayed and the variance of reaction times is slightly increased (see Fig 8B). In principle, this observed robustness against deviations of KC activity from baseline allows the model to process odors even when they follow each other quickly such that the odor recognition has no time to relax to 16 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 On the x-axis, we plotted the number of noisy PNs; on the y-axis, the percentage of trials in which the KCs displayed the correct representation (100 trials for each number of noisy PN/noise combination). Left: performance of an inverted Single-neuron SHS generative model. We used a modified version of our generative model, in which single KCs, as opposed to clusters of KCs, are activated sequentially. This is then projected to the PNs as described for our proposed model and inverted with the described unscented Kalman filter. Right: performance of proposed model with Cluster SHS. The large differences in the performance of the two models can be attributed to the within-cluster lateral input that the KCs receive (i.e. intelligent coincidence detector) in the Cluster SHS model: The performance of the single-neuron SHS model degrades for smaller number of noisy PNs, for all noise levels. In particular, for high noise rather small numbers of noisy PNs lead to poor performance. doi:10.1371/journal.pcbi.1004528.g010 doi:10.1371/journal.pcbi.1004528.g010 Intelligent coincidence detector. It has been found that a KC activates when the number of active and connected PNs surpasses a threshold [5,8,37]. For example, in honeybees this threshold was calculated to be around six out of the ten PNs that connect to a single KC [8]. This sort of coincidence-detector mechanism is thought to provide the brain with robustness against noise by responding to a range of PN responses instead of just a specific one [38]. Here, we will show that our model implements a coincidence detector which goes beyond simple thresholding of active PNs. We suggest that a similar mechanism may be used by the insect brain. The idea of simple thresholding is that the KCs evaluate how many of its con- nected PNs are active and only gets activated if a specific threshold of active PNs is surpassed. The way the proposed model improves on this intuitive procedure is as follows: Since each KC is part of a cluster, the activation state of the other within-cluster KCs and the temporally pre- ceding KCs is highly relevant for the activation state of that KC. Modelling Insect Olfaction with Bayesian Inference Fig 10. Performance of the model with noisy PN input. The performance of our model was measured in terms of displaying the expected sequence in response to the input while a number of PNs that connect to a single KC display large amounts of noise. Three noise levels were used (different colors); with high noise (SNR of only 1), the activity of a PN is mostly noise-driven, because the fluctuations due to noise are comparable to activity of the noise-free PNs. With an SNR of 2 (called here low noise), the noise variations are about one third of the maximum noise-free PN activity. Medium noise had an SNR of 1.5. On the x-axis, we plotted the number of noisy PNs; on the y-axis, the percentage of trials in which the KCs displayed the correct representation (100 trials for each number of noisy PN/noise combination). Left: performance of an inverted Single-neuron SHS generative model. We used a modified version of our generative model, in which single KCs, as opposed to clusters of KCs, are activated sequentially. This is then projected to the PNs as described for our proposed model and inverted with the described unscented Kalman filter. Right: performance of proposed model with Cluster SHS. The large differences in the performance of the two models can be attributed to the within-cluster lateral input that the KCs receive (i.e. intelligent coincidence detector) in the Cluster SHS model: The performance of the single-neuron SHS model degrades for smaller number of noisy PNs, for all noise levels. In particular, for high noise rather small numbers of noisy PNs lead to poor performance. Fig 10. Performance of the model with noisy PN input. The performance of our model was measured in terms of displaying the expected sequence in response to the input while a number of PNs that connect to a single KC display large amounts of noise. Three noise levels were used (different colors); with high noise (SNR of only 1), the activity of a PN is mostly noise-driven, because the fluctuations due to noise are comparable to activity of the noise-free PNs. With an SNR of 2 (called here low noise), the noise variations are about one third of the maximum noise-free PN activity. Medium noise had an SNR of 1.5. The reason why the inferred KC activity can closely follow the time-jittered input is that the Bayesian inference can easily delay or speed up KC activity to match the time-jittered PN activity. This finding indicates that the insect brain may use similar computations as used by the model to adjust KC activations to the ongoing time-jittered PN activity. Importantly, such a computational strategy to deal with time-jitter means that the internal model of the insect as encoded by the KC dynamics can be kept simple as typical deviations imposed by nature can be dealt with due to the robustness of neuronal sequences against such deviations. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 17 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference coincidence detector. To compute these results using our model we removed the cluster aspect by replacing clusters with single KCs. In essence, this single-neuron stable heteroclinic sequence (SHS) model is the Laurent-Rabinovich model combined with Bayesian inference and an observation equation for PNs. One can clearly see that recognition performance was dependent on both the number of noisy PNs and the SNR of the input. For high SNR, perfor- mance rate stayed high (i.e. > 90%) for as many as 7 out of 20 PN failures. For medium and low SNR, this threshold was 3 and just 1 PN failures, respectively. When we used the proposed cluster-SHS model (Fig 10B), the performance for high/medium/low SNR remained high (>90%) for up to 13/7/5 out of 20 PN failures. Another way of looking at this is to observe that the cluster-SHS model could still recognize at >90% performance when the single-SHS model was completely lost at 0% performance. This means that a key advantage of the cluster-SHS encoding over using just sequences of single KCs is that roughly double the amount of PNs may fail until recognition ventures below 90%. In sum, the model acts as an intelligent coincidence detector based on lateral influence at the KC level, as implemented by Bayesian inference. This lateral influence emerges because the generative model, following experimental observations, packs multiple (here, three) KCs into one cluster. Although speculative, we here presented a quantitative functional reason why few, but more than one KC, may encode the same odor: to be better guarded against noise, either in the stimulus and/or at the PN level. Projections of high-dimensional trajectories. In this section, we show that the model, without having been designed to do so, replicates another important experimental finding in insect olfaction. With experimental data, a visualization of high-dimensional PN population activity is achieved by a reduction to three dimensions, with a minimum loss of information; see [15] for a review. This is often done using linear local embedding [6,25,39] or principal components analysis (PCA) [7,40]. The response to a stimulus of the measured PN population is typically observed as closed trajectories in the phase space of the firing rate of the observed neurons and visualized using the first three components of the reduced system, see Fig 11A. One striking feature of these three-dimensional trajectories is that there are multiple points in which the trajectory makes sharp changes in direction. From [7] one can estimate the number of these points at around 5 or 6 during the dynamic phase. To our knowledge, a functional explanation for these sharp direction changes has not been presented yet. To address this question, we computed the same three-dimensional trajectory plots using PCA for the synthetic PN activity of our model during recognition (see Fig 11B). As can be seen from a comparison of Fig 11A and 11B, the resulting model plots look qualitatively similar to the experimentally measured plots. Although speculative, we posit that these sharp turning points represent the different elements of a KC cluster sequence. We found that, in our model, the number of turning points equals the number of clusters in a sequence (see Fig 11B). In other words, the three-dimensional trajectory plots gained from measured PNs may present vicarious evidence of a sequential activation of KC clusters. Note that we did not design the model to generate these trajectories. Rather, these trajectories are a consequence of the sequen- tial neuronal activity of KCs expressed in the high-dimensional PN activity. For example, if in our model a KC sees only a limited number of its PNs active, but its within-cluster KCs are fully activated at the same time and transmit this information by lateral connections, this is a strong sign for the KC that the low number of active PNs is just due to noise and it should activate as well, despite the contradicting activity of its input. However, if the within-cluster KCs are not activated, and a high number of PNs is inactive as well, this would be a clear sign that a KC should not acti- vate. In other words, for the proposed ‘intelligent’ coincidence detector, a KC receives both bottom-up sensory and lateral information. This intelligent coincidence detector is automati- cally done during recognition. A simple threshold detector would only receive bottom-up information. The improvement in recognition performance of an intelligent coincidence detector as com- pared to a simple one can be seen in Fig 10. In Fig 10A we show the performance of a simple PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 18 / 31 Discussion We modelled the time-evolving spatial patterns observed in the projection neurons (PN) of the antennal lobe and the Kenyon cells (KC) in the mushroom body of the insect brain by using a Bayesian model based on specific neuronal sequences. We were able to replicate several key experimental results, namely, the sparseness of the KC response patterns, the epochs of inhibi- tion and excitation in the response of the PNs, the fast recognition of odors and the remarkable PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 19 / 31 Modelling Insect Olfaction with Bayesian Inference Fig 11. Dimension reduction of the PN response to a stimulus. (A) Data as presented in [7] showing the trajectories in the first three components of principal component analysis for three different odors (different plots) and three stimulus durations for each odor (blue, red and green for 0.3s, 1s and 3s, respectively). The dynamic phase of activity lies between baseline activity (represented by B) and the fixed point (FP), where a number of turning points can be seen, between 2 and 4 for different cases. (B) First three elements of principal components analysis of the data obtained with the proposed model of the 30 PNs during the first phase of the full response, for different number of clusters in a sequence (4, 6 and 10 clusters). The number of elements equals the number of sharp turning points in the trajectory (big circles in the left-most plot). Around each turning point, the data points agglomerate, reflecting the perio in which a cluster remains active. Since we used data from only the onset sequential phase, the trajectories we present are not closed as in experimental data where the data acquisition is done up until the system returns to baseline, a few seconds after odor offset. The points B and FB, as before, represent th baseline (at odor onset) and the fixed point (end of onset sequential phase). Fig 11. Dimension reduction of the PN response to a stimulus. (A) Data as presented in [7] showing the trajectories in the first three components of principal component analysis for three different odors (different plots) and three stimulus durations for each odor (blue, red and green for 0.3s, 1s and 3s, respectively). Modelling Insect Olfaction with Bayesian Inference The proposed model was based on experimental findings and physiological data. The hier- archical nature of the odor processing in the proposed model was based on the traditional view of the way in which the insect brain processes odors [28]. The sequential activity in the PN and KC populations has been previously observed in vivo as part of the response to an odor [4,5,7]; additionally, experimental evidence has been found for the connections between the KC and PN populations used in the model [26,28]. In addition, the connections among KCs and among PNs in the proposed model are consistent with experimental findings [27,41]. The hierarchical processing of the proposed model appears to work in the same way as what is known about the insect brain: The traditional view is that the first step in processing the incoming signal happens in the antennal lobe, where the PNs process the information sent by the olfactory receptor neurons and transform it into a dense spatiotemporal coding. This infor- mation is sent to the mushroom body (among other targets), where the spatiotemporal coding is read. The KCs in the mushroom body exhibit very sparse spatiotemporal coding (for a review on the current view of the insect brain’s processing, see [28]). Importantly, although the model was built following experimental observations at hand, there were three findings which concur closely with experimental findings but emerged as unexpected features. These emergent findings indicate that the model captures some of the essence of what is important in insect olfaction. Firstly, we found that the steady state response may be explained by a temporally stretched version of the last sequence element of an odor. If one wanted to model this ‘directly’, one would probably formulate a model which transitions after a sequential phase to a fixed point attractor and back to a sequence for the experimentally observed phase. We found that this is not necessary; rather the same dynamic sequence model can be used. One requirement for this to work is that one uses Bayesian inference to model how the sensory input guides the internal KC states. Secondly, we found that the model replicated the typical three-dimensional state-space tra- jectories of PN activity. It came as a surprise to us how accurately the model response replicated the experimentally observed trajectories. Discussion The dynamic phase of activity lies between baseline activity (represented by B) and the fixed point (FP), where a number of turning points can be seen, between 2 and 4 for different cases. (B) First three elements of principal components analysis of the data obtained with the proposed model of the 30 PNs during the first phase of the full response, for different number of clusters in a sequence (4, 6 and 10 clusters). The number of elements equals the number of sharp turning points in the trajectory (big circles in the left-most plot). Around each turning point, the data points agglomerate, reflecting the period in which a cluster remains active. Since we used data from only the onset sequential phase, the trajectories we present are not closed as in experimental data where the data acquisition is done up until the system returns to baseline, a few seconds after odor offset. The points B and FB, as before, represent the baseline (at odor onset) and the fixed point (end of onset sequential phase). doi:10.1371/journal.pcbi.1004528.g011 robustness of the model to different types of noise and unexpected deviations. In addition, as an emerging behavior of the model, we found that the model delivered novel, mechanistic explanations for two experimentally observed but yet unexplained phenomena: (i) the exis- tence of a steady state phase in the response of the PN and KC populations and (ii) the experi- mentally-observed points of sudden direction changes in the trajectories of the first three response components. In addition, we found that the KCs in our model behave as intelligent coincidence detectors of PN activity, which, as we showed, helps odor recognition in the pres- ence of noise. Finally, we showed that the model can actually decode PN firing rate activity with high recognition performance. Although a future application, we anticipate that the pro- posed model for insect olfactory recognition may serve as a first step towards building real- world machines for artificial odor recognition. 20 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Bayesian inference as a decoding mechanism A key component of our model is the Bayesian inference, which helps the KCs decode the information contained in the PN activity, making the proposed model a recognition model. This decoding mechanism goes beyond a simple feedforward connection from PNs to KCs, in that it balances the expectations that the brain has about the internal dynamics of the KCs with the information contained in the PN activity. The connections that are created between PNs and KCs by the Bayesian inference through the Kalman gain (see Methods) are Bayes-optimal, in the sense that they minimize the so-called precision-weighted prediction error to obtain the best results possible. There is an underlying assumption in the proposed model: that PNs and KCs perform com- putations similar to what Bayesian inference would prescribe given a generative model (of how PN activity arises from odor reception). As we proposed a firing rate model, there is no link yet to the single neuron level. It is unclear how the proposed model can be implemented by net- work of single spiking neurons, but the idea that the brain behaves as an optimal Bayesian observer in perceptual decision-making tasks has already been substantiated and possible neu- ral circuits have been suggested [18,42–45]. In this work, we demonstrate how Bayesian infer- ence, along with an appropriate generative model, can act as a decoding mechanism and explain the performance of the insect brain in recognizing odors, as well as the remarkable robustness, and other phenomena observed in experimental data. Modelling Insect Olfaction with Bayesian Inference Metastable states as a coding mechanism The exact way in which the insect brain’s PNs encode the incoming olfactory information is still unclear. Based on the comparison between our model and experimental data, in particular the tri-dimensional trajectories obtained with PCA (section ‘Projections Of High-Dimensional Trajectories’), we suggest that the antennal lobe encodes the identity of an odor as a sequence of a small number of metastable states in the configuration space of PN firing rates. This means that an odor is represented in the antennal lobe as a collection of points both in the phase space of PN firing rates and the transitions between these points (i.e. as a stable heterocli- nic sequence). The exact number of these points may be odor-dependent, but given the speed at which PN activity changes, which has been reported from experimental data to be around 50–300ms, this number could be as low as four. Thus the odor representation could comprise, during the onset sequential phase, five metastable points and the transitions between them. The close match of these PN trajectories suggests that their cause is the sequential and sparse KC activity. Although speculative, the congruence of the computational model and experimental observation may be seen as evidence that the underlying KC activity is structured spatiotemporally as elements of a sequence. If this were not the case, one would not observe the sharp turns in our model plots. If the number of sharp turning points in experimental data is fixed at around four or five during the dynamical phase, as indicated by experimental results, our model results suggest that the evolution of the PN population’s response to a stimulus can be divided in four or five main stages that are well dis- tinct from one another (each one representing an unstable equilibrium point in the KC firing rate space), making the readout (by downstream neurons) a relatively easy task. This interpre- tation is supported by experimental findings in locusts [7,29], where the PN population changes completely every 50–300ms. Thirdly, we found that the model can decode (synthetic) PN firing rates with ceiling perfor- mance and is rather robust against noise and deviations (see Figs 8 and 10). One further finding was that the model implements at the KC level a smart coincidence detector on PN activity. Taken together, these findings are remarkable, because we achieved this high performance and robustness only by closely following the experimentally established blue-print of the olfactory insect brain. Without these ‘experimental’ instructions on how to build an olfactory recogni- tion model, we would have not arrived at the high robustness of recognition. For example, by using sequences of KC clusters, as opposed to sequences of single KCs, we found a stark improvement in robustness (Fig 10). We believe that this finding is important because it sug- gests that the insect brain may actually use the hypothesized cluster sequence mechanism to optimize its recognition performance and robustness. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 21 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 How many odors can the model recognize? The insect brain is able to recognize many odors and even mixtures of odorants with different ratios [6,25] and for each one of these, a representation exists in the brain. In the proposed model, a high number of these representations can be encoded in a single connectivity matrix, like in the insect brain. Exactly how many sequences can be stored and recognized by the model will depend on a number of factors, most notably on the size of the KC population. Here, we discuss the capacity of the model in terms of the KC population’s size. To explore the capacity of the model, we ran simulations with bigger population sizes to test how many sequences can be embedded while maintaining the model’s ability to accurately rec- ognize them. We ran multiple simulations and found that the maximum number of sequences in a model grows faster than the size of the KC population and this growth is non-linear (e.g. polynomial). Importantly, we found that for KC numbers below 100, the system can store and accurately recognize only as many different odors as there are KCs (90). However, when going beyond 100 KCs, the number of odors that can be stored and recognized grows faster than the number of KCs. For example, for 500 KCs, 900 odors could be stored and recognized accurately (success rate > 95%). Simulations with more than 500 KCs are technically possible but we had to abandon these simulations because the computer run times became prohibitive (> 1 week on a modern desktop computer to assess the 500 KCs recognition performance). We are confi- dent that the current Matlab implementation can be improved upon using an implementation using parallel computing (as used by the insect brain). It is an open question how many differ- ent odors could be recognized with 50,000 KCs (as in the locust). The proposed model would be an ideal tool to address this question, once the implementation is fast enough. For future work, there are at least three ways in which one can improve the model’s capacity further, i.e. increase the number of recognizable odors while the number of KCs remains the same. Firstly, in this work we chose the sequences randomly, which means that some KCs can belong to many clusters. Modelling Insect Olfaction with Bayesian Inference One interesting feature of the Lotka-Volterra equations as a modelling device of sequential activation of neurons is their remarkable robustness against unexpected input and noise and initial conditions different from zero, their trial-by-trial reproducible dynamics and the mathe- matical depth at which they have been studied previously [22,56]. Here, we incorporated both the sequential neuronal activations in the generative model, as introduced in [11,21], and the decoding mechanism to identify in an online fashion the odors with Bayesian inference. The SHS in the generative model grants the model with robustness against noise in the neuronal dynamics, which is further improved by the Bayesian inference which gives the model additional robustness against neuronal and sensory noise. The inclusion of clusters of KCs in our model, coupled with the ability of the Bayesian inference to reconcile conflicting data, gives the model a reliable and flexible mechanism as implemented by PN and KC activity. Previous models of neuronal sequential activations Sequential spiking of neurons or groups of neurons has been modelled before. A well-estab- lished model for spiking neurons are synfire chains, where groups of synchronously firing neu- rons can be set to fire in a specific and reproducible sequence, using feedfoward connections [46,47]. In another study, the parameters in the Hodgkin-Huxley model were modified to allow for the sequential firing of neurons in a desired sequence [48] and in [49], the authors show how sequential switching can be obtained with random connectivity. For decoding, hid- den Markov models have been used for identifying clusters of neurons firing together in a trial- invariant sequence as a response to a taste stimulus in rodents, e.g. [50]. In firing-rate models, an influential model for sequential activation is the Laurent-Rabino- vich model [11,21], where the connectivity between the neurons establishes a particular sequence of neuronal activations which is followed consistently across different trials. This model introduced the use of the Lotka-Volterra equations for sequential neuronal activations, which have been subsequently used successfully in areas as different as birdsong recognition [51,52], visual perception [53], handwriting recognition [54] and dendritic dynamics [55]. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 22 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Generative model Lotka-Volterra equations for KC activity. The Lotka-Volterra equations were introduced as a model for sequential dynamics in olfaction in [11,21]. For a population of N neurons they are: _xi ¼ xiðsi X N j¼1 rijxjÞ þ Z 3 3 where i = 1,2,. . ., N, xi is the state of the i-th neuron, σi is a parameter, η is noise and ρij is the connectivity matrix between the neurons of the space. Under the condition ρii = 1, for all i = 1,2,. . ., N, the system possesses a collection of equilibrium points of the form Qi = (0,0,. . ., σi,0,. . .,0), where the non-zero entry is at the i-th position. For our generative model, we dropped the dependence of σi and ρij on the system’s input that was used in [11], making them instead a ﬁxed parameter of the system. A stable heteroclinic sequence (SHS) is a collection of equilibrium points and segments of trajec- tories that join them pairwise, i.e., a trajectory that goes from the first equilibrium point to the next, until the last one in a given sequence is reached. A stable heteroclinic channel (SHC) is a neighbor- hood around the SHS such that any trajectory that enters it will not leave it until the sequence ends at the last equilibrium point. A formal definition of these concepts can be found in [22]. Under the conditions in [22], the system in Eq 3 exhibits an SHS (and SHC). These condi- tions set the values of the connectivity matrix and of the constant terms σi in such a way that a neuron excites the next neuron in a sequence and highly inhibits all others. With this, the neu- rons are activated in a reproducible sequence. For our model, we modified these conditions to enable us to have more than one compo- nent xi be non-zero in an equilibrium point. Therefore, the equilibrium points in our system have the form Qi = (0,0,. . .,ai,0,. . .,0,ai,. . .), where the non-zero entries are in positions mij, with j = 1,. . .,Ai, and Ai is the number of non-zero entries in this equilibrium point. Methods The model consists of two parts. The first one, called the generative model, is the part that sets the dynamics of neuron populations (both PNs and KCs). The second part is the Bayesian inference, which takes the output of the generative model (the PN population activity) and infers the hidden KC activity that is consistent with this input, identifying the presented odor, if it has been encountered before. For the generative model we used the Lotka-Volterra equa- tions, to generate activity qualitatively similar to that observed in the mushroom body’s Ken- yon cells (KC) and the projection neurons (PN) of the antennal lobe. Modelling Insect Olfaction with Bayesian Inference How many odors can the model recognize? A more careful selection of the clusters in the sequences will most likely lead to an increase in the number of recognizable sequences. We expect that this is pre- cisely what is happening in an insect brain, where KC connections are optimized, probably both during lifetime and by evolutionary processes. Secondly, the exact shape of the expected sequences (both at the PN and KC levels) plays an important role in the recognition. For our simulations, we used sequences similar to those in Fig 5B, where all KCs in a cluster rise at the same time and to the same maximum firing rate. A less stringent definition of a “sequence” will most likely lead to a much larger number of embeddable sequences. Thirdly, the exact weights of the connections between KCs may be improved further. While those used throughout this paper (see Fig 1) give accurate recognition results, a further optimization of the connectivity scheme could lead to a further improvement in model capacity. 23 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference We found that to preserve the stability of the SHS the connectivity matrix must obey Eq 4 and the within-cluster inhibition must be stronger than the inhibition from a cluster to the pre- vious cluster in the sequence. Additionally, the number of embedded sequences (depending on the size of the population) must be low enough. We ensured that these conditions hold by using the reported values of σi and ρij. Projection to PN space. The Lotka-Volterra equations in Eq 3 are used to model the behavior of the KC population in the insect brain. The neuronal states (firing rates) from these equations are then passed through an observation equation, which transforms these KC firing rates into PN firing rates, and is given by: Y ¼ YX þ G 5 Y ¼ YX þ G 5 where Y is the N×1 vector of PN ﬁring rates, X is the M×1 vector of KC ﬁring rates from the Lotka-Volterra system, Θ is a N × M observation matrix, and Γ is Gaussian noise. For every KC, we chose randomly a set of PNs that connect to it. The components of the observation matrix Θ are all ones and zeroes and implement the connections between KCs and PNs, i.e., if a KC connects to a PN, the corresponding component of Θ is 1, otherwise it is 0. The generative model can be written in terms of prior and posterior distributions over the PNs, i.e., if a KC connects to a PN, the corresponding component of Θ is 1, otherwise it is 0. The generative model can be written in terms of prior and posterior distributions over the hidden states (KC firing rates) and observed states (PN firing rates) as follows: The generative model can be written in terms of prior and posterior distributions over the hidden states (KC firing rates) and observed states (PN firing rates) as follows: Pðx1:T; y1:TÞ ¼ Pðx1Þ Y T t¼2 Pðxtjxt1Þ Y T t¼1 PðytjxtÞ Pðxtjxt1Þ ¼ N ðFðxt1; QINNÞÞ PðytjxtÞ ¼ N ðgðxtÞ; RIMMÞ where x is an N-dimensional vector of real numbers that represents the KC dynamics (ﬁring rate) and y is an M-dimensional vector for the PN dynamics; x1:T is a sequence of values for x obtained at times 1,2,. . .,T; F(x) is a discretized version of Eq 3 (see below), g(x) is the right- hand side of the observation equation (Eq 5) and Q and R are the state and observation noise precisions, respectively; IN×N and IM×M are identity matrices. N ð; Þ is the Gaussian distribution. To be able to work with Eqs 3 and 5, we must first discretize Eq 3 to obtain the form xt+1 = F (xt). We do this by integrating using the forward Euler method [58], transforming Eq 3 into: xtþ1 ¼ xt þ Dtf ðxtÞ 6 6 where f(xt) is the right-hand side of Eq 3. Given the small time steps used during our simula- tions (Δt 0.1), the forward Euler method provides a stable and precise solution to these dif- ferential equations. Similarly, the discretization of Eq 5 is: where f(xt) is the right-hand side of Eq 3. Given the small time steps used during our simula- tions (Δt 0.1), the forward Euler method provides a stable and precise solution to these dif- ferential equations. Similarly, the discretization of Eq 5 is: yt ¼ gðxtÞ 7 7 where g(xt) is the right-hand side of Eq 5. Note that g(xt) from Eq 5 contains a term of Gaussian noise, so it is not included in Eq 7. Generative model The values of ai are set by the parameters in Eq 3: for example, in the case where all the KCs in a cluster connect to each other with the same strength ρ, then all values ai equal σi / ρ. We now present conditions under which these points are equilibrium points of the system and an SHS arises. First, to guarantee that the points Qi are equilibrium points, the following condition has to be met: For x = Qi, si X Ai j¼1 rimijxmij ¼ 0 4 si X Ai j¼1 rimijxmij ¼ 0 4 For simplicity, we set σi = 1 for all i = 1,2,. . ., N, and to satisfy the condition in Eq 4, we set rimij ¼ ðAiÞ 1. The values for the connectivity matrix ρ were set as presented in ‘Model parame- ters used for simulations’ and Fig 1B. 24 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference the observed states, Bayesian inference balances the observed data with what it knows of the dynamics of the generative model in order to estimate the values of the hidden states that best fit the observed variables. We made use of the unscented Kalman filter (UKF) as a Bayesian inference implementation [57]. The UKF works by comparing the data being observed at a given time step with a prediction made by the UKF in the previous step. These predictions are made using the generative model’s equations, so they follow its dynamics, and are done both for the observation and hidden vari- ables. The difference between the observation and the prediction, called prediction error, is com- puted for both levels. These two prediction errors and the equations in the generative model are used to make the next-step prediction for both levels, such that the prediction error of one level can affect the prediction of the other. This back and forth exchange of information is what creates the extra connections in the model (i.e. the connections from PNs to KCs). At every time step in the process, these calculations and predictions are made and the expectations adjusted. More specifically, the Bayesian inference continuously infers the states of the KCs from observed PN firing rates that were generated by Eq 5. We used the unscented Kalman filter (UKF) to estimate the states of the hidden variables xt (the firing rates of the KCs) from the observed data yt (the PN firing rates). At every time step t, the current estimates xt and yt (start- ing with some initial conditions for the first step, which we set to all zeroes or randomly), and a minimal set of points surrounding them, called sigma points, are propagated through the non- linear equations of the generative system (Eq 6); assuming that the distribution of the hidden variables is Gaussian, a prediction for the mean and the variance of the hidden variables (KCs) and the observed variables (PNs) is calculated as a weighted sum of the propagated sigma points. In the next time step, these predictions are used to estimate the current states of the hid- den variables with the update equation xt ¼ xt þ Kðyt ytÞ, where the Kalman gain K repre- sents the precision expected from the data relative to the precision expected from the nonlinear dynamics of the system and is computed with the covariance matrices of the hidden and observed variables as calculated with the sigma points. In preparation for the next time step, a new prediction is calculated using the current estimation and the process is repeated. The filter takes as input the PN activity and as priors an initial condition of the KC popula- tion and the covariance matrix for the noise vectors (H)i = ηi from Eq 3 and Γ from Eq 5. We set the initial state of the KCs to zeroes (or randomly, for testing for robustness) and the noise covariance matrices (priors) are unit matrices multiplied by two different constants (Q for the hidden states, R for the observed states). These constants, called precisions, determine the rela- tive importance given either to the observations (PN readings) or to the internal dynamics of the KCs. For all our simulations, we used values of Q = 0.1 and R = 0.001. These values were chosen because they provide the best balance between following the observed PN data and fol- lowing the expected KC dynamics. Given that Bayesian inference does not require the entire data set, but only the data for the given step and the prediction from the previous one, this process can be done online, that is, it can be done as data acquisition is happening, without having to wait for the entire process to be over. This characteristic makes the Bayesian inference a plausible mechanism through which the brain can decode the information encoded in the PN responses. Bayesian inference We implemented a Bayesian inference scheme that allows the system to identify a perceived stimulus. Similar setups, with Bayesian inference working on a generative model based on SHS, have proven fruitful in other applications [14,51,54]. In general, Bayesian inference observes variables whose states are known to be caused by other, hidden variables. Through the nonlin- ear equations of a generative model, which describe the way in which the hidden states cause PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 25 / 31 Raster plots To generate the illustrative raster plots shown in Fig 3, we adapted our generative model to generate spikes while keeping its fundamental structure. We created a hierarchical three-level model, where the third (top) level employs the proposed cluster-encoding Lotka-Volterra equations (see Fig 1). These are output to the second level, representing the KCs (Fig 3A, right panel), which are modeled using FitzHugh-Nagumo equations to generate spikes [59]. The input from the third to the second level raises the membrane potential in the FitzHugh- Nagumo equations to the threshold of spiking. Spikes are generated by adding noise to pass the firing threshold. For simplicity, we used Gaussian noise. The states of the second level (representing the membrane potential of the KCs) are the input to the first (lowest) level, which represents the PNs, also modeled with the FitzHugh- Nagumo equations (see Fig 3A, left panel) in the same way: Only when the input from the sec- ond level takes the membrane potential close to threshold, spikes are occasionally generated both by Gaussian noise and by the spikes of the second level. The connections between the PNs and the KCs in this model are made in the same way as in our model: by a random 1 to 20 projection from KCs to PNs with equal weights. This model was only used to generate the data to create the plots in Fig 3. Because of this, we did not apply Bayesian inference to it. For a full description of the three-level hierarchical model see S1 Text. Modelling Insect Olfaction with Bayesian Inference For the data generation phase, the following steps were followed: (1) Set-up the parameters of the system, e.g. population sizes. (2) Select the clusters that will form the sequences. We did this randomly, minimizing repetition of neurons in different sequences. (3) Create the connec- tivity matrix, embedding all the desired sequences in it. (4) Set-up the initial conditions for all the neurons. This step determines which sequence will be generated: to generate data with a sequence, the initial conditions must be around the first equilibrium point of that sequence, i.e. the first cluster of the sequence must be activated. (5) Integrate Eq 3 to obtain KC activity. (6) Randomly generate an observation matrix and generate PN activity using Eq 5. Of special note are the algorithms for creating the clusters and sequences, and for generating the connectivity matrix. These can be seen in the files cgenerate_clusters.m and cget_rho.m, respectively. Additionally, the generation of the observation matrix can be seen in the file cge- nerate_neurons.m. For the inversion phase, the steps are the following: (1) Set the precision parameters. (2) Set the initial conditions, which were typically set to zeroes. (3) Call the UKF using the PN data generated previously as input. The implementation of the UKF is contained in the file UKF.m. Simulation setup In this section, we present an overview of the steps necessary to implement our model. To com- plement this, source code for the implementation, as well as examples, can be found at [https:// github.com/dcuevasr/Olfaction/]. The simulations are divided in two steps: firstly, data genera- tion; secondly, inversion. 26 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Full stimulus data We created PN activity which presents the three phases of the response to an odor (see Fig 7). To do this, we generated the response in the KCs for one sequence for the first phase, then repeated the last point in the sequence for a certain time to generate the response in the second phase; finally, we added the response of a second sequence (embedded in the same connectivity matrix) at the end for the third phase (Fig 7B). We created the PN response with a 1:20 projec- tion (i.e. each KC is connected to 20 PNs), as used in the other simulations. Using this full response PN activity as input to the Bayesian inference, we measured the inferred KC responses. Author Contributions Conceived and designed the experiments: DCR SJK. Analyzed the data: DCR SB SJK. Contrib- uted reagents/materials/analysis tools: DCR SB SJK. Wrote the paper: DCR SB SJK. Conceived and designed the experiments: DCR SJK. Analyzed the data: DCR SB SJK. Contrib- uted reagents/materials/analysis tools: DCR SB SJK. Wrote the paper: DCR SB SJK. Modelling Insect Olfaction with Bayesian Inference In the section ‘Intelligent coincidence detector’, we make use of this criterion to compare the performances of our model and the single-neuron modification in order to quantify the effect of the lateral inhibition received by KCs from those KCs with which it shares a cluster. For each model, we ran a hundred trials each for all combinations of the number of extra-noisy PNs (between one and twenty) and extra noise added to these PNs (with SNRs of 2, 1.25 and 1). For each of these trials, we considered the odor to be properly identified if the Euclidean dis- tance between the inferred and expected responses was lower than the 0.1 threshold. In Fig 6, the variable Recognition, which goes from -1 for the incorrect representation to 1 for the correct (expected) one, was calculated using the following formula: R ¼ Dc Di Dri 8 R ¼ Dc Di Dri 8 where R is the recognition, Dc is the distance between the observed KC activity and the correct (expected) one; Di is the distance between the observed activity and the incorrect one (the other sequence in the system) and Dri is the distance between the two sequences embedded in the connectivity matrix. Supporting Information S1 Text. Hierarchical spiking model. Description of a hierarchical spiking model used in the generation of Fig 4. (PDF) S1 Text. Hierarchical spiking model. Description of a hierarchical spiking model used in the generation of Fig 4. Decision making: Choice and reaction time When making decisions and to generate reaction times, we compare the KC response obtained by the Bayesian inference with the expected KC response (used to generate the data). In partic- ular, we compute the Euclidean distance between the two trajectories in the firing rate phase space of the KCs and define recognition of an odor as correct, if the Euclidean distance drops below a threshold anytime during the trial. We further defined reaction time as the time at which the threshold is crossed. We set a threshold of 0.1 for our simulations, which is 0.1 times the maximum value of the firing rate of a KC. We found that once this threshold is crossed, the two trajectories (inferred and the data) do not drift apart for the rest of the response, making this threshold a good assessment of whether the inference is choosing the correct representation. 27 / 31 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004528 October 9, 2015 Modelling Insect Olfaction with Bayesian Inference 6. Stopfer M, Jayaraman V, Laurent G. Intensity versus Identity Coding in an Olfactory System. Neuron. 2003; 39: 991–1004. doi: 10.1016/j.neuron.2003.08.011 PMID: 12971898 7. 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https://openalex.org/W4361937425	https://figshare.com/articles/journal_contribution/Figure_S6_from_Single-Cell_Derived_Primary_Rectal_Carcinoma_Cell_Lines_Reflect_Intratumor_Heterogeneity_Associated_with_Treatment_Response/22477115/1/files/39928613.pdf	English	null	Figure S1 from Single-Cell–Derived Primary Rectal Carcinoma Cell Lines Reflect Intratumor Heterogeneity Associated with Treatment Response	null	2,023	cc-by	187	Supplementary Figure S6 SUPPLEMENTARY FIGURE S6: Volcano plots of gene expression analysis Resistant (1G10) vs. sensitive AA. Resistant (2D2) vs. sensitive B B. Resistant (2G4) vs. sensitive C C. Supplementary Figure S6 SUPPLEMENTARY FIGURE S6: Volcano plots of gene expression analysis Resistant (1G10) vs. sensitive AA. Resistant (2D2) vs. sensitive B B. Resistant (2G4) vs. sensitive C C. plementary Figure S6 LEMENTARY FIGURE S6: Volcano plots of gene expression analysis based on total sequencing. Differential expression of genes between the three most sensitive SCDCLs 1D2 and 2F1) and the three resistant SCDCLs 1G10 (A), SCDCL 2D2 (B), and SCDCL 2G4 Resistant (1G10) vs. sensitive AA. Resistant (2D2) vs. sensitive B B. Resistant (2G4) vs. sensitive C C. Resistant (1G10) vs. sensitive AA. Resistant (2D2) vs. sensitive B B. Resistant (2D2) vs. sensitive B B Resistant (2G4) vs. sensitive C C. Resistant (2G4) vs. sensitive C SUPPLEMENTARY FIGURE S6: Volcano plots of gene expression analysis based on total RNA sequencing. Differential expression of genes between the three most sensitive SCDCLs (2F4, 1D2 and 2F1) and the three resistant SCDCLs 1G10 (A), SCDCL 2D2 (B), and SCDCL 2G4 (C).
https://openalex.org/W3185254526	https://periodicos.uem.br/ojs/index.php/rbhe/article/download/52914/751375151345	Portuguese	null	"A mulher soldado" nos palcos (São João Del-Rei, Minas Gerais, 1916)	Revista Brasileira de História da Educação	2,020	cc-by	12,917	A MULHER SOLDADO ON STAGE (SÃO JOÃO DEL REI, MINAS GERAIS, 1916): AN OPERET IN SCENE EDUCATING SENSIBILITIES A MULHER SOLDADO EN LOS ESCENARIOS (SÃO JOÃO DEL-REI, MINAS GERAIS, 1916): UNA OPERETA EN ESCENA EDUCANDO SENSIBILIDADES Carolina Mafra de Sá1, Ana Maria de Oliveira Galvão2 1Universidade Federal do Agreste de Pernambuco, Garanhuns, PE, Brasil. 2Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil. Autor para correspondência. E-mail: mafradesa@gmail.com Carolina Mafra de Sá1, Ana Maria de Oliveira Galvão2 1Universidade Federal do Agreste de Pernambuco, Garanhuns, PE, Brasil. 2Universidade Fede Belo Horizonte, MG, Brasil. Autor para correspondência. E-mail: mafradesa@gmail.com R e s u m o : As apresentações da opereta A mulher soldado em São João del-Rei - MG (1916) foram muito apreciadas pelo público e pela imprensa e se configuraram como performances que educaram sensibilidades. Este artigo analisa o manuscrito da peça, jornais locais e uma peça são-joanense do período, fontes encontradas no acervo que pertencia ao grupo amador responsável pelos espetáculos. Buscamos compreendera educação das sensibilidades que teria ocorrido durante essas encenações. Apoiadas nos estudos sobre a história da educação das sensibilidades, identificamos que os espetáculos ao mesmo tempo em que reforçavam sensibilidades ligadas à dominação masculina, impostas por uma elite dominante, davam margem para que pessoas simpáticas à luta feminista experimentassem novas sensibilidades. P a l a v r a s - c h a v e : história da educação das sensibilidades, gênero, história dos esp A b s t r a c t : The presentations of the operetta A mulher soldado in São João del-Rei - MG (1916) were highly appreciated by the public and the press and were configured as performances that educated sensibilities. This article analyzes the play's manuscript, local newspapers and a local author play, from sources found in the collection that belonged to the amateur group responsible for the shows. Weseek to understand the education of the sensibilities that would have occurred during these performances. Supported by studies of the History of Education of the Sensibilities, we identified that the shows, while reinforcing sensibilities linked to male domination, imposed by a dominant elite, allowed people sympathetic to the feminist struggle to experience new sensibilities. K e y w o r d s : history of education of the sensibilities, gender, history of spectacles. R e s u m e n : Las presentaciones de la opereta A mulher soldado en São João del-Rei (1916) fueron muy apreciadas por el público y por la prensa y se configuraron como performances que educaron sensibilidades. R E V I S T A B R A S I L E I R A D E H I S T Ó R I A D A E D U C A Ç Ã O ( v . 2 1 , 2 0 2 1 ) A R T I G O O R I G I N A L R E V I S T A B R A S I L E I R A D E H I S T Ó R I A D A E D U C A Ç Ã O ( v . 2 1 , 2 0 2 1 ) A R T I G O O R I G I N A L 1 Trata-se da tese de doutorado intitulada Do convento ao quartel: a educação das sensibilidades nos espetáculos teatrais realizados pelo Club Dramático Arthur Azevedo, em São João del-Rei – MG (1915- 1916), realizada por Carolina Mafra de Sá, sob orientação da professora Ana Maria de Oliveira Galvão. A pesquisa foi financiada pela Capes, assim como o doutorado sanduíche na Université Paris Ouest Nanterre La Défense que foi de grande importância para o desenvolvimento da investigação. A MULHER SOLDADO ON STAGE (SÃO JOÃO DEL REI, MINAS GERAIS, 1916): AN OPERET IN SCENE EDUCATING SENSIBILITIES A MULHER SOLDADO EN LOS ESCENARIOS (SÃO JOÃO DEL-REI, MINAS GERAIS, 1916): UNA OPERETA EN ESCENA EDUCANDO SENSIBILIDADES Analizamos el manuscrito de lapieza, periódicos locales y una pieza sanjoanense del período, fuentes encontradas en el acervo que pertenecía al grupo responsable por los espectáculos. Buscamos comprender la educación de las sensibilidades que podrían Haber tenido lugar en esas presentaciones. Apoyadas en los estudios sobre la historia de la educación de las sensibilidades, identificamos que los espectáculos al mismo tiempo que reforzaban sensibilidades vinculadas a la dominación masculina, daban margen para que las personas simpatizantes conla lucha feminista experimentas en nuevas sensibilidades. P a l a b r a s c l a v e : historia de la educación de las sensibilidades, género, historia de los espectáculos. http://dx.doi.org/10.4025/rbhe.v21.2021.e144 e-ISSN: 2238-0094 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades 2 “Si bien huellas de estos temas son rastreables en trabajos previos - por ejemplo, en historias que se ocupan de la infancia, del currículum, e de la formación docente – los efectos del llamado ‘giro afectivo’ (Lara & Enciso Domínguez, 2013; Macon & Solana, 2015) de los últimos tempos permitieron nuevos recortes y profundizaciones mediante la construcción de abordajes específicos. Este ‘giro’ temático de las ciências sociales - con especial mención del impulso liderado por la antropología – supuso un cambio em la producción de conocimiento sobre tópicos tales como los afectos, la sensibilidad, las emociones y los gustos, principalmente reconociendo su variabilidad cultural e histórica (Lutz & White, 1986)”. INTRODUÇÃO A pesquisa que deu origem ao presente artigo dedicou-se a compreender a educação das sensibilidades que se deu a partir de apresentações teatrais realizadas por um grupo de teatro amador, na cidade mineira de São João del-Rei, no ano de 19161. Segundo Oliveira (2018), nos últimos 20 anos, a História da Educação experimentou “[...] uma inflexão em direção aos sentidos e às sensibilidades [...]”, oportunizada “[...] pela renovação dos estudos em História da Educação e a sua intensa aproximação do campo da História na América Latina” (Oliveira, 2018, p. 120). Embora o interesse pelo tema dos sentidos e sensibilidades não seja algo novo entre os historiadores, Oliveira (2018), assim como Pineau (2018) identificam, mais recentemente, o que para este último se trata de uma ‘virada afetiva’ do campo. Pineau (2018), em balanço das investigações do campo da história da educação sobre estética e sensibilidades na América Latina, observa uma mudança em relação aos objetos de estudos. Para o autor, há uma ‘virada afetiva’; pesquisadores voltam-se à análise de sujeitos, discursos, formas de distribuição, produção e apropriação de conhecimentos e práticas vinculados ao mundo sensível. Pineau (2018, p. 2, grifo do autor, tradução nossa) 2 afirma que Embora traços desses temas sejam rastreáveis em trabalhos anteriores - por exemplo, em histórias que tratam da infância, do currículo e da formação de professores - os efeitos da chamada ‘virada afetiva’ (Lara & Enciso Domínguez, 2013; Macon & Solana, 2015) dos últimos tempos permitiram novos recortes e aprofundamentos através da construção de abordagens específicas. Essa ‘virada’ temática das ciências sociais - com especial ênfase no impulso liderado pela antropologia – configurou uma mudança na produção de conhecimento sobre questões como os afetos, a sensibilidade, as emoções e os gostos, principalmente reconhecendo sua variabilidade cultural e histórica (Lutz & White, 1986). 2 “Si bien huellas de estos temas son rastreables en trabajos previos - por ejemplo, en historias que se ocupan de la infancia, del currículum, e de la formación docente – los efectos del llamado ‘giro afectivo’ (Lara & Enciso Domínguez, 2013; Macon & Solana, 2015) de los últimos tempos permitieron nuevos recortes y profundizaciones mediante la construcción de abordajes específicos. INTRODUÇÃO Este ‘giro’ temático de las ciências sociales - con especial mención del impulso liderado por la antropología – supuso un cambio em la producción de conocimiento sobre tópicos tales como los afectos, la sensibilidad, las emociones y los gustos, principalmente reconociendo su variabilidad cultural e histórica (Lutz & White, 1986)”. p. 2 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. A investigação aqui apresentada está afinada com a tendência da ‘virada afetiva’ nas ciências sociais e, como nos ensina Oliveira (2018), diante do reconhecimento dos limites das histórias de caráter generalizante, busca compreender a materialidade da vida, as respostas que as pessoas dão às diferentes situações vividas, as sensibilidades envolvidas nessas situações. Objetiva apreender, ainda, além dos discursos, como se reagia a eles, ou seja, “[...] compreender as respostas emocionais de diferentes indivíduos aos imperativos sociais” (Oliveira, 2018, p. 121). Analisamos, portanto, os processos de educação das sensibilidades vivenciados em espetáculos teatrais que ocorreram em São João del-Rei no ano de 1916. É importante destacar que, entre final do século XIX e início do século XX, aquela cidade vivia intensas transformações. Novidades tecnológicas, como a estrada de ferro, a eletricidade, o telégrafo, a indústria, a tipografia e o desenvolvimento da imprensa local modificaram o espaço público, os modos de sociabilidades3. Os tempos e os espaços do trabalho, do lazer e do convívio se transformavam. Além da tradicional religiosidade, representada pelos cultos católicos e pelas festas religiosas tradicionais, em São João del-Rei existiam diversos espaços de sociabilidade e variadas formas de lazer. Os são-joanenses assistiam e participavam de campeonatos de futebol4, frequentavam cafés e o cinematógrafo5, assistiam a espetáculos de circo e de teatro apresentados por companhias itinerantes, por grupos amadores e por profissionais da cidade. Neste contexto, o Club Dramático Arthur Azevedo tinha por objetivo realizar apresentações de destaque dando provas do ‘alto grau de civilidade’ daquela gente6. Liderado por militares de alta patente, homens de letras e do governo, o grupo de teatro amador era constituído por artistas locais, homens, mulheres e crianças das elites são-joanenses. Tal agremiação realizava apresentações no Teatro Municipal de operetas que, embora criticadas pelos literatos do período, foram muito aplaudidas em cidades consideradas ‘adiantadas em civilização’ como Paris, Lisboa, Rio de Janeiro. 6 Para mais informações sobre o Club Dramático Arthur Azevedo, seus membros e seu repertório ver Sá (2015). Para mais informações ver Guerra (1968). Para mais informações ver Adão, Lima, Campos e Silva (2009), Couto e Barros (2011), Sadi e A (2011). Para mais informações sobre São João Del-Rei na virada do século XIX para o XX, ver Guilarduci (20 3 Para mais informações sobre São João Del-Rei na virada do século XIX para o XX, ver Guilarduci (2009). 4 Para mais informações ver Adão, Lima, Campos e Silva (2009), Couto e Barros (2011), Sadi e Adão (2011). 3 Para mais informações sobre São João Del-Rei na virada do século XIX para o XX, ver Guilarduci (2009). 4 Para mais informações ver Adão, Lima, Campos e Silva (2009), Couto e Barros (2011), Sadi e Adão 3 Para mais informações sobre São João Del-Rei na virada do século XIX para o XX, ver Guilarduci (2009). 4 Para mais informações ver Adão, Lima, Campos e Silva (2009), Couto e Barros (2011), Sadi e Adão (2011). 5 Para mais informações ver Guerra (1968). 6 Para mais informações sobre o Club Dramático Arthur Azevedo, seus membros e seu repertório ver Sá (2015). (2011). 5 Para mais informações ver Guerra (1968). INTRODUÇÃO Do repertório encenado pelo grupo foi selecionada, para o escopo deste artigo, a versão portuguesa da opereta francesa A mulher soldado, que teve grande sucesso de público nas referidas cidades e foi bastante aclamada pela imprensa local. Embora bastante encenada e aplaudida, essa peça, ao lado de outras, foi severamente criticada pelos homens de letras franceses do século XIX, por se caracterizar como ‘peça ligeira’, de ‘gênero fácil’ e ‘demasiadamente obscena’. 3 Para mais informações sobre São João Del-Rei na virada do século XIX para o XX, ver Guilarduci (2009). 4 Para mais informações ver Adão, Lima, Campos e Silva (2009), Couto e Barros (2011), Sadi e Adão (2011). p. 3 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades Contudo, no início do século XX, o gênero opereta passa a simbolizar uma arte tipicamente francesa7, um patrimônio daquela nação, mas ainda enfrentava as críticas de alguns que prezavam por um ‘teatro civilizador’ em Lisboa e no Rio de Janeiro. Contudo, no início do século XX, o gênero opereta passa a simbolizar uma arte tipicamente francesa7, um patrimônio daquela nação, mas ainda enfrentava as críticas de alguns que prezavam por um ‘teatro civilizador’ em Lisboa e no Rio de Janeiro. A escolha por analisar as encenações desta opereta se fundamentou na noção de ‘poética’ elaborada por Zumthor (2007). Segundo o autor, a prática discursiva da ‘poética’ se diferencia por ser capaz de gerar efeitos, de proporcionar prazer. Tomamos o teatro como um discurso poético e os espetáculos teatrais como performances, único modo vivo de comunicação poética para o autor. O sucesso de público, registrado pela imprensa, e as repetidas apresentações são indícios de que esses espetáculos se constituíram como performances, momentos que proporcionaram prazer aos envolvidos. Para Zumthor (2007, p. 68) a performance, uma “[...] presença plena, carregada de poderes sensoriais, simultaneamente em vigília [...]”, modifica o conhecimento e não apenas comunica. Acrescentamos à definição do autor, a presença de diferentes sensibilidades durante a comunicação poética. Dessa forma, as encenações da opereta se configuraram como fenômenos educativos, momentos em que ouvintes e intérpretes, em interação, ao mesmo tempo em que realizaram, constituíram uma poética. Inspiradas na noção cunhada por Pesavento (2007)8, criamos distinções entre as sensibilidades individuais, coletivas e hegemônicas. 8 Pesavento (2007, p. 14-15) define sensibilidades como as “[...] operações imaginárias de sentido e de representação do mundo, que conseguem tornar presente uma ausência e produzir, pela força do pensamento, uma experiência sensível do acontecimento”. INTRODUÇÃO Definimos como sensibilidades individuais as operações imaginárias de sentido e representação do mundo, que permitem reviver, pela força do pensamento, experiências sensíveis singulares, sobre um acontecimento vivido por determinado sujeito; as sensibilidades coletivas são as operações imaginárias de sentido e de representação do mundo relacionadas com experiências comuns. Operações estas que produzem, pela força do pensamento, modos se sentir comuns a grupos sociais específicos; e as sensibilidades hegemônicas são operações imaginárias de sentido e de representação do mundo que grupos dominantes almejam impor aos demais sujeitos sociais. 7 Para mais informações sobre o teatro francês do século XIX, ver Yon (2000) e Yon (2012). METODOLOGIA E FONTES O principal acervo utilizado para a realização da pesquisa é mantido pelo Grupo de Pesquisa em Artes Cênicas – GPAC, da Universidade Federal de São João del-Rei - UFSJ. Rocha Junior (2006), coordenador do grupo, afirma que o acervo foi constituído por duas coleções: a primeira pertenceu ao Clube Teatral Arthur 8 Pesavento (2007, p. 14-15) define sensibilidades como as “[...] operações imaginárias de sentido e de representação do mundo, que conseguem tornar presente uma ausência e produzir, pela força do pensamento, uma experiência sensível do acontecimento”. p. 4 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. Azevedo, associação de amadores da cidade, que atuou de 1905 até 1985, possuía uma biblioteca com cerca de 8.000 volumes sobre os mais variados assuntos e “[...] aproximadamente cento e vinte textos manuscritos e/ou datilografados e outros cento e oitenta textos, com numerosos vestígios de montagem” (Rocha Junior, 2006, p. 71). A esse conjunto de fontes foi acrescida uma segunda coleção: o acervo particular de Antônio Manoel de Souza Guerra, mais conhecido como Antônio Guerra, que esteve envolvido com o teatro desde seus 13 anos, atuou como ator, ensaiador, ponto, fundou e participou de alguns grupos de teatro amador em São João del-Rei e em outras cidades de Minas Gerais. Antônio Guerra foi secretário do Club Dramático Arthur Azevedo, na época de sua inauguração em 1915. Segundo os estatutos dessa associação, o secretário tinha a obrigação de manter organizada a escrituração do clube, seu livro de atas, “[...] cópias de ofícios remetidos, álbum com recortes de opinião da imprensa e coleção de programas, arquivo de documentos [...]”9. Assim, Guerra o fez e parece ter tomado gosto pelo ofício, pois, por onde passou, em função de seu trabalho como gerente da Singer, colecionou notas da imprensa e programas de teatro. Com este material, confeccionou 13 álbuns que hoje estão disponíveis na UFSJ. Esses álbuns guardam fotografias, postais, ingressos e programas dos espetáculos, recortes de jornais da região com críticas e notícias sobre o teatro, além de exemplares dos jornais publicados por dois clubes de teatro amador dos quais ele fez parte: o jornal O Theatro – Órgão Oficial do Club Dramático Arthur Azevedo, publicado pela referida organização de São João del-Rei e o jornal O Theatro, publicado pelo Club Dramático Familiar de Lavras10. 10 Para mais detalhes sobre a constituição deste acervo e para uma análise aprofundada em relação à materialidade dos álbuns ver Lima (2006), que se dedicou ao estudo do teatro amador no início do século XX, no interior de Minas Gerais, a partir dos álbuns confeccionados por Antônio Guerra. 9 Estatutos do Club Theatral Arthur Azevedo, disponível no acervo do GPAC – UFSJ. 12 A Opinião, A Reforma, A Tribuna, Ação Social, Gaumont, Jornal do Povo, Minas Jornal, O Dia, O Florete, O Reporter, O Zuavo. , p J 10 Para mais detalhes sobre a constituição deste acervo e para uma análise aprofundada em relação à materialidade dos álbuns ver Lima (2006), que se dedicou ao estudo do teatro amador no início do século XX, no interior de Minas Gerais, a partir dos álbuns confeccionados por Antônio Guerra. 11 No álbum 13 estão os exemplares do número 1 ao 8 e o número 10, que compreendem o período de 28 de agosto de 1915 a 24 de agosto de 1916. 12 A Opinião A Reforma A Tribuna Ação Social Gaumont Jornal do Po o Minas Jornal O Dia O , , p p 11 No álbum 13 estão os exemplares do número 1 ao 8 e o número 10, que compreendem o período de 28 de agosto de 1915 a 24 de agosto de 1916. 9 Estatutos do Club Theatral Arthur Azevedo, disponível no acervo do GPAC – UFSJ. 10 Para mais detalhes sobre a constituição deste acervo e para uma análise aprofundada em relação à materialidade dos álbuns ver Lima (2006), que se dedicou ao estudo do teatro amador no início do século XX, no interior de Minas Gerais, a partir dos álbuns confeccionados por Antônio Guerra. 11 No álbum 13 estão os exemplares do número 1 ao 8 e o número 10, que compreendem o período de 28 de agosto de 1915 a 24 de agosto de 1916. 12 A Opinião, A Reforma, A Tribuna, Ação Social, Gaumont, Jornal do Povo, Minas Jornal, O Dia, O Florete, O Reporter, O Zuavo. METODOLOGIA E FONTES Em 1968, o amador publicou seu livro: Pequena história de teatro, circo, música e variedades em São João del-Rei 1717 a 1967, escrito a partir de suas coleções e memórias. Neste artigo, tomamos o livro de Guerra como fonte, além do álbum 13, composto por um material datado de 1915 a 1929. Nesse álbum estão os exemplares do jornal publicado pelo clube amador, que era distribuído gratuitamente nos dias dos espetáculos11, além de 101 recortes de jornais, retirados de 11 periódicos diferentes12, publicados em São João del-Rei. Outro conjunto de fontes importante é o dos textos teatrais analisados, que se encontram, dispostos em ordem alfabética, em uma das prateleiras destinadas às 10 Para mais detalhes sobre a constituição deste acervo e para uma análise aprofundada em relação à materialidade dos álbuns ver Lima (2006), que se dedicou ao estudo do teatro amador no início do século XX, no interior de Minas Gerais, a partir dos álbuns confeccionados por Antônio Guerra. p. 5 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades peças do acervo.Utilizamos a cópia manuscrita da opereta13A mulher soldado, ou Vinte e oito dias de Clarinha, que é uma versão de Gervasio Lobato e Acacio Antunes da vaudeville-opérette Lesvingt-huitjours de Clairettes, em quatro atos, do compositor francês Victor Roger e dos escritores Hippolyte Raymond e Antony Mars Vapereau (1893). Também foi utilizado ovaudevile Santo Antônio nas Águas, escrito em 1911 pelo são-joanense Severiano de Resende, que se encontra no acervo em uma versão datilografada. Com o objetivo de nos aproximarmos da educação das sensibilidades que ocorreu durante as encenações da opereta A mulher soldado, inspiramo-nos no ‘método de análise dramática’ de Williams (2010). O autor criticava seus contemporâneos14 que supunham que literatura e cena existissem em separado, “[...] embora o teatro seja, ou possa ser, tanto literatura quanto cena, não um em sacrifício do outro, mas um por causa do outro” (Williams, 2010, p. 38). Neste estudo, portanto, analisamos o texto da opereta como texto escrito para ser encenado. Além disso, para analisar a educação que ocorria durante as encenações, consideramos as circunstâncias da produção e apresentação dos espetáculos e as sensibilidades envolvidas. 13 No acervo existem duas cópias manuscritas de versões distintas da opereta A mulher soldado. O manuscrito utilizado nesta pesquisa está em um caderno brochurão, no formato A4, de capa dura, azul marinho. Na capa há a informação de que ele compunha o ‘Repertório Teatral de Antônio Guerra’ e o título das duas peças ali manuscritas: n. 15 e 16. A mulher soldado – Arnaldo e a vida de um jornalista. METODOLOGIA E FONTES Partimos do texto teatral e cruzamos os dados com as informações sobre as encenações15 e sobre a dimensão sociocultural e sensível daquele contexto.Os dados foram encontrados nas críticas e notícias dos jornais locais e, aqueles relacionados ao contexto, na peça são-joanense – Santo Antônio nas Águas, em pesquisas sobre a cidade de São João del-Rei do período, sobre a história do teatro, história das mulheres, história do corpo e sobre a história do exército brasileiro. Ao abordar algumas cenas a partir de diversos aspectos e ao considerar os sujeitos envolvidos nas apresentações e suas sensibilidades, buscamos elencar algumas sensações e sentimentos que possivelmente teriam afetado os espectadores. 15 A mulher soldado foi encenada cinco vezes em São João Del-Rei no ano de 1916. 14 Williams publicou esse texto pela primeira vez em 1954. ç Williams publicou esse texto pela primeira vez em 1954. A MULHER SOLDADO EM CENA TENSIONANDO SENSIBILIDADES HEGEMÔNICAS E PROVOCANDO SENSAÇÕES E SENTIMENTOS DIVERSOS A estrutura narrativa da opereta estudada tensionava as sensibilidades hegemônicas da sociedade são-joanense provocando sensações e sentimentos distintos, que possivelmente teriam afetado os espectadores. Ao analisar o enredo da opereta no conjunto de obras encenadas pelo clube dramático, observamos que existem algumas semelhanças entre elas. Tais semelhanças não são gratuitas, elas p. 6 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. estão relacionadas com as motivações que levaram os amadores são-joanenses a escolhê-las: as possibilidades materiais e artísticas do grupo e a capacidade dos enredos de dialogar e proporcionar prazer ao público local, ou seja, o potencial poético das peças. Um dos elementos semelhantes entre as operetas encenadas pelos amadores que certamente contribuiu para estabelecer uma empatia imediata com a plateia são-joanense é o ambiente em que se passavam as narrativas16. Em uma cidade ondeas pessoas conviviam já há algum tempo com corporações do exército brasileiro, possivelmente o público se veria nos palcos e/ou a seus filhos e maridos. Dos cinco espetáculos em que se encenou a opereta A mulher soldado, dois homenageavam militares, o presidente do clube, capitão José Pimentel e o general Napoleão Felipe Achê, de Niterói17. Uma ‘opereta militar’ que se passava em um quartel seria ideal à ocasião. Outra semelhança observada é a estrutura narrativa das operetas que é inspirada na fórmula das ‘peças bem feitas’ defendida pelo importante crítico francês Sarcey e apreciada por Artur Azevedo. As ‘peças bem feitas’, padrão inventado por Scribe, caracterizou grande parte da dramaturgia francesa, dos gêneros ligeiros, produzida na Belle Époque. Segundo Flores (2008), Scribe desenvolveu uma fórmula de escrita dramática que respondia às demandas de expansão do ‘teatro como entretenimento’. Tal expansão era resultado das influências de um mundo industrial em que a produção dramática voltada para o mercado, para a comercialização, se ampliava. Flores (2008) afirma que a invenção de Scribeteria possibilitado o aquecimento de um mercado teatral, em que a profissão de escritor dramático passou a ser bem paga. A fórmula das ‘peças bem feitas’ tinha como foco os efeitos da dramaturgia no público e baseava-se “[...] em sentimentos que todos compreendem e que interessam a todos porque são os sentimentos comuns à natureza humana; é exposto com clareza, desenvolve-se logicamente e tem um desfecho feliz” (Carlson, 1997, p. 277)18. 16 Para construir essa afirmativa comparamos as duas operetas mais encenadas pelo clube amador e mais celebradas pela imprensa local, são elas: A mulher soldado e O periquito. Para mais informações sobre O periquito, ver Sá (2015). 17 José Pimentel foi homenageado no dia 16 de maio de 1916 e o general Achê em 21 de junho do mesmo ano. 18 Esta é a avaliação de Sarcey sobre o drama Le maître de foges de Georges Ohnet (1883) que preenchia os critérios do que ele denominava uma ‘peça bem feita’. A MULHER SOLDADO EM CENA TENSIONANDO SENSIBILIDADES HEGEMÔNICAS E PROVOCANDO SENSAÇÕES E SENTIMENTOS DIVERSOS Desse modo, analisar a peça tendo em vista essa fórmula nos aproxima das sensações e sensibilidades envolvidas no momento da performance. Segundo Flores (2008, p. 27), as ‘peças bem feitas’ têm uma “[...] estrutura bastante fixa do começo ao fim: apresentação do problema, desenvolvimento do mesmo, clímax e resolução”. A opereta analisada distribui essas etapas em três atos, 16 Para construir essa afirmativa comparamos as duas operetas mais encenadas pelo clube amador e mais celebradas pela imprensa local, são elas: A mulher soldado e O periquito. Para mais informações sobre O periquito, ver Sá (2015). p. 7 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades apresentando o problema no primeiro, o desenvolvimento no segundo e o clímax e resolução no terceiro e último ato. Distribuída dessa maneira, a narrativa desenvolve-se em torno do problema e as ações dramáticas encadeiam-se logicamente tornando-o cada vez mais complexo. Assim, a tensão aumenta gradativamente e evidencia conflitos latentes. O ponto culminante do problema é também o momento em que todos os conflitos gerados ao longo da narrativa são solucionados. Ainda que a opereta encenada tenha sido escrita por franceses em contexto distinto do estudado, podemos dizer que o problema tensionado pela peça com o objetivo de envolver a plateia é indício dos sentimentos e das sensibilidades das pessoas envolvidas com os espetáculos ocorridos em São João del-Rei (1916), na medida em que a encenação deu lugar à performance. Cabe-nos, então, analisar os elementos que geravam tensões ao longo da narrativa, com o objetivo de perceber que sensações eles causaram ao público são-joanense. Na opereta A mulher soldado, o problema é, em parte, gerado por um mal- entendido. O sargento Gabriel, casado com Clarinha, consentiu em receber a visita de Alice no quartel e aguardava-a ansiosamente, pois desejava distrair-se um pouco. A mulher que ele esperava era “[...] uma conquista de rapaz solteiro [...]” (p. 9)19, que ele reencontrou quando levou sua esposa à costureira (coincidentemente a costureira era Alice). Ela foi ao quartel sem saber que Gabriel era casado. O mal- entendido começa quando o sargento Villar e o Capitão pressupõem que Alice fosse a esposa legítima de Gabriel. 19 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. A MULHER SOLDADO EM CENA TENSIONANDO SENSIBILIDADES HEGEMÔNICAS E PROVOCANDO SENSAÇÕES E SENTIMENTOS DIVERSOS A situação vai se complicando a cada cena: um ruído de uma conversa com Villar torna-se verdade para o Capitão, e se espalha para todos os soldados e oficiais. Clarinha, a verdadeira esposa legítima, chega ao quartel para visitar o marido e fica sabendo por Villar (que supõe ser ela uma amante de Gabriel), que ele não estava disponível, pois visitava o quartel acompanhado de sua ‘esposa legítima’, Alice. Clarinha, para vingar-se do marido, se disfarça de soldado e toma o lugar do reservista Ventura. Assim, o problema que costura toda a ação dramática é apresentado no primeiro ato: a presença de uma mulher, disfarçada de soldado, no quartel. No segundo ato, a tensão em torno da presença de uma mulher em um ambiente masculino, sendo vista e tratada como um homem, envolve o público e se intensifica na medida em que Clarinha enfrenta situações corriqueiras para um reservista, mas que para uma senhora, poderiam ser consideradas inadequadas. A performance, como comunicação poética, aconteceu durante esses espetáculos, pois as sensações que a ação dramática provocou dialogavam com as sensibilidades da plateia, de modo a evidenciar e a provocar conflitos relacionados com a maneira como aquelas pessoas significavam o que era ser homem e ser mulher, como elas p. 8 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. experimentavam a sexualidade e suas maneiras de pensar o convívio entre homens e mulheres. Dividir o quarto e a cama com o reservista Thomé foi uma das situações complicadas que Clarinha enfrentou, exposta durante grande parte do segundo ato. Thomé agiu como de costume, despindo-se antes de deitar-se. Como fazia muito frio, ele propôs dividir a cama com o colega, mas Clarinha recusou-se. O reservista achou muito estranho o constrangimento do ‘rapaz’ ao vê-lo tirar a roupa, além de estranhar sua teimosia ao preferir morrer de frio e o desconforto de dormir, vestido, na cadeira. Que sensações uma cena como essa poderia causar ao público são-joanense daquele período? A situação, mesmo nos dias de hoje, causaria certo constrangimento à mulher que estivesse vestida de soldado. Sabemos que, com relação às sensibilidades, as rupturas se dão em longo prazo e que, ao investigar um período histórico distante de nós apenas um século, corremos o risco de cometer anacronismos. Segundo Pesavento (2007, p. 20 Versão datilografada da peça Santo Antônio nas Águas escrita por Severiano de Resende, localizada no acervo do GPAC. Escrito por um são-joanense, para ser encenado por um grupo de amadores de São João del-Rei em 1911, tomamos este vaudevile como fonte para compreender as sensibilidades, se não das pessoas daquela cidade, ao menos daquelas que frequentavam e/ou produziam espetáculos teatrais naquele local e período. A MULHER SOLDADO EM CENA TENSIONANDO SENSIBILIDADES HEGEMÔNICAS E PROVOCANDO SENSAÇÕES E SENTIMENTOS DIVERSOS 15), As sensibilidades são sutis, difíceis de capturar, pois se inscrevem sob o signo da alteridade, traduzindo emoções, sentimentos e valores que não são mais os nossos. Mais do que outras questões a serem buscadas no passado, elas evidenciam que o trabalho da história envolve sempre uma diferença no tempo, uma estrangeiridade com relação ao que se passou por fora da experiência do vivido. E esta, no caso, insere o conceito das sensibilidades sob o signo da alteridade, sem o que não é possível a reconfiguração do passado, meta imprescindível do historiador, como assinala Paul Ricoeur. Devemos, então, nos perguntar qual a intensidade e o significado da tensão gerada pela cena em questão, no público são-joanense de 1916. Que relação as mulheres e os homens da época tinham com o próprio corpo? O que representava a nudez feminina e a nudez masculina? Que significados envolviam a coexistência de corpos de sexos opostos em um mesmo ambiente? Quais eram os limites do olhar, do cheirar, do tocar o outro? A elite são-joanense vivia, durante as primeiras décadas do século XX, entre a devoção à Igreja Católica e o desejo pelo progresso e pela civilização. Como já mencionamos, a atenção dessas pessoas voltava-se para as novas ideias, os novos hábitos, as técnicas, tecnologias e produtos que vinham do Rio de Janeiro e de Paris, ao mesmo tempo em que preservavam fortemente os costumes e rituais católicos. Encontramos indícios dessa dualidade vivida pela elite da cidade, no vaudevile escrito, em 1911, pelo são-joanense Severiano de Resende. A narrativa se passa em um balneário e em torno da crença que explicaria as propriedades milagrosas de suas águas. Localizado no território do município de Tiradentes, vizinho a São João del- Rei, o balneário era frequentado, também, pela elite do período para seus passeios p. 9 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades e deleites da vida no campo. No vaudevile Santo Antônio nas Águas20, existem dois personagens emblemáticos o doutor Polybio e o padre Zuquim. O primeiro acabava de chegar de Paris e estava encantado pela cidade, ‘luz da civilização e do progresso’, ‘centro fulgurante’, ‘capital do universo intelectual’. 21 Referência ao romance Viagens de Gulliver, do escritor inglês Jonathan Swift (1667-1745). “Falto de grandeza, mesquinho, medíocre” (Houaiss & Villas, 2009, p. 463). A MULHER SOLDADO EM CENA TENSIONANDO SENSIBILIDADES HEGEMÔNICAS E PROVOCANDO SENSAÇÕES E SENTIMENTOS DIVERSOS Segundo Polybio, as luzes de Paris ainda não tinham chegado à São João del-Rei, “[...] nesta terra vegeta-se [...] é grande a natureza, as montanhas altivas, os rios soberbos [...] O homem pequeno, liliputiano21, os horizontes morais limitados para os voos do talento; o espírito asfixia-se e debate-se num meio acanhado. On ne vive pás [...]”. O comendador, um terceiro personagem, censura Polybio por aquelas ‘caraminholas’ e pelo desprezo das coisas da ‘nossa terra’. Para ele, jovens como Polybio exibiam-se como “[...] propagandistas das doutrinas mais subversivas e disparatadas”. O padre Zuquim, porém, é sucinto e bem mais radical que o comendador; para ele Paris é a ‘terra da perdição’ a ‘moderna Gomorra’. Diante do quadro pintado pelo vaudevile são- joanense, podemos afirmar que a elite vivia essa dualidade e que, certamente, as pessoas tinham opiniões que variavam em uma escala que ia do encantamento de Polybio ao radicalismo do padre Zuquim. Essa reflexão nos ajuda a imaginar o lugar, ou a repercussão, na cidade mineira, de algumas transformações que ocorreram em relação às sensibilidades dos parisienses. Segundo Sohn (2008), durante o século XX, viveu-se na França um progressivo recuo do pudor. Anteriormente tido como uma virtude aconselhada às meninas e moças, o significado do pudor se transforma na Belle Époque. Tal transformação é acelerada no período entreguerras com a superação de tradições seculares como as interdições à exibição do corpo das mulheres e suas curvas, o hábito de fazer a toalete sem despir-se e a relação dos casais com o sexo e a sexualidade. Sohn (2008, p. 110, grifo do autor) afirma que “[...] no final do século XIX ainda se faz amor ‘totalmente nus, só com camisola’ e a alcova é inimiga da luz’. Esses interditos remetem a uma concepção cristã de sexualidade, circunscrita ao casal legítimo, destinada essencialmente à reprodução e inimiga da concupiscência”. É plausível imaginar que os casais são-joanenses tinham diversos modos de se relacionar sexualmente, o que dependia do envolvimento com a religião e da relação de cumplicidade que construíam entre si, visto que os obstáculos para a concretização dos desejos sexuais, neste caso, eram apenas o julgamento de Deus e do cônjuge. Esses matizes surgem pela gradual falência dos casamentos arranjados p. 10 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. e a valorização do casamento por amor22. 22 O vaudevile Santo Antônio nas Águas expõe justamente essa questão: o Comendador, viúvo bem- sucedido, manifestou interesse em casar-se com a filha de sua amiga Helena, a jovem Geni. Helena hesita, pois percebe que sua filha e Thomazinho estavam apaixonados. Em vários momentos da peça, os personagens discutem a questão, sempre defendendo que os jovens deveriam ficar juntos porque se amavam. A história se resolve com a intervenção de Santo Antônio, casando o jovem casal e o comendador com sua amiga Helena, que também era viúva. 23 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. A MULHER SOLDADO EM CENA TENSIONANDO SENSIBILIDADES HEGEMÔNICAS E PROVOCANDO SENSAÇÕES E SENTIMENTOS DIVERSOS No início do século XX, também ocorreram mudanças em relação à exposição dos corpos no espaço público. De acordo com Sohn (2008), o corpo começa a ser exposto ‘sob o efeito combinado da moda e do turismo balneário’; em 1900 as competições aquáticas também contribuíram para esse processo. As roupas de banho, como as do Rio e de Paris, possivelmente permitiam “[...] associar o banho de mar e decência” (Sohn, 2008, p. 110); no caso são-joanense, associava-se o banho em Águas Santas à decência. Dessa forma, a nudez de uma mulher diante de seu marido já não era algo impensável para o público são- joanense. Porém, a exposição do corpo da mulher a um estranho certamente o era. No caso da opereta A mulher soldado, a nudez de Clarinha diante de Thomé também significava a revelação do disfarce da mulher soldado, o enfrentamento das consequências disso diante das regras do quartel e o fim da vingança da protagonista. Poderíamos nos perguntar: por que o disfarce de Clarinha se configuraria como uma vingança de uma mulher traída? A protagonista não estaria se penalizando ao passar pela experiência de servir ao exército? Em quê, ou como, isso afetaria seu marido Gabriel? O que significava a presença de uma mulher no quartel para as pessoas daquele contexto? No fim do segundo ato, Gabriel escuta a discussão entre Thomé e Clarinha, percebe que ela estava no quarto ao lado e vai ao seu encontro com a intenção de convencê-la a acabar com o disfarce. Ele dispensa Thomé, a fim de ter uma conversa particular com Clarinha: Gabriel Clarinha é preciso acabar com isto. Eu não posso sofrer mais estes vexames. Clarinha Pois não sofra; não sou eu a culpada. [...] Gabriel Mas tu não vês que de um momento para o outro descobrem que és mulher? Clarinha [À lápis ao lado: Pois q desc...] Não se incomode. Eu tenho quem me defenda se for preciso (p. 36-37)23. p q ] Não se incomode. Eu tenho quem me defenda se for preciso (p. 36-37)23. 22 O vaudevile Santo Antônio nas Águas expõe justamente essa questão: o Comendador, viúvo bem- sucedido, manifestou interesse em casar-se com a filha de sua amiga Helena, a jovem Geni. Helena hesita, pois percebe que sua filha e Thomazinho estavam apaixonados. A MULHER SOLDADO EM CENA TENSIONANDO SENSIBILIDADES HEGEMÔNICAS E PROVOCANDO SENSAÇÕES E SENTIMENTOS DIVERSOS Em vários momentos da peça, os personagens discutem a questão, sempre defendendo que os jovens deveriam ficar juntos porque se amavam. A história se resolve com a intervenção de Santo Antônio, casando o jovem casal e o comendador com sua amiga Helena, que também era viúva. Gabriel Clarinha é preciso acabar com isto. Eu não posso sofrer mais estes vexames. Clarinha Pois não sofra; não sou eu a culpada. [...] Gabriel Mas tu não vês que de um momento para o outro descobrem que és mulher? Clarinha [À lápis ao lado: Pois q desc...] Não se incomode. Eu tenho quem me defenda se for preciso (p. 36-37)23. 24 Segundo Maria Ligia Prado e Stella Scatena Franco, na segunda metade do século XIX, foram publicados dicionários biográficos, compêndios com histórias de vida de mulheres ‘célebres’ e ‘ilustres’. “Josefina Álvares de Azevedo, que editou, em 1897, Galeria ilustre, sobre antigas heroínas estrangeiras, e Inês Sabino, cujo livro Mulheres ilustres do Brasil saiu do prelo em 1899. Outras referências de peso são o livro de Joaquim Norberto de Sousa e Silva, Brasileiras célebres (editado em 1862), e o de Joaquim Manuel de Macedo, Mulheres célebres (publicado em 1878), sobre figuras femininas estrangeiras ‘em todos os tempos’. Macedo também produziu um anuário de biografias de brasileiros (1876) que relata a trajetória de várias mulheres. A revista do Instituto Histórico e Geográfico Brasileiro, por sua vez, manteve (entre 1839 e 1889) uma coluna, publicada em quase todas as edições, denominada ‘Biografia de Brasileiros Distintos por Letras, Armas e virtudes’, na qual aparecem as biografias de quatro mulheres que viveram no período colonial” (Prado & Franco, 2013, p. 195). 25 Segundo Prado & Franco (2013), Maria Quitéria nasceu em 1792 na Bahia, era filha única criada no sertão. Sabia montar, caçar e manejar armas de fogo. Não sabia ler nem escrever. Já Wolff (2013) afirma que, Maria Quitéria ficou órfã de mãe aos nove anos e quando jovem “[...] assumiu o comando da casa e dos irmãos mais novos até seu pai casar-se novamente e a nova madrasta desaprovar seus ‘modos independentes’ e seus hábitos de montar a cavalo e caçar como um homem” (Wolff, 2013, p. 425). 26 Segundo Prado & Franco (2013), Jovita Feitosa nasceu no Ceará em 1848, perdeu sua mãe ainda criança e foi criada pelo pai. Quando jovem foi morar com o tio no Piauí. Jovita sabia ler, escrever, atirar e era p. 11 de 25 O. suas atitudes usadas como exemplo de coragem e de amor à pátria e como incentivo aos homens para fazerem o mesmo, embora nem todos aprovassem a presença de mulheres nos quarteis. Machado de Assis publicou no Diário do Rio de Janeiro, de 7 de fevereiro de 1865, um longo artigo que se dedicava a “[...] demarcar o espaço de ação da mulher durante uma guerra: rezar, cuidar dos feridos, costurar para os soldados” (Prado & Franco, 2013, p. 201). Wolff (2013) acrescenta que a história de Jovita causou grande polêmica na imprensa da época. A autora cita uma publicação no Jornal do Commercio de 1865 que afirmou ser a presença da jovem no exército uma ofensa grave à dignidade dos homens. Segundo Wolff (2013, p. 430, grifo do autor), elas usavam um saiote sobre a farda, o que para a autora, deixava claro “[...] que o exército não seria lugar ideal para as mulheres e que estas ‘soldadas’ eram exceções”. Na segunda metade do XIX, as mulheres acompanhavam as tropas, seguindo seus filhos e/ou maridos e cumprindo os papéis sublinhados por Machado de Assis. Elas cuidavam do abastecimento e do preparo da comida, costuravam, lavavam e cuidavam dos feridos e doentes. No momento das apresentações da opereta A mulher soldado em São João del-Rei (1916), as mulheres já tinham sido afastadas dos quartéis, por meio das reformas realizadas no Exército. Elas não mais atuavam nas atividades indicadas ao seu sexo, menos ainda, como soldadas. As possibilidades das mulheres se alistarem disfarçadas de homens tornaram-se nulas depois de instituído o exame médico obrigatório antes do engajamento do soldado. A presença delas somente era tolerada quando eram esposas dos soldados ou oficiais (Wolff, 2013). Nesse período, embora as atenções estivessem mais voltadas para a Primeira Guerra Mundial do que para o conflito do Contestado, corriam algumas notícias sobre as ‘virgens’, mulheres seguidoras do líder dos rebeldes (o monge José Maria, morto na primeira batalha) que diziam ter visões do líder falecido, momentos em que recebiam orientações sobre como proceder na luta. Teodora e Maria Rosa lideraram os rebeldes por um tempo, por meio dessas visões. Visto que a unidade do Exército, lotada em São João del-Rei, participou desta guerra entre 1914 e 1915, possivelmente histórias circularam sobre as guerreiras do Contestado. Mas essas mulheres eram vistas, assim como os rebeldes que lideraram, como fanáticas, ignorantes, humildes sertanejas27. 27 Para mais informações sobre esta guerra e as críticas dos oficiais reformistas da revista A Defesa Nacional à campanha e ao exército, ver Frank d. McCann (2009). costureira. Apresentou-se para a guerra do Paraguai seguindo os passos do irmão em julho de 1865. De acordo com Wolff (2013) alguns biógrafos afirmam que ela teria se desentendido com o tio, por isso fugiu da fazenda onde morava. p. 11 de 25 p. 11 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades Fica evidente que a preocupação de Gabriel não é com a integridade da esposa, ou com os problemas que ela estava enfrentando como reservista. O sargento se preocupava apenas com sua própria ‘honra’. Gabriel afirma não poder mais sofrer aqueles ‘vexames’. O sargento era, portanto, a vítima da história, ele estava sendo molestado e corria o risco de ter sua esposa descoberta vestida como soldado no quartel. Clarinha demonstra não se importar com a revelação de seu disfarce – “Pois que descubram!” –, mostra-se ciente de que o marido seria o maior prejudicado naquela situação e, para concretizar sua vingança, ela parece desejar ter sua identidade revelada. Por que Gabriel sentia tamanha vergonha daquela situação? Por que essa ideia fazia sentido para o público são-joanense? Que sensibilidades estavam envolvidas nessa trama? Na segunda metade do século XIX, com o objetivo de despertar o patriotismo entre os brasileiros foram escritas biografias de ‘heróis’ e ‘heroínas’ nacionais24. Entre as ‘heroínas’, duas tiveram destaque nessas publicações por lutarem pela pátria, disfarçadas de soldados. A primeira, Maria Quitéria, apresentou-se como o soldado Medeiros e lutou em batalhas pela independência do Brasil. Depois de descoberto seu disfarce, foi condecorada pessoalmente pelo Imperador em 1823. Antonia Alves Feitosa, conhecida como Jovita Feitosa, vestiu-se de homem e apresentou-se como voluntário para lutar na guerra do Paraguai; foi descoberta rapidamente e, segundo Wolff (2013, p. 429), “[...] ao invés de ser imediatamente descartada, tornou-se curiosidade nacional, percorrendo vários estados conclamando os homens a apresentarem-se como voluntários. Chegou a ser promovida a sargento”. Ainda que as duas mulheres tivessem motivos pessoais para apresentarem-se como voluntários nas guerras, Maria Quitéria25 e Jovita Feitosa26 foram saudadas e 26 Segundo Prado & Franco (2013), Jovita Feitosa nasceu no Ceará em 1848, perdeu sua mãe ainda criança e foi criada pelo pai. Quando jovem foi morar com o tio no Piauí. Jovita sabia ler, escrever, atirar e era 26 Segundo Prado & Franco (2013), Jovita Feitosa nasceu no Ceará em 1848, perdeu sua mãe ainda criança e foi criada pelo pai. Quando jovem foi morar com o tio no Piauí. Jovita sabia ler, escrever, atirar e era p. 12 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. 28 Versão datilografada da peça Santo Antônio nas Águas, escrita por Severiano de Resende, localizada no acervo do GPAC. p. 11 de 25 É importante considerar também a luta das mulheres por direitos que se intensificava nas primeiras décadas do século XX, gerando fortes reações, como críticas expressas em peças de teatros, charges e na imprensa. Segundo Rachel costureira. Apresentou-se para a guerra do Paraguai seguindo os passos do irmão em julho de 1865. De acordo com Wolff (2013) alguns biógrafos afirmam que ela teria se desentendido com o tio, por isso fugiu da fazenda onde morava. p. 13 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades Soihet (2013), as mulheres dos setores médios e da alta burguesia reivindicavam o direito à capacitação profissional e ao trabalho remunerado, o acesso pleno à educação de qualidade, direito de voto e de elegibilidade, considerados essenciais para alcançar os outros objetivos. A luta feminina ganhava força desde a proclamação da República. Apesar de a Constituição ter sido promulgada em 1891 sem afirmar o direito das mulheres ao voto, o tema foi discutido na Assembleia Constituinte e continuou na pauta dos intelectuais e da imprensa. Soihet (2013) conta que a oposição ao sufrágio feminino se fundamentava no argumento ‘científico’ da fragilidade da mulher, de sua inteligência limitada, de sua inadequação para realizar atividades públicas. A mulher deveria ocupar-se da família. Soihet (2013), as mulheres dos setores médios e da alta burguesia reivindicavam o direito à capacitação profissional e ao trabalho remunerado, o acesso pleno à educação de qualidade, direito de voto e de elegibilidade, considerados essenciais para alcançar os outros objetivos. A luta feminina ganhava força desde a proclamação da República. Apesar de a Constituição ter sido promulgada em 1891 sem afirmar o direito das mulheres ao voto, o tema foi discutido na Assembleia Constituinte e continuou na pauta dos intelectuais e da imprensa. Soihet (2013) conta que a oposição ao sufrágio feminino se fundamentava no argumento ‘científico’ da fragilidade da mulher, de sua inteligência limitada, de sua inadequação para realizar atividades públicas. A mulher deveria ocupar-se da família. No vaudevile Santo Antônio nas Águas, já referido, fica evidente que o debate sobre as reivindicações das mulheres estava, de alguma maneira, presente na cidade. Os personagens, comendador, doutor Polybio e Thomazinho conversam sobre a qualidade do serviço de telégrafos: Comendador Os telegramas são todos entregues tarde e a más horas, se não chegam estropiados. Dr Polybio Como deu-se há pouco comigo em viagem para Belo Horizonte. Telegrafei de Lafaiete aos amigos, comunicando-lhes minha chegada, isto às 6h30 da manhã, para me esperarem com o almoço. Cheguei a capital mineira às 10 horas, e quando jantava às 6 da tarde, é que vieram trazer o despacho telegráfico. Em Paris sim a eletricidade é a eletrio cidade. Thomazinho Entre nós esse serviço anda todo atrapalhado. 29 Versão datilografada da peça Santo Antônio nas Águas, escrita por Severiano de Resende, localizada no acervo do GPAC. 30 Ver Figuras 1 e 2. Dr Polybio Os seus direitos são iguais aos meus. O senhor faz o que entende não é assim? Eu também tenho o mesmo direito, pago-lhe na mesma moeda. [Fala modificada pelo Club Dramático Arthur Azevedo] Os seus direitos são iguais aos meus. O Sr. tem uma amante não é verdade? Pois eu também quero ter um amante. Estou me guiando por si. [Fala original do manuscrito]30. Dr Polybio No Rio, então, dão-se trocadilhos engraçados. Incroyable. Efeito das conquistas do feminismo; desde que meteram as mulheres nos telégrafos e correios, as comunicações andam atrapalhadas, como tudo os homens rasparam as barbas e o bigode, enroscam-se ao braço das mulheres, quando deviam servir-lhes de ponto de apoio e elas vestem calças. O homem vira mulher e a mulher torna-se homem. Moderníssimo28. p. 14 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. Diante do comentário do amigo comendador, Helena, que até então apenas escutava, protesta e Geni complementa: Helena Protesto. As mulheres fazem o serviço muito direitinho. Geni E com muito capricho DrPolybio Helena Protesto. As mulheres fazem o serviço muito direitinho. Geni E com muito capricho DrPolybio Helena Protesto. As mulheres fazem o serviço muito direitinho. Geni E com muito capricho DrPolybio As mulheres. As novas ideias emancipam-nas e dão-lhes acesso as mais nobres funções. Em Paris as mulheres vão se introduzindo em todos os serviços: - Le monde marche29. A conversa então toma outro rumo e deixa no ar a observação do doutorPolybio, jovem estudado que acabava de voltar de Paris. O autor do vaudevile parece representar algumas posições de são-joanenses a respeito das conquistas feministas e das mudanças ‘moderníssimas’ que a sociedade vivia naquele momento. Tais posicionamentos iam desde a oposição às mudanças que aconteciam e o temor em relação à inversão dos papéis sociais de homens e mulheres, até aqueles que apostavam no desenvolvimento, no progresso inspirado na ‘civilizada’ Paris, onde as mulheres já ocupavam nobres funções. As personagens Helena e Geni, apesar de timidamente, posicionaram-se em defesa das conquistas do movimento feminista. Diante desse panorama, podemos imaginar as inúmeras sensações que a encenação de uma mulher no quartel, disfarçada de soldado, poderia causar na plateia são-joanense. Na cena 4 do segundo ato, que começamos a descrever acima, quando Gabriel pede à Clarinha para dar fim a sua vingança, ele fica furioso com a resposta negativa, com o ‘atrevimento’ da esposa ao deixar claro que sabia que ele seria o maior prejudicado com toda aquela história, e por insinuar que não se importava. Então, Gabriel reage da seguinte maneira: Gabriel (furioso) Clarinha! Clarinha! Eu sou seu marido. Ainda me não desobriguei dos meus direitos. Gabriel (furioso) Clarinha! Clarinha! Eu sou seu marido. Ainda me não desobriguei dos meus direitos. Clarinha Os seus direitos? Clarinha Os seus direitos? Os seus direitos são iguais aos meus. O senhor faz o que entende não é assim? Eu também tenho o mesmo direito, pago-lhe na mesma moeda. [Fala modificada pelo Club Dramático Arthur Azevedo] Os seus direitos são iguais aos meus. O Sr. tem uma amante não é verdade? Pois eu também quero ter um amante. Estou me guiando por si. [Fala original do manuscrito]30. p. 15 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades Gabriel (avança para Clarinha) Tu não me ofendas! Olha que eu [...] (agarra-a por um braço) Clarinha (gritando) Deixe-me! Deixe-me! Villar (acorda) Não se pode dormir! Que inferneira! Gabriel (furioso) Tu queres enlouquecer-me queres um escândalo? (p. 36-37)31. A discussão do casal em relação aos direitos do marido e da esposa certamente remeteu o público são-joanense às discussões sobre as reivindicações das mulheres que ocorriam no período. Gabriel cobrava seus direitos à esposa Clarinha, expressos na ideia da obediência. Ainda que alguns e algumas discordassem de que as mulheres deveriam ser subservientes em relação aos maridos, a atitude do sargento fazia todo o sentido naquele contexto. De acordo com Cortês (2013), o Código Civil promulgado em janeiro de 1916 continuava tratando a mulher casada como ser inferior ‘relativamente incapaz’, que deveriam viver sob a proteção de seus maridos o ‘chefe da sociedade conjugal’, responsável por definir os destinos da esposa e de toda a família. É interessante notar, como podemos ver nas Figuras 1 e 2, que a fala de Clarinha, no original do manuscrito da opereta, foi modificada. No texto original, Clarinha afirmava que tinha direitos iguais aos do marido e que, portanto, se ele tinha uma amante ela também queria gozar desse direito. Embora as leis fossem mais rígidas para as mulheres, o adultério era condenado para ambos os sexos pela justiça32 e pela Igreja Católica. Dessa forma, esse elemento foi retirado da fala da protagonista. Não seria recomendável a uma elite que se dizia virtuosa e civilizada atentar contra as duas instituições (Estado e igreja) de maneira tão contundente. No entanto, mesmo com a mudança, a reivindicação por direitos iguais não é apagada. A nova fala de Clarinha abre-se à interpretação, ao gosto do espectador: “Os seus direitos? Os seus direitos são iguais aos meus. 32 O Código Civil de 1916 cria a ‘ação de desquite’ e o adultério era um dos motivos que a justificavam. Porém, o artigo 319 abria alguns precedentes para que os adúlteros fossem perdoados, tornando ilegítima a ação de desquite. “Art. 319. O adultério deixará de ser motivo para desquite: I. Se o autor houver concorrido para que o réu o cometesse. II. Se o cônjuge inocente lhe houver perdoado. Parágrafo único. Presume-se perdoado o adultério, quando o cônjuge inocente, conhecendo-o, coabitar com o culpado” (Brasil, 1916). 31 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. 33 Cópia manuscrita da opereta A Mulher Soldado, acervo GPAC. 34 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. Clarinha O senhor faz o que entende não é assim? Eu também tenho o mesmo direito, pago-lhe na mesma moeda” (p. 36-37)33. p. 16 de 25 p. 16 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. Figura 1 - Fala original do manuscrito da opereta. Fonte: Cópia manuscrita da opereta A mulher soldado, acervo GPAC - UFSJ (p. 37). Figura 1 - Fala original do manuscrito da opereta. Fonte: Cópia manuscrita da opereta A mulher soldado, acervo GPAC - UFSJ (p. 37). Figura 2 - Fala modificada. Fonte: Cópia manuscrita da opereta A mulher soldado, acervo GPAC - UFSJ (p. 37). Figura 2 - Fala modificada. Fonte: Cópia manuscrita da opereta A mulher soldado, acervo GPAC - UFSJ (p. 37). Fonte: Cópia manuscrita da opereta A mulher soldado, acervo GPAC - UF Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades Sentindo-se ofendido por Clarinha, Gabriel devolve a ofensa respondendo ao amigo Villar, que se aproximou atraído pelo barulho da discussão do casal: Villar Continuas na mesma? Queres a viva força que ela diga que é tua mulher?! Gabriel (Com ironia) Não, esta senhora não é minha mulher. É [...] minha amante. (Thomé entra) Estásatisfeita? Clarinha Infame! (dá-lhe uma bofetada) (p. 37-38)34. Ao homem seria tão ofensivo o fato de sua mulher exigir ter os mesmos direitos que ele, quanto à mulher ofendia ser colocada no lugar da amante. Thomé testemunha a agressão feita ao sargento Gabriel e chama os soldados para prender o reservista que cometera a infração. Clarinha, então, é presa. No terceiro ato, o clímax se dá pela prisão de Clarinha que, disfarçada de soldado, foi condenada ao fuzilamento por agredir um sargento. Thomé, Gabriel e Villar ficam aflitos para viabilizar a fuga de Clarinha. O primeiro por culpa (pois foi ele o delator do companheiro reservista) e os outros dois por saberem do disfarce de Clarinha. Thomé fica de guarda na porta da prisão onde ela estava. Arrependido por ter colocado Ventura/Clarinha naquela situação, Thomé resolve ajudá-lo a fugir. Ele consegue um vestido com a empregada da estalagem para que o colega fugisse disfarçado de mulher. Gabriel e Villar chegam antes que o plano de Thomé fosse concretizado e o enxotam. Ele, para não ver seu plano fracassado, resiste em deixar o serviço de sentinela, mas acaba acatando as ordens dos dois sargentos, que encontram Clarinha vestida como mulher e entendem a insistência de Thomé em continuar de guarda. Logo que Clarinha dá as costas, o verdadeiro Ventura aparece para recuperar sua farda que estava com o sargento Villar. Gabriel e Villar entregam a farda ao reservista e o prendem no lugar de Clarinha. O capitão recebe a denúncia de que haveria uma mulher vestida como um militar e Clarinha é impedida de sair do quartel. O capitão decide interrogar o prisioneiro Ventura, acusado de ser uma mulher disfarçada de soldado, e depois interroga Clarinha, acusada de ser um soldado vestido como uma senhora. Toda a situação é revelada e o sargento Gabriel perdoado pelo capitão. Clarinha, como um reservista, acata as ordens do capitão de perdoar seu marido. Esta era uma virtude dos militares adequada às mulheres, a obediência aos superiores. Supomos que compunham o público que assistia à opereta encenada pelo ‘Club Dramático Arthur Azevedo’, mulheres da classe média e da elite, que mesmo que mantivessem uma conduta condizente com uma sociedade bastante religiosa, como era a são-joanense, apreciavam os ‘avanços’ franceses e, no mínimo, eram p. 18 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. Villar Continuas na mesma? Queres a viva força que ela diga que é tua mulher?! Gabriel (Com ironia) Não, esta senhora não é minha mulher. É [...] minha amante. (Thomé entra) Estásatisfeita? Clarinha Infame! (dá-lhe uma bofetada) (p. 37-38)34. simpáticas às reivindicações do movimento feminista, como Helena e Geni (personagens do vaudevile são-joanense de Severiano de Resende). Essas mulheres possivelmente vibraram com a vingança de Clarinha e, certamente, sentiram-se vingadas por meio da atitude da protagonista ao disfarçar-se de soldado e ao servir no quartel onde o marido era sargento. Estas eram formas de ‘ofender a dignidade’ do militar: vestir calças, montar cavalos, enfrentar, sem ser descoberta, as mesmas situações que um reservista enfrentava, mostrar-se tão capaz quanto um homem, reivindicar direitos iguais. Naquela noite, assistindo à opereta, podemos imaginar que seus corpos se aprumaram, os pulmões se encheram de ar, os olhos sorriram, elas sentiram o prazer de ser, por instantes, Clarinhas, Marias Quitérias e Jovitas, sentiram o prazer de estar no comando da situação. Tais sensações podem ter sido elaboradas de inúmeras maneiras por cada uma das espectadoras, constituindo sentimentos, valores e sensibilidades. Seria impossível dizer ao certo que sensibilidades essas mulheres aprenderam ou elaboraram naquela noite, até porque esse é um processo que se dá em longo prazo e não apenas em um único evento, mas podemos imaginar que, ao experimentar o prazer de sentirem-se fortes e capazes como as heroínas citadas, elas criavam o gosto pela sensação, o que pode ter sido motor de transformações pessoais e, quem sabe, de transformações sociais. Já os homens que, como Polybio (personagem do vaudevile são-joanense), acreditavam que as conquistas feministas faziam parte do desenvolvimento e do progresso da sociedade, possivelmente riram de si mesmos, achando ridículas as atitudes de personagens como Villar que subjugava as mulheres se achando no controle da situação quando afirmou que tinha conquistado Clarinha no bonde. Homens, como Polybio, viam, na situação em que uma mulher não era notada em meio aos soldados, a confirmação de suas convicções, a prova de que elas eram, sim, capazes de desempenhar atividades realizadas pelos homens. De outro modo, homens que, como o comendador (também personagem do vaudevile de Severiano de Resende), acreditavam que as mulheres eram frágeis, pouco inteligentes e que sua função era cuidar da família, possivelmente se remexeram nas cadeiras incomodados com o fato de, naquele quartel, ninguém notar a presença de uma mulher. Thomé, que passou um bom tempo tão próximo de Clarinha, se revelou incapaz, por nem imaginar que estava diante de uma senhora, mesmo quando a viu vestida como mulher, no final do terceiro ato. Villar Continuas na mesma? Queres a viva força que ela diga que é tua mulher?! Gabriel (Com ironia) Não, esta senhora não é minha mulher. É [...] minha amante. (Thomé entra) Estásatisfeita? Clarinha Infame! (dá-lhe uma bofetada) (p. 37-38)34. É certo que, por ele ser um homem simples, sem instrução, essa imagem se desvincularia dos homens cultos e instruídos. A cena que expõe a teimosia do capitão em obter de Ventura (vestido como soldado) uma confissão de que ele era uma mulher e de chegar a afirmar com convicção que Clarinha era um homem, quando ela já vestia saias, possivelmente provocaria um riso nervoso e coraria as faces daqueles que tinham como verdade a inadequação e a incapacidade das mulheres ocuparem esses lugares e funções tradicionalmente masculinas. Contudo, um traço do personagem Ventura p. 19 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades justificava a confusão do Capitão, que não poderia ser desmoralizado em cena, por um clube liderado por um capitão, que encenava para uma plateia composta, também, por militares. Ventura era um homem afeminado. No final do primeiro ato, Ventura procura o sargento Villar para recuperar sua farda. O texto da opereta faz a seguinte indicação para o momento em que ele se apresenta aos sargentos: “Ventura – Eu sou pasteleiro, e chamo-me Ventura de Sá. (com um todo afeminado)” (p. 21, 1º ato, cena XI)35. Ao ser acusado de ser mulher, no terceiro ato, Ventura se defende: “Por aparências não me tomem / Palavra d’honra que sou homem” (p. 46, 3º ato, cena VI)36. A imprensa local elogiou a interpretação do personagem Ventura feita pelo amador Humberto Preda. Ele teria arrancado “[...] boas gargalhadas da plateia” (O Theatro, p. 28). Segundo um autor anônimo que teve seu texto publicado no periódico O Zuavo (p. 34, grifo do autor): “Seus trejeitos afeminados, seu andar, sua voz adocicada, seu bem estudado ‘remelexo’ também, a princípio, me deixou confuso quanto ao verdadeiro sexo que possui! Muito bem!”. O autor desse texto corroborava a percepção dos militares em cena, que acusavam Ventura de ser mulher, atribuindo à encenação do amador a perfeição. Ao mesmo tempo, ele livrava o capitão do ridículo de não ser capaz de distinguir entre um homem e uma mulher. A comicidade conferida ao personagem Ventura ridicularizava sua característica afeminada e deixava intocada a reputação do capitão, que teria razão de se confundir em relação ao sexo de Ventura. 35 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. 36 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. 36 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. 35 Cópia manuscrita da opereta A mulher soldado, acervo GPAC. Villar Continuas na mesma? Queres a viva força que ela diga que é tua mulher?! Gabriel (Com ironia) Não, esta senhora não é minha mulher. É [...] minha amante. (Thomé entra) Estásatisfeita? Clarinha Infame! (dá-lhe uma bofetada) (p. 37-38)34. Os homens da plateia, que acreditavam que as mulheres eram seres inferiores, possivelmente voltaram para suas casas satisfeitos e convencidos de suas ideias. No final da opereta, tudo voltou para seu ‘devido lugar’, com a obediência de Clarinha e seu perdão concedido a Gabriel. Já as mulheres que acreditavam em sua natureza frágil e em sua inadequação para espaços e funções consideradas masculinas, possivelmente ficaram apreensivas diante de cenas que demonstravam as situações que Clarinha precisou enfrentar como um reservista. Elas temeram pela integridade da protagonista, julgaram sua atitude impensada e vibraram com o final feliz em que Clarinha acatou as ordens do capitão de perdoar o marido Gabriel. É claro que esses homens e mulheres são modelos que nos ajudam a perceber possíveis sensações e sentimentos gerados na encenação da opereta A mulher soldado e, como modelos, eles simplificam a realidade que, certamente, é muito mais complexa. Não temos a pretensão de afirmar que essas pessoas existiram tal e qual as descrevemos, mas os modelos aqui imaginados foram construídos a partir de dados sobre os homens e p. 20 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 Sá, C. M., & Galvão, A. M. O. mulheres daquela sociedade, naquele período, portanto, eles são instrumentos que nos auxiliaram na aproximação da realidade investigada. mulheres daquela sociedade, naquele período, portanto, eles são instrumentos que nos auxiliaram na aproximação da realidade investigada. CONSIDERAÇÕES FINAIS A análise realizada no presente artigo revela que a estrutura narrativa da opereta A mulher soldado, condizente com a fórmula das ‘peças bem feitas’, provocava efeitos no público reforçando sensibilidades hegemônicas, ao mesmo tempo em que possibilitava sensações e sentimentos distintos e mesmo opostos àqueles legitimados pela elite dominante.O desfecho da trama nos indica quais eram, para os membros da diretoria do Club Dramático, os sentimentos que ‘todos compreenderiam’, comuns a todos os são-joanenses que potencialmente frequentavam as casas teatrais. Tornam-se evidentes as sensibilidades hegemônicas, aquelas que, a partir das relações de poder estabelecidas naquela sociedade,buscavam silenciar outras sensibilidades (coletivas e individuais). A raiva da esposa traída em alguma medida é compreendida e legitimada, pois Clarinha não é punida por seu disfarce. Mas a ela não cabe questionar a ordem de perdoar o marido. A obediência, o sentimento de subalternidade diante de um capitão do exército, é destacado, mas também é exposto e valorizado o sentimento de subalternidade da mulher diante de outros homens e mesmo do marido adúltero. Na cena em que o capitão interroga Clarinha e Ventura a fim de verificar a denúncia sobre a presença, no quartel, de uma mulher que estaria vestida como um militar, o sentimento de fragilidade, inadequação, incapacidade das mulheres de ocupar uma função no exército é reforçado. Porém, esses sentimentos não são direcionados apenas às mulheres, mas também à feminilidade, ao modo de ser feminil que é julgado como inadequado. Homens ‘afeminados’, como Ventura, deveriam se sentir tão frágeis, inadequados e incapazes quanto as mulheres. Contudo, a estrutura narrativa da opereta, ao tensionar essas sensibilidades ao longo de toda a peça, acaba por possibilitar, a construção de outros modos de sentir, outras formas de responder às tensões expostas no palco. Cada sujeito presente nas apresentações provavelmente se afetou de maneira diferente, conforme seu lugar social, suas experiências anteriores, suas sensibilidades individuais e coletivas. Diante da impossibilidade de investigar sujeitos específicos e o modo como se sentiram nas encenações, criamos possíveis modelos de espectadores a partir do estudo das questões que foram postas em cena, em relação ao contexto histórico, social e cultural das apresentações. Foi possível inferir que na plateia existia uma diversidade de sujeitos que pensavam e sentiam diferentemente. Ali estavam homens e mulheres brancos, brancas, negros, negras, que se posicionavam quanto às relações de gênero, mais próximos à defesa da emancipação das mulheres, até homens e mulheres que p. CONSIDERAÇÕES FINAIS In Anais do 11º Congresso Nacional de História do Esporte, Educação Física, Lazer e Dança (p. 11- 26). Viçosa, MG. CONSIDERAÇÕES FINAIS 21 de 25 Rev. Bras. Hist. Educ., 21, e144 2021 A mulher soldado nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades acreditavam nas distinções que colocavam o homem no lugar da autoridade e a mulher no lugar da subalternidade. A reação do sargento Gabriel diante de sua esposa disfarçada como soldado reforçava sensibilidades ligadas às relações de gênero em que as mulheres deveriam sentir-se inferiores, impossibilitadas de fazer escolhas, incapazes em relação aos homens. Por outro lado, o disfarce evidenciava uma esposa ousada que colocava em xeque a autoridade do marido, que deveria sentir-se envergonhado. O fato de o disfarce não ser percebido pelos militares agravava a situação. Dessa forma, na medida em que a peça tensionava a autoridade do marido, ela também expunha uma mulher que pensava por si, que tomava decisões de acordo com seus julgamentos e desejos, e que por muito tempo em cena teve seus planos bem sucedidos. Clarinha mostra-se perspicaz ao escolher vingar-se de forma a fragilizar o marido, corajosa ao enfrentar Gabriel a ponto de dar-lhe uma bofetada e capaz de sobreviver às adversidades impostas aos reservistas daquele destacamento. Por um lado, aqueles e aquelas que defendiam o lugar de dominação do homem sentiram-se incomodados com a situação em que Gabriel se encontrava e compartilharam seu sentimento de vergonha, raiva e medo. Por outro, aqueles e aquelas simpáticas à luta pela emancipação das mulheres sentiram-se esperançosos, capazes de enfrentar a dominação masculina. Por fim, podemos afirmar que os resultados deste estudo contribuem para compreender a educação das sensibilidades como um fenômeno complexo, dialógico e plural. O estudo evidencia as diversas respostas possíveis e modos de sentir que se constituíram em um mesmo momento (durante os espetáculos teatrais) a partir das experiências de cada sujeito presente. Essas experiências estão relacionadas ao modo como esses sujeitos particulares se relacionavam com o contexto histórico, social e cultural em que estavam inseridos. O trabalho também contribui para o campo da história da educação, evidenciando outras possibilidades de abordagem dos fenômenos educativos, somando-se ao movimento do campo de ampliar objetos de estudos, fontes de pesquisa e métodos de análises de dados. REFERÊNCIAS Adão, K. S., Lima, A. W., Campos, A. E. D., & Silva, T. J. B. 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São Paulo, SP: Cosac Naify CAROLINA MAFRA DE SÁ é professora adjunta do curso de Licenciatura em Pedagogia, da Universidade Federal do Agreste de Pernambuco (UFAPE). Possui graduação em Pedagogia (2005), pela Universidade Federal de Minas Gerais, mestrado (2009) e doutorado (2015) em Educação pela UFMG, com estágio de doutorado sanduíche (PDSE) na Université Paris-Ouest Nanterre la Défense, França. Recebido em: 01.04.2020 Aprovado em: 03.11.2020 Publicado em: 02.12.2020 Editor-associado responsável: Evelyn de Almeida Orlando (PUC-PR) Email: evelynorlando@gmail.com https://orcid.org/0000-0001-5795-943X Como citar este artigo: Como citar este artigo: Sá, C. M., & Galvão, A. M. O. “A mulher soldado” nos palcos (São João Del-Rei, Minas Gerais, 1916): uma opereta em cena educando sensibilidades. (2021). Revista Brasileira de História da Educação, 21. DOI: http://dx.doi.org/10.4025/rbhe.v21.2021.e144 E-mail: mafradesa@gmail.com https://orcid.org/0000-0002-3871-0716 E-mail: mafradesa@gmail.com https://orcid.org/0000-0002-3871-0716 https://orcid.org/0000-0002-3871-0716 ANA MARIA DE OLIVEIRA GALVÃO é professora titular da Faculdade de Educação da Universidade Federal de Minas Gerais (UFMG). Possui graduação em Pedagogia (1990), pela Universidade Federal de Pernambuco, mestrado (1994) e doutorado (2000) em Educação pela UFMG, com estágio de doutorado sanduíche (PDEE) no Institut National de Recherche Pédagogique (INRP), França. Realizou estágio sênior (com bolsa Capes), na Northern Illinois University, Estados Unidos, em 2012-2013. Este artigo é publicado na modalidade Acesso Aberto sob a licença Creative Commons Atribuição 4.0 (CC-BY 4). E-mail: anamgalvao@uol.com.br https://orcid.org/0000-0001-9063-8267 E-mail: anamgalvao@uol.com.br https://orcid.org/0000-0001-9063-8267 p. 25 de 25 Rev. Bras. Hist. Educ., 21, e144 2021
https://openalex.org/W4321509934	http://ojs.ihar.edu.pl/index.php/biul/article/download/974/844	Polish	null	Występowanie Streptomyces scabies na bulwach ziemniaka uprawianego w warunkach pielęgnacji mechaniczno-chemicznej	Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin	2,013	cc-by-sa	4,307	BIULETYN INSTYTUTU HODOWLI I AKLIMATYZACJI ROŚLIN KRYSTYNA ZARZECKA 1 MAREK GUGAŁA 1 HONORATA DOŁĘGA 1 ALICJA BARANOWSKA 2 1 Katedra Szczegółowej Uprawy Roślin, Uniwersytet Przyrodniczo-Humanistyczny, Siedlce 2 Instytut Rolnictwa, Państwowa Szkoła Wyższa, Biała Podlaska 1 Katedra Szczegółowej Uprawy Roślin, Uniwersytet Przyrodniczo-Humanistyczny, Siedlce 2 Instytut Rolnictwa, Państwowa Szkoła Wyższa, Biała Podlaska DOI: 10.37317/biul-2013-0019 DOI: 10.37317/biul-2013-0019 ∗ Redaktor prowadzący: Wojciech Nowacki Key words: common scab, cultivars, potato, weed control methods Key words: common scab, cultivars, potato, weed control methods WSTĘP Choroby występujące na bulwach ziemniaka mogą prowadzić do obniżenia ich jakości i wartości handlowej. Parch zwykły ziemniaka jest najczęściej występującą chorobą skórki, którą powodują bakterie Streptomyces scabies. Objawia się on strupowatymi zgrubieniami, które, gdy występują w znacznych ilościach, mogą eliminować ziemniak z obrotu handlowego (Atiq i in., 2013; Sadowski, 2006). Choroby tej nie można całkowicie wyeliminować, ale można częściowo ograniczyć jej rozwój stosując prawidłową agrotechnikę (Baldo i Babiker, 2013; Kurzawińska, 1992; Lenc, 2009). Stan zdrowotny bulw zależy od wielu czynników, takich jak: warunki pogodowe, glebowe, odporność odmian, zabiegi agrotechniczne na plantacji (Boligłowa i in., 2004; Głuska, 2002; Gugała i in., 2007; Rębarz i Borówczak, 2009; Singhai i Sarma, 2011). Nowacki (2006) stwierdził, że straty plonu ogólnego spowodowane przez porażenie bulw parchem zwykłym przekraczały nawet 8%. Udział w plonie bulw porażonych przez Streptomyces scabies może być duży i osiągać nawet 60–100% (Lenc, 2009; Sawicka i Krochmal-Marczak, 2009). Burgieł i Gleń (1997), Gruczek (2001), Klikocka (2000) uważają, że herbicydy chroniąc rośliny ziemniaka przed chwastami, niszczą jednocześnie źródła infekcji różnych chorób. Przeprowadzone badania miały na celu określenie, czy zastosowanie zabiegów mechaniczno-chemicznych regulujących zachwaszczenie przyczynia się do poprawy ilości, a tym samym jakości plonu bulw ziemniaka. Occurrence of common scab (Streptomyces scabies) on potato tubers in conditions of mechanical and chemical crop cultivation Wyniki badań pochodzą z doświadczenia polowego przeprowadzonego w latach 2008–2010 w Rolniczej Stacji Doświadczalnej należącej do Uniwersytetu Przyrodniczo-Humanistycznego w Siedlcach. Doświadczenie założono metodą losowanych podbloków jako dwuczynnikowe w trzech powtórzeniach. Czynnikiem I rzędu były odmiany ziemniaka: Cekin, Satina, Tajfun, czynnikiem II rzędu — sposoby odchwaszczania z udziałem herbicydów i ich mieszanin: Command 480 EC, Command 480 EC i Afalon Dyspersyjny 450 SC, Stomp 400 SC, Stomp 400 SC i Afalon Dyspersyjny 450 SC oraz obiekt kontrolny — pielęgnacja mechaniczna. Objawy parcha zwykłego oceniano w 9- stopniowej skali na 100 bulwach pobranych losowo z poszczególnych obiektów doświadczenia. Badania dowiodły, że zastosowane sposoby odchwaszczania z udziałem herbicydów w istotny sposób wpłynęły na ograniczenie występowania parcha zwykłego na bulwach oraz na średni stopień porażenia próby i średni stopień porażenia bulw porażonych. Słowa kluczowe: odmiany, parch zwykły, sposoby odchwaszczania, ziemniak The results reported in this work come from a field experiment carried out at the University of Natural Sciences and Humanities in Siedlce in the years 2008–2010. The experiment was set in the split-plot design as a two-factorial, three-replication trial. The factors examined were as follows: factor I — potato cultivars: Cekin, Satina, Tajfun, factor II — weed control methods for application herbicides and their mixtures: Command 480 EC, Command 480 EC and Afalon Dyspersyjny 450 SC, Stomp 400 SC, Stomp 400 SC and Afalon Dyspersyjny 450 SC and control object — mechanical weeding. Symptoms produced by the common scab were evaluated using a nine-grade scale on 100 tubers chosen at random from the experimental objects. The results demonstrated that weed control methods with herbicides significantly reduced the infection of tubers with common scab, mean degree of sample infestation and mean degree of tubers infestation. ∗ Redaktor prowadzący: Wojciech Nowacki 79 79 Krystyna Zarzecka ... MATERIAŁ I METODY Doświadczenie polowe przeprowadzono w latach 2008–2010 metodą losowanych podbloków, w układzie split-plot, w trzech powtórzeniach, jako dwuczynnikowe. Eksperyment założono na glebie zaliczanej do kompleksu żytniego bardzo dobrego, klasy IVa, o odczynie lekko kwaśnym (2009) i kwaśnym (lata 2007 i 2008). Czynnikiem I rzędu były odmiany ziemniaka — Cekin, Satina, Tajfun, a czynnikiem II rzędu — sposoby odchwaszczania: 1. pielęgnacja mechaniczna do i po wschodach rośliny uprawnej, 2. pielęgnacja mechaniczno-chemiczna z herbicydem Command 480 EC w dawce 0,2 dm3∙ha- 1, 3. pielęgnacja mechaniczno-chemiczna z mieszaniną herbicydów Command 480 EC w dawce 0,2 dm3∙ha-1 i Afalon Dyspersyjny 1,0 dm3∙ha-1, 4. pielęgnacja mechaniczno- chemiczna z herbicydem Stomp 400 SC stosowanym w dawce 3,5 dm3∙ha-1, 5. pielęgnacja mechaniczno-chemiczna z mieszaniną herbicydów Stomp 400 SC w dawce 3,5 dm3∙ha-1 i Afalon Dyspersyjny 1,0 dm3∙ha-1. Odmiany uprawiane w doświadczeniu charakteryzowały się podwyższoną odpornością na parcha zwykłego: Cekin — ocena 6,5, Satina — ocena 6,5, Tajfun — ocena 6,0 w skali 1–9 (Chotkowski i Stypa, 2010; Lista opisowa odmian, 2011). ) Oceny porażenia parchem zwykłym dokonano bezpośrednio po zbiorze na 100 bulwach pobranych losowo z każdego obiektu doświadczenia (45 prób). Określono procentowy udział bulw porażonych, średni stopień porażenia próby i średni stopień porażenia bulw 80 Krystyna Zarzecka ... porażonych w skali 1–9, gdzie 9 oznacza bulwy zdrowe, 6 — 16–20% powierzchni chorej, a 1 — ponad 50% powierzchni opanowanej przez parcha (Roztropowicz i in., 1999). Wyniki badań przekształcono za pomocą transformacji Blissa i opracowano statystycznie za pomocą analizy wariancji, a ocenę istotności różnic, przy poziomie p = 0,05 pomiędzy porównywalnymi średnimi testowano za pomocą wielokrotnych przedziałów Tukeya. MATERIAŁ I METODY Tabela 1 Warunki meteorologiczne w okresie maj — lipiec w latach 2008–2010 Meteorological conditions over the period May — July in the years 2008–2010 Miesiące Months Parametr Dekady — Decades Średnia/Suma Mean/Sum Wielolecie Multiyear I II III 2008 Maj May Temperatura — Temperature (ºC) 11,9 13,2 13,1 12,7 12,5 Opady — Rainfall (mm) 53,5 26,2 5,9 85,6 44,1 Czerwiec June Temperatura — Temperature (ºC) 18,3 16,0 17,8 17,4 17,2 Opady — Rainfall (mm) 0,1 36,3 12,6 49,0 52,4 Lipiec July Temperatura — Temperature (ºC) 17,6 18,3 19,4 18,4 19,2 Opady — Rainfall (mm) 44,3 9,9 15,6 69.8 49,8 2009 Maj May Temperatura — Temperature (ºC) 12,3 12,3 14,0 12,9 12,5 Opady — Rainfall (mm) 4,8 14,5 49,6 68,9 44,1 Czerwiec June Temperatura — Temperature (ºC) 13,9 14,3 19,0 15,7 17,2 Opady — Rainfall (mm) 35,6 43,4 66,2 145,2 52,4 Lipiec July Temperatura — Temperature (ºC) 19,2 19,7 19,4 19,4 19,2 Opady — Rainfall (mm) 14,9 3,9 7,6 26,4 49,8 2010 Maj May Temperatura — Temperature (ºC) 12,7 14,8 14,6 14,0 12,5 Opady — Rainfalls (mm) 30,3 41,2 21,7 93,2 44,1 Czerwiec June Temperatura — Temperature (ºC) 18,6 16,7 16,9 17,4 17,2 Opady — Rainfalls (mm) 12,5 47,3 2,8 62,6 52,4 Lipiec July Temperatura — Temperature (ºC) 20,2 23,9 20,6 21,6 19,2 Opady — Rainfalls (mm) 0,0 14,3 62,7 77,0 49,8 Tabe Warunki meteorologiczne w okresie maj — lipiec w latach 2008–2010 Meteorological conditions over the period May — July in the years 2008–2010 WYNIKI I DYSKUSJA Analiza średnich trzyletnich wyników badań wykazała istotny wpływ sposobów odchwaszczania, uprawianych odmian i warunków pogodowych podczas wegetacji na procentowy udział bulw porażonych, średni stopień porażenia próby i średni stopień porażenia bulw porażonych (tab. 2–5). Zastosowanie zabiegów odchwaszczających z udziałem herbicydów i ich mieszanin zmniejszało występowanie parcha zwykłego na bulwach wszystkich uprawianych odmian ziemniaka. Istotnie mniejszy udział bulw porażonych, stopień porażenia próby oraz średni stopień porażenia bulw porażonych, w porównaniu z obiektem kontrolnym, stwierdzono na wszystkich obiektach doświadczenia, na których aplikowano herbicydy i ich mieszaniny (tab. 2–4). Udowodnione statystycznie różnice, w obrębie wszystkich oznaczanych cech porażenia parchem, wykazano także pomiędzy obiektami odchwaszczanymi pojedynczym herbicydem i mieszaninami herbicydów, tj. 2 i 3 oraz 4 i 5. Według Boligłowy i in. (2004) stan zdrowotny bulw ziemniaka po zbiorze zależał od rodzaju użytego środka chwastobójczego. Z testowanych 81 Krystyna Zarzecka ... substancji aktywnych: linuron, fluorochloridon, cyjanazyna, mieszanina metrybuzyna + fluorochloridon najsilniej ograniczały występowanie parcha zwykłego na bulwach. Natomiast inne preparaty (rimsulfuron + metrybuzyna, linuron + cyjanazyna) zwiększały procentowy udział bulw z objawami Streptomyces scabies. Burgieł i Gleń (1997) stwierdzili mniejsze nasilenie tej choroby pod wpływem stosowania linuronu. W badaniach Gugały i in. (2007) sposoby pielęgnacji nie miały istotnego wpływu na udział bulw z objawami parcha i stopień porażenia próby, tym nie mniej zaobserwowano tendencję do zmniejszania udziału bulw zainfekowanych po zastosowaniu herbicydów i ich mieszanin do odchwaszczania ziemniaka. WYNIKI I DYSKUSJA Tabela 2 Procentowy udział bulw porażonych parchem zwykłym w zależności od odmiany i sposobów odchwaszczania Percentage of tubers infested by a common scab depending on the cultivar and weed control method Sposoby odchwaszczania Weed control methods Procent bulw porażonych — Percentage of infested tubers odmiany — cultivars średnio mean Cekin Satina Tajfun Obiekt kontrolny — pielęgnacja mechaniczna Control object — mechanical weeding 10,5 18,1 3,0 10,6 Command 480 EC 7,3 12,1 2,5 7,3 Command 480 EC + Afalon Dyspersyjny 450 SC 6,9 9,7 0,4 5,7 Stomp 400 SC 6,5 13,5 2,2 7,4 Stomp 400 SC i Afalon Dyspersyjny 450 SC 5,0 9,5 1,1 5,2 Średnio — Mean 7,3 12,6 1,8 7,2 NIR — LSD0.05 odmiany — cultivars 1,4 sposoby odchwaszczania — weed control methods 2,3 interakcja — interaction 3,4 Tabela 3 Średni stopień porażenia próby parchem zwykłym w zależności od odmiany i sposobów odchwaszczania Mean degree of sample infestation by a common scab depending on the cultivar and weed control method Sposoby odchwaszczania Weed control methods Średni stopień porażenia próby (skala 1-9) Mean degree of sample infestation (scale 1-9) odmiany — cultivars średnio mean Cekin Satina Tajfun Obiekt kontrolny — pielęgnacja mechaniczna Control object — mechanical weeding 8,57 8,18 8,90 8,55 Command 480 EC 8,70 8,52 8,92 8,72 Command 480 EC + Afalon Dyspersyjny 450 SC 8,78 8,65 8,99 8,81 Stomp 400 SC 8,77 8,47 8,94 8,72 Stomp 400 SC i Afalon Dyspersyjny 450 SC 8,84 8,65 8,97 8,82 Średnio — Mean 8,73 8,50 8,95 8,72 NIR — LSD0.05 odmiany — cultivars 0,05 sposoby odchwaszczania –—weed control methods 0,09 interakcja — interaction 0,13 Tabela 2 Procentowy udział bulw porażonych parchem zwykłym w zależności od odmiany i sposobów odchwaszczania Percentage of tubers infested by a common scab depending on the cultivar and weed control method Sposoby odchwaszczania Weed control methods Procent bulw porażonych — Percentage of infested tubers odmiany — cultivars średnio mean Cekin Satina Tajfun Obiekt kontrolny — pielęgnacja mechaniczna Control object — mechanical weeding 10,5 18,1 3,0 10,6 Command 480 EC 7,3 12,1 2,5 7,3 Command 480 EC + Afalon Dyspersyjny 450 SC 6,9 9,7 0,4 5,7 Stomp 400 SC 6,5 13,5 2,2 7,4 Stomp 400 SC i Afalon Dyspersyjny 450 SC 5,0 9,5 1,1 5,2 Średnio — Mean 7,3 12,6 1,8 7,2 NIR — LSD0.05 odmiany — cultivars 1,4 sposoby odchwaszczania — weed control methods 2,3 interakcja — interaction 3,4 Tabela 2 Procentowy udział bulw porażonych parchem zwykłym w zależności od odmiany i sposobów odchwaszczania Tabela 3 Średni stopień porażenia próby parchem zwykłym w zależności od odmiany i sposobów odchwaszczania Mean degree of sample infestation by a common scab depending on the cultivar and weed control method Sposoby odchwaszczania Weed control methods Średni stopień porażenia próby (skala 1-9) Mean degree of sample infestation (scale 1-9) odmiany — cultivars średnio mean Cekin Satina Tajfun Obiekt kontrolny — pielęgnacja mechaniczna Control object — mechanical weeding 8,57 8,18 8,90 8,55 Command 480 EC 8,70 8,52 8,92 8,72 Command 480 EC + Afalon Dyspersyjny 450 SC 8,78 8,65 8,99 8,81 Stomp 400 SC 8,77 8,47 8,94 8,72 Stomp 400 SC i Afalon Dyspersyjny 450 SC 8,84 8,65 8,97 8,82 Średnio — Mean 8,73 8,50 8,95 8,72 NIR — LSD0.05 odmiany — cultivars 0,05 sposoby odchwaszczania –—weed control methods 0,09 interakcja — interaction 0,13 Ta Średni stopień porażenia próby parchem zwykłym w zależności od odmiany i sposobów odchwaszczania 82 Krystyna Zarzecka ... WYNIKI I DYSKUSJA Tabela 4 Średni stopień porażenia bulw porażonych parchem zwykłym w zależności od odmiany i sposobów odchwaszczania Mean degree of tuber infestation by a common scab depending on the cultivar and weed control method Sposoby odchwaszczania Weed control methods Średni stopień porażenia bulw porażonych (skala 1–9) Mean degree of tuber infestation (scale 1–9) odmiany — cultivars średnio mean Cekin Satina Tajfun Obiekt kontrolny — pielęgnacja mechaniczna Control object — mechanical weeding 5,47 4,87 6,53 5,62 Command 480 EC 6,20 5,92 7,45 6,52 Command 480 EC + Afalon Dyspersyjny 450 SC 6,50 5,94 8,33 6,93 Stomp 400 SC 5,98 5,71 7,28 6,32 Stomp 400 SC i Afalon Dyspersyjny 450 SC 6,42 5,85 8,22 6,83 Średnio — Mean 6,12 5,65 7,56 6,45 NIR — LSD0.05 odmiany — cultivars 0,33 sposoby odchwaszczania — weed control methods 0,57 interakcja — interaction r.n.-n.s. r.n. — różnice nieistotne, n.s. — not significant differences Tabela Średni stopień porażenia bulw porażonych parchem zwykłym w zależności od odmiany i sposobów odchwaszczania Najmniejszy udział bulw z objawami parcha oraz najmniejszy stopień porażenia bulw wystąpił u odmiany Tajfun, istotnie większy u odmiany Cekin, a największy u odmiany Satina, co świadczy o dużym zróżnicowaniu odmian, a jednocześnie wskazuje, że przy doborze odmian do produkcji należy kierować się ich odpornością na choroby. Znaczne różnice w stopniu porażenia odmian obserwowali również Atiq i in. (2013), Gawińska- Urbanowicz (2007), Lenc (2009), Sadowski (2006), Wróbel (2003). Ponadto Sawicka i Krochmal-Marczak (2009) oraz Sadowski i in. (2004) stwierdzili, że zróżnicowanie w nasileniu parcha zwykłego w większym stopniu zależy od roku prowadzenia badań aniżeli od odmian. Wyniki uzyskane z oceny występowania parcha zwykłego na badanych odmianach nie w pełni były zgodne z odpornością na tę chorobę podaną przez IHAR i COBORU (Chotkowski i Stypa, 2010; Lista opisowa odmian, 2011). Do podobnych spostrzeżeń doszli Lenc i in. (2006). Również Wróbel (2006) wykazał, że dwie odmiany, których odporność według COBORU była jednakowa, różniły się udziałem bulw porażonych o ok. 30%. Zainfekowanie bulw ziemniaka parchem zwykłym jest uwarunkowane genetycznie, ale jak wynika z badań własnych i wyżej wymienionych autorów, w znacznym stopniu zależy też od warunków środowiskowych. Wielu autorów uważa, że na występowanie parcha zwykłego duży wpływ mają przede wszystkim warunki klimatyczne. Mała wilgotność gleby, szczególnie w czasie tuberyzacji, stwarza korzystne warunki dla rozwoju Streptomyces scabies (Gugała i in., 2007; Kurzawińska, 1992; Lenc, 2009; Szutkowska i Lutomirska, 2002; Wróbel, 2003). WYNIKI I DYSKUSJA Kapsa i Gawińska-Urbanowicz (2013) obserwowały corocznie przez cztery lata występowanie głównych chorób ziemniaka w 9-11 województwach i odnotowały, że średnie porażenie roślin parchem zwykłym wahało się między 10,8-21,2% w zależności od lat i terenu badań. Ponadto stwierdziły, że monitorowanie plantacji pod kątem chorób wywoływanych przez bakterie jest niezwykle ważne z powodu nowych źródeł infekcji i dużych zmian w składzie sprawców tych chorób. Lenc i in. (2006) stwierdzili, że prawidłowa pielęgnacja, wykonany w optymalnym terminie i warunkach pogodowych zbiór, ograniczały porażenie bulw Streptomyces scabies i Fusarium spp Tabela 5 Porażenie bulw ziemniaka parchem zwykłym w latach badań (w % i skali 1–9) Infestation of potato tubers by a common scab in the years of study (in % and scale 1–9) Wyszczególnienie Specification Lata Years Średnio Mean NIR0,05 LSD0.05 2008 2009 2010 Procent bulw porażonych Percentage of infested tubers 7,6 11,7 2,4 7,2 1,4 Średni stopień porażenia próby (skala 1–9) Mean degree of sample infestation (scale 1–9) 8,75 8,48 8,94 8,72 0,05 Średni stopień porażenia bulw porażonych (skala 1–9) Mean degree of tuber infestation (scale 1–9) 5,97 5,60 7,77 6,45 0,33 Porażenie bulw ziemniaka parchem zwykłym w latach badań (w % i skali 1–9) Infestation of potato tubers by a common scab in the years of study (in % and scale 1–9 Również Gawińska-Urbanowicz (2007) stwierdziła niewielkie zagrożenie odmian średnio wczesnych parchem zwykłym, a ich udział w plonie wynosił przeciętnie 8,2%. Natomiast Sawicka i Krochmal-Marczak (2009) analizując wpływ biostymulatorów, odmian i lat badań wykazały, że średni udział bulw porażonych Streptomyces scabies wynosił 68,3% i w największym stopniu zależał od warunków pogodowych podczas wegetacji. W prowadzonych badaniach wykazano, że procentowy udział bulw porażonych, średni stopień porażenia próby jak i średni stopień porażenia bulw porażonych zależały od warunków meteorologicznych w latach badań. Najmniejsze nasilenie choroby wystąpiło w 2010 roku, w którym temperatury powietrza w okresie początkowego wzrostu i wiązania bulw były największe (czerwiec, lipiec). Potwierdzają to spostrzeżenia Lutomirskiej (2008), że pomiędzy temperaturą a udziałem bulw porażonych parchem zwykłym występuje zależność prostoliniowa ujemna. Oznacza to, że wraz ze wzrostem temperatury w zakresie ok. 19–25°C następuje pogorszenie warunków dla rozwoju patogena. Warunki wilgotnościowe w okresie badawczym były zróżnicowane. Największe opady w maju i lipcu wystąpiły w 2010 roku, natomiast w czerwcu w 2009 roku (145,2 mm) i były one aż trzykrotnie większe w porównaniu do okresu wieloletniego. WYNIKI I DYSKUSJA Natomiast Lutomirska (2008) stwierdziła, że głównym czynnikiem meteorologicznym, który determinował porażenie bulw parchem zwykłym była temperatura gleby w okresie wiązania i wczesnego wzrostu bulw. W ocenianym okresie udział bulw porażonych w zebranym plonie był niewielki, wynosił średnio 7,2% (tab. 5). 83 Krystyna Zarzecka ... Tabela 5 Porażenie bulw ziemniaka parchem zwykłym w latach badań (w % i skali 1–9) Infestation of potato tubers by a common scab in the years of study (in % and scale 1–9) Wyszczególnienie Specification Lata Years Średnio Mean NIR0,05 LSD0.05 2008 2009 2010 Procent bulw porażonych Percentage of infested tubers 7,6 11,7 2,4 7,2 1,4 Średni stopień porażenia próby (skala 1–9) Mean degree of sample infestation (scale 1–9) 8,75 8,48 8,94 8,72 0,05 Średni stopień porażenia bulw porażonych (skala 1–9) Mean degree of tuber infestation (scale 1–9) 5,97 5,60 7,77 6,45 0,33 Również Gawińska-Urbanowicz (2007) stwierdziła niewielkie zagrożenie odmian średnio wczesnych parchem zwykłym, a ich udział w plonie wynosił przeciętnie 8,2%. Natomiast Sawicka i Krochmal-Marczak (2009) analizując wpływ biostymulatorów, odmian i lat badań wykazały, że średni udział bulw porażonych Streptomyces scabies wynosił 68,3% i w największym stopniu zależał od warunków pogodowych podczas wegetacji. W prowadzonych badaniach wykazano, że procentowy udział bulw porażonych, średni stopień porażenia próby jak i średni stopień porażenia bulw porażonych zależały od warunków meteorologicznych w latach badań. Najmniejsze nasilenie choroby wystąpiło w 2010 roku, w którym temperatury powietrza w okresie początkowego wzrostu i wiązania bulw były największe (czerwiec, lipiec). Potwierdzają to spostrzeżenia Lutomirskiej (2008), że pomiędzy temperaturą a udziałem bulw porażonych parchem zwykłym występuje zależność prostoliniowa ujemna. Oznacza to, że wraz ze wzrostem temperatury w zakresie ok. 19–25°C następuje pogorszenie warunków dla rozwoju patogena. Warunki wilgotnościowe w okresie badawczym były zróżnicowane. Największe opady w maju i lipcu wystąpiły w 2010 roku, natomiast w czerwcu w 2009 roku (145,2 mm) i były one aż trzykrotnie większe w porównaniu do okresu wieloletniego. Liczni autorzy (Lenc, 2009; Sawicka i Krochmal-Marczak, 2009; Szutkowska, 2002; Wróbel, 2003, 2006) uważają, że choroba bardziej się nasila w warunkach mniejszej wilgotności, a nawet suchej aury w okresie tuberyzacji. Badania Jankowskiej i Lutomirskiej (2013) prowadzone na 125 genotypach różnej wczesności wykazały, że wraz ze wzrostem sumy opadów w okresie kwitnienia następowało ograniczenie nasilenia zmian chorobowych powodowanych przez parcha zwykłego na bulwach. LITERATURA Atiq M., Khalid A. R., Hussian W., Nawaz A., Asad S., Ahmad T. M. 2013. Genetic potential of potato germplasm against common scab disease caused by Streptomyces scabies. Pak. J. Phytopathol. 25 (01): 27 — 30. Baldo N. H., Babiker M. Y. 2013. Effect of some cultural practices on common scab (Streptomyces scabies [Thaxter]) of potato at two production sites in Khartoum State. Int. J. of Sudan Res. 3 (2): 149 — 166. Boligłowa E., Gleń K., Pisulewski P. 2004. Wpływ stosowania herbicydów na plonowanie i niektóre cechy jakości bulw ziemniaka. Zesz. Probl. Post. Nauk Rol. 500: 391 — 397. Burgieł Z., Gleń K. 1997. Wpływ herbicydu Afalon na zdrowotność bulw ziemniaka. Pestycydy 3–4: 85 — 91. Chotkowski J., Stypa I. 2010. Odmiany ziemniaków. Charakterystyka tabelaryczna. Wyd. IHAR, Bonin:1 — 12. Gawińska-Urbanowicz H. 2007. Ocena występowania chorób grzybowych i bakteryjnych ziemniaka w warunkach polowych. Biul. IHAR 243: 191 — 197. Głuska A. 2002. Wpływ warunków glebowych i rozkładu opadów na plon i niektóre cechy jakości bulw jako ograniczenia w produkcji ekologicznej ziemniaka. Zesz. Probl. Post. Nauk Rol. 489: 113 — 121. k f k b lki h i i i h ł j k b l i i k i l Głuska A. 2002. Wpływ warunków glebowych i rozkładu opadów na plon i niektóre cechy jakości bulw jako ograniczenia w produkcji ekologicznej ziemniaka. Zesz. Probl. Post. Nauk Rol. 489: 113 — 121. Gruczek T. 2001. Efektywne sposoby walki z chwastami i ich wpływ na jakość bulw ziemniaka. Biul. IHAR 217: 221 — 231. ograniczenia w produkcji ekologicznej ziemniaka. Zesz. Probl. Post. Nauk Rol. 489: 113 121. Gruczek T. 2001. Efektywne sposoby walki z chwastami i ich wpływ na jakość bulw ziemniaka. Biul. IHAR 217: 221 — 231. Gugała M., Zarzecka K., Rymuza K. 2007. Wpływ uprawy roli i sposobu odchwaszczania plantacji ziemniaka na porażenie bulw parchem zwykłym (Streptomyces scabies Thaxt). Biul. IHAR 246: 127 — 133. Ś Jankowska J., Lutomirska B. 2013. Środowiskowa i genotypowa zmienność występowania parcha zwykłego na bulwach zaawansowanych materiałów hodowlanych ziemniaka. Prog. Plant Prot./Post. Ochr. Roślin 53 (3): 538 — 544. ( ) Kapsa J., Gawińska-Urbanowicz H. 2013. Nasilenie sprawców chorób ziemniaka na terenie Polski w latach 2009-2013. Prog. Plant Prot./Post. Ochr. Roślin 53 (3): 545 — 551. Klikocka H. 2000. Wpływ stosowania różnych metod pielęgnowania i uprawy roli na porażenie bulw ziemniaka parchem zwykłym (Streptomyces scabies (Thaxt.) Waksman et Henrici). Prog. Plant Prot./Post. Ochr. WYNIKI I DYSKUSJA Liczni autorzy (Lenc, 2009; Sawicka i Krochmal-Marczak, 2009; Szutkowska, 2002; Wróbel, 2003, 2006) uważają, że choroba bardziej się nasila w warunkach mniejszej wilgotności, a nawet suchej aury w okresie tuberyzacji. Badania Jankowskiej i Lutomirskiej (2013) prowadzone na 125 genotypach różnej wczesności wykazały, że wraz ze wzrostem sumy opadów w okresie kwitnienia następowało ograniczenie nasilenia zmian chorobowych powodowanych przez parcha zwykłego na bulwach. Kapsa i Gawińska-Urbanowicz (2013) obserwowały corocznie przez cztery lata występowanie głównych chorób ziemniaka w 9-11 województwach i odnotowały, że średnie porażenie roślin parchem zwykłym wahało się między 10,8-21,2% w zależności od lat i terenu badań. Ponadto stwierdziły, że monitorowanie plantacji pod kątem chorób wywoływanych przez bakterie jest niezwykle ważne z powodu nowych źródeł infekcji i dużych zmian w składzie sprawców tych chorób. L i i (2006) t i d ili ż idł i l j k t l Lenc i in. (2006) stwierdzili, że prawidłowa pielęgnacja, wykonany w optymalnym terminie i warunkach pogodowych zbiór, ograniczały porażenie bulw Streptomyces scabies i Fusarium spp. 84 Krystyna Zarzecka ... WNIOSKI 1. Udział bulw z objawami parcha, średni stopień porażenia próby i średni stopień porażenia bulw porażonych parchem zwykłym były istotnie determinowane przez sposoby odchwaszczania, uprawiane odmiany i warunki pogodowe podczas wegetacji ziemniaka. 2. Herbicydy i ich mieszaniny zastosowane do odchwaszczania plantacji ograniczały porażenia bulw wywołane przez Streptomyces scabies, co przyczyniło się do poprawy jakości plonu ziemniaka. 2. Herbicydy i ich mieszaniny zastosowane do odchwaszczania plantacji ograniczały porażenia bulw wywołane przez Streptomyces scabies, co przyczyniło się do poprawy jakości plonu ziemniaka. 3. Najmniejszy udział bulw z objawami parcha oraz najmniejszy stopień porażenia bulw stwierdzono u odmiany Tajfun, a istotnie większy u pozostałych uprawianych w doświadczeniu odmian, co sugeruje, że właściwy dobór odmian stwarza możliwości ograniczenia występowania parcha zwykłego. 3. Najmniejszy udział bulw z objawami parcha oraz najmniejszy stopień porażenia bulw stwierdzono u odmiany Tajfun, a istotnie większy u pozostałych uprawianych w doświadczeniu odmian, co sugeruje, że właściwy dobór odmian stwarza możliwości ograniczenia występowania parcha zwykłego. g y ęp p y g 4. Warunki atmosferyczne w okresie wegetacji wpływały istotnie na występowanie parcha zwykłego. W 2010 roku, w którym temperatury powietrza w okresie początkowego wzrostu i wiązania bulw były największe (czerwiec, lipiec) infekcja bulw parchem zwykłym była najmniejsza. LITERATURA Roślin 38 (2): 634 — 636. ( ) Kurzawińska H. 1992. Wpływ zróżnicowanego nawożenia mineralnego oraz trzech terminów sadzenia wybranych odmian ziemniaka na występowanie parcha zwykłego (Streptomyces scabies (Thaxt) Waksman et Henrici). Zesz. Nauk. AR w Krakowie 267: 149 — 158. 85 Krystyna Zarzecka ... Lenc L. 2009. Występowanie Streptomyces scabies na bulwach ośmiu odmian ziemniaka uprawianego w systemie ekologicznym i integrowanym. Ochrona Środowiska i Zasobów Naturalnych 40: 669 — 676. y g y g y y Lenc L., Sadowski Cz., Nowacki W. 2006. Wpływ podkiełkowywania sadzeniaków na występowanie parcha zwykłego (Streptomyces scabies) i suchej zgnilizny bulw (Fusarium spp.) sześciu odmian ziemniaka uprawianego systemem ekologicznym. Zesz. Probl. Post. Nauk Rol. 511: 337 — 345. p g y g y Lista opisowa odmian. Rośliny rolnicze. 2011. Cz. 2. Wyd. COBORU, Słupia Wielka: 1 — 151. Lutomirska B. 2008. Wpływ czynników meteorologicznych na porażenie bulw ziemniaka parchem zwykłym. Prog. Plant Prot./Post. Ochr. Roślin 48 (1): 216 — 220. Lutomirska B. 2008. Wpływ czynników meteorologicznych na porażenie bulw ziemniaka parchem zwykłym. Prog. Plant Prot./Post. Ochr. Roślin 48 (1): 216 — 220. Nowacki W. 2006. Straty plonu handlowego ziemniaków powodowane przez choroby i szkodniki w 2005 roku. g ( ) Nowacki W. 2006. Straty plonu handlowego ziemniaków powodowane przez choroby i szkodniki w 2005 roku. Prog. Plant Prot./Post. Ochr. Roślin 46 (1): 193 — 201. Rębarz K., Borówczak F. 2009. Porażenie patogenami bulw ziemniaków odmiany Satina w zależności od deszczowania, technologii uprawy i nawożenia azotowego. Prog. Plant Prot./Post. Ochr. Roślin 49 (4): 1762 — 1766. Roztropowicz S., Czerko Z., Głuska A., Goliszewski W., Gruczek T., Lis B., Lutomirska B., Nowacki W., Rykaczewska K., Sowa-Niedziałkowska G., Szutkowska M., Wierzejska- Bujakowska A., Zarzyńska K., Zgórska K. 1999. Metodyka obserwacji, pomiarów i pobierania prób w agrotechnicznych doświadczeniach z ziemniakiem. Wyd. IHAR Radzików, Oddział Jadwisin: 1 — 50. Sadowski Cz. 2006. Stan zdrowotności polskiego ziemniaka i jej zagrożenia. Zesz. Probl. Post. Nauk Rol. 511: 37 — 51. Sadowski Cz., Pańka D., Lenc L. 2004. Porównanie zdrowotności bulw i kiełków wybranych odmian ziemniaka uprawianych w systemie ekologicznym. Zesz. Probl. Post. Nauk Rol. 500: 373 — 381. p y y g y Sawicka B., Krochmal-Marczak B. 2009. Wpływ stosowania nawozu dolistnego Insol 7 i bioregulatora Asahi SL na zdrowotność bulw kilku odmian ziemniaka. Annales UMCS, E 64 (2): 29 — 38. Singhai P. K., Sarma B. K. 2011. Lenc L. 2009. 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https://openalex.org/W2107637490	https://europepmc.org/articles/pmc3530464?pdf=render	English	null	Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production	PloS one	2,012	cc-by	6,894	Abstract Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4- phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a b-sesquiphellandrene synthase as it was demonstrated to be functional in producing b- sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing b-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme- A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of b-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction. Citation: Song AA-L, Abdullah JO, Abdullah MP, Shafee N, Othman R, et al. (2012) Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production. PLoS ONE 7(12): e52444. doi:10.1371/journal.pone.0052444 Editor: Paul Jaak Janssen, Belgian Nuclear Research Centre SCK/CEN, Belgium Received September 21, 2012; Accepted November 13, 2012; Published December 26, 2012 ceived September 21, 2012; Accepted November 13, 2012; Published December 26, 2012 Copyright: 2012 Song et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by ‘‘The Genomics and Molecular Biology Initiative of the Malaysia Genome Institute, Ministry of Science, Technology and Innovation, Malaysia (MOSTI) (07-05-MGI-GMB 004)’’ and National Science Fellowship, MOSTI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: raha@biotech.upm.edu.my Adelene Ai-Lian Song1, Janna Ong Abdullah2, Mohd. Puad Abdullah1, Norazizah Shafee2, Roohaida Othman4, Ee-Fun Tan5, Normah Mohd. Noor4, Abdul Rahim Raha1,3* Adelene Ai-Lian Song1, Janna Ong Abdullah2, Mohd. Puad Abdullah1, Norazizah Shafee2, Roohaida Othman4, Ee-Fun Tan5, Normah Mohd. Noor4, Abdul Rahim Raha1,3* 1 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor Darul Ehsan, Malaysia, 2 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor Darul Ehsan, Malaysia, 3 Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor Darul Ehsan, Malaysia, 4 Institute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan, Malaysia, 5 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor Darul Ehsan, Malaysia December 2012 \| Volume 7 \| Issue 12 \| e52444 tation: Song AA-L, Abdullah JO, Abdullah MP, Shafee N, Othman R, et al. (2012) Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme e Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production. PLoS ONE 7(12): e52444. doi:10.1371/journal.po Introduction sesquiterpene precursor for the anti-malarial drug artemisinin [5]. Isoprenoids may be synthesized via two pathways, either the mevalonate pathway or the non-mevalonate pathway, also known as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway [6]. All organisms only have either one of the pathways except plants which possess both pathways, although they are localized in different organelles of the cell. The mevalonate pathway is usually associated with eukaryotes while the MEP pathway is more commonly found in prokaryotes. However, some Gram-positive bacteria such as Staphylococcus aureus, Lactobacillus plantarum and L. lactis have adopted the mevalonate pathway through evolution for isoprenoid biosynthesis [7,8]. Lactococcus lactis is a food grade lactic acid bacterium which has been used as a starter culture in food fermentation for centuries. However, over the last decade, L. lactis has found other new and exciting applications as it has been developed into cell factories for the production of bioactive compounds such as enzymes, peptides and vaccine antigens [1,2]. Recently, Hernandez et al. [3] reported on the first attempt to utilize L. lactis for the production of the isoprenoids linalool and nerolidol from strawberry (Fragaria6ana- nassa) by heterologous expression of the FaNES gene. Isoprenoids are a large and diverse group of naturally occurring metabolites found in the primary metabolism of all living organisms. However, some organisms especially plants also produce isoprenoids as secondary metabolites, and these isopre- noids have interesting properties such as flavour, fragrance and toxicity [4]. Some of the most valuable isoprenoids known so far include the diterpene, taxadiene, which is the precursor for paclitaxel, a potent anti-cancer drug as well as amorphadiene, the E. coli and yeasts have been extensively studied and engineered as heterologous hosts for the production of plant isoprenoids. These include many different metabolic engineering strategies employed to increase the production of the heterologous isoprenoids in the host cells [9]. However, L. lactis is also a potential heterologous host for isoprenoid production with certain advan- December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org 1 Overexpressing HMGR for Sesquiterpene Production contained 16 reaction buffer, 2 mM MgCl 2, 0.2 mM dNTP, 5 units of Taq polymerase or High-Fidelity PCR Enzyme Mix (Fermentas, USA), 0.5 mM of forward and reverse primers and approximately 20 ng of template DNA. The reaction was run for 30 cycles using a 94uC for 1 min, 60uC for 1 min and 72uC for 2 min temperature-time profile. Bacterial Strains and Culture Conditions L. lactis NZ9000 strain, a derivative of the nisin-negative L. lactis MG1363 which has the nisR and nisK genes from the nisin gene cluster inserted into its chromosome was used as the host in this study [14]. The nisR and nisK genes enable nisin induction of the PnisA promoter on the pNZ8048 plasmid which was used for all L. lactis cloning purposes in this study. All L. lactis strains were cultured in M17 broth or agar [15] supplemented with 0.5% (w/v) glucose (GM17) and 7.5 mg/mL chloramphenicol whenever necessary. L. lactis was typically grown at 30uC as a stand culture. When screening for L. lactis transformants, M17 agar supplemen- ted with 0.5% (w/v) glucose, 0.5 M sucrose and 7.5 mg/mL chloramphenicol was used. Expression of Recombinant Proteins For protein expression, overnight culture of L.lactis NZ9000 harbouring pNZ:PMSTS or pNZ:PMSTS:mvaA were inoculated into fresh GM17 medium at 5% (v/v) and grown to an OD600 of 0.4. The culture was induced with 40 ng/mL nisin for 2 h which was pre-determined to be the optimum induction condition (data not shown). After induction, the cells were harvested in an assay buffer (20 mM Tris-HCl, pH 8.0, 5 mM MgCl2 and 2 mM dithiothreitol), modified from Chen et al. [17], by centrifugation at 14386g for 10 min at 4uC. Crude extracts were prepared by subjecting the cells to sonication treatment using the Omni Ruptor 4000 (Omni International, GA, USA) set to 10% power and pulsed for 2 min per sample. Then, the samples were centrifuged at 16,0006g, for 10 min, at 4uC with the final aqueous phase collected for further purification. For recombinant protein purification, His SpinTrap (GE Healthcare, WI, USA) columns equilibrated with ten column volumes of binding buffer containing 20 mM imidazole were used according to the manufacturer’s protocol to trap recombinant protein via the his-tag fusion. Subsequently, the column was washed with another ten column volumes of binding buffer and the final elution was performed in the presence of 300 mM imidazole. After purification, the proteins were desalted using HiTrap Column (GE Healthcare, WI, USA) according to the manufacturer’s protocol. At each step, the protein was analyzed by SDS-PAGE according to Laemmli [18]. Introduction To construct the pNZ:PMSTS plasmid, the digested PMSTS gene was ligated into pNZ8048 digested with PstI and KpnI and then transformed into the L. lactis NZ9000 host via electroporation [16]. To construct the pNZ:PMSTS:mvaA plasmid, the amplified PMSTS and mvaA genes digested with their respective REs were allowed to ligate with pNZ8048 digested with PstI and HindIII and then transformed into L. lactis NZ9000. This allowed the mvaA gene to be ligated downstream of the PMSTS gene via the KpnI site in the pNZ8048 plasmid, thereby yielding the pNZ:PMSTS:mvaA plasmid. tages such as its GRAS (Generally Regarded as Safe) status and the absence of inclusion bodies [3,10]. Apart from that, L. lactis offers a food-grade alternative to the other more common and well-studied hosts for heterologous isoprenoid production such as E. coli and S. cerevisiae. Whilst E. coli uses the MEP pathway and yeast uses the mevalonate pathway, respectively, for the pro- duction of isoprenoids, the study of L. lactis as a prokaryotic host which uses the mevalonate pathway for isoprenoid production is appealing. Previously, we reported the cloning and expression of a sesqui- terpene synthase from orchid (Vanda Mimi Palmer) in L. lactis [10]. Here we describe the cloning and expression of another sesquiterpene synthase, isolated from Persicaria minor (synonym Polygonum minus), a local Malaysian herbaceous fragrant plant with biological activities including anti-oxidant [11] and anti-ulcer [12] properties. The recombinant L. lactis developed was able to produce P. minor sesquiterpene in vivo thereby making it suitable as a microbial cell factory for P. minor sesquiterpene production. We also attempted to metabolically engineer the mevalonate pathway of L. lactis by overexpressing the endogenous 3-hydroxy-3- methylglutaryl coenzyme-A reductase (HMGR) enzyme, an established rate-limiting enzyme in the eukaryotic mevalonate pathway [13]. This is the first reported attempt to metabolically engineer the endogenous host’s prokaryotic mevalonate pathway for an increased isoprenoid production. In vitro Enzymatic Assays The P. minor sesquiterpene synthase (PMSTS) [Accession no: JX025008] was amplified using the PMSTS forward and reverse primers 59- CTCTGCAGAACATCACCATCACCAT CACATG- TATTCCATGATCATG-39 and Rev: 59- CTGGTACCTTA- TATCAGTATGGG ATCG-39 primers respectively. The re- striction enzyme (RE) sites, PstI and KpnI are underlined while the his-tag sequence incorporated into the forward primer is shown in italic. The mvaA gene was amplified using nested PCR from the genome of L. lactis MG1363 [Accession no: YP_001032255]. Due to the highly rich A-T sequence in the mvaA gene, a longer mvaA fragment which included flanking regions were first amplified and the PCR product was susbsequently used as template for a second amplification of the actual mvaA gene using the forward and reverse primers 59- CTGGTACCGGAGGCAAAAAATGATGA- GAA AAAAATTTTATCAAATGTC 39 and 59-CTAAGCTTC- TAATGGTGATGGTGATG GTGTTTTCTCAAATTTTTTAG- TAAATTTTGG- 39 respectively. As with the PMSTS primers, the RE sites, KpnI and HindIII, are underlined and the his-tag sequence incorporated into the reverse primer is italicized. A ribosomal binding site (RBS), highlighted in grey is also in- corporated into the forward primer. The PCR reaction mixture Enzymatic assay for the recombinant sesquiterpene synthase was performed as previously described [17]. Briefly, the purified protein extract in the assay buffer was mixed with 20 mL farnesyl diphosphate substrate from a 1 mg/mL stock (Sigma Aldrich, MO, USA) in a total volume of 1 mL. The mixture was then incubated with shaking at 30uC for 3 h and the reaction was stopped with 1 mL of 4 M CaCl 2. All the above reactions were performed in a 20 mL headspace vial which was then subjected to solid phase microextraction (SPME) sampling using a 100 mm polydimethyl- siloxane (PDMS) coated fiber. Extraction time used was 15 min at 60uC and desorption time was 5 min. Blank tests were run in between sampling runs to ensure no carryover from the SPME fiber or the GC-MS system. HMGR enzymatic assays were performed according to Wilding et al. [19] with minor modifications. His-tag purified protein from pNZ:PMSTS:mvaA was added to the assay buffer (50 mM NaCl, I mM EDTA, 25 mM KH2PO4, pH 7.5, 5 mM DTT, 0.25 mM NADPH and 0.25 mM HMG-CoA). The protein sample in assay buffer mixture was diluted to a final volume of 1 mL. Materials and Methods Bacterial Strains and Culture Conditions December 2012 \| Volume 7 \| Issue 12 \| e52444 In vitro Enzymatic Assays Spectro- photometric assays were performed using a Hitachi U-2900 Spectrophotometer (Hitachi, Tokyo, Japan) set to read OD December 2012 \| Volume 7 \| Issue 12 \| e52444 2 PLOS ONE \| www.plosone.org Overexpressing HMGR for Sesquiterpene Production Production of Recombinant PMSTS and HMGR Enzymes The recombinant PMSTS from the pNZ:PMSTS plasmid and PMSTS together with HMGR from the pNZ:PMSTS:mvaA plasmid were analysed by SDS-PAGE after protein purification and were found to be successfully produced (Figure 2). As expected, the pNZ:PMSTS plasmid produced only one intense PMSTS protein band of ,63 kDa. However, some host protein contaminations can be seen from the presence of faint bands of different sizes in Figure 2A, lane 1. These faint bands were undetected after the desalting process due to their low concentra- tions and only the PMSTS band remained (Figure 2A, Lanes 2 and 3). On the other hand, the pNZ:PMSTS:mvaA plasmid produced two separate intense protein bands ,63 kDa for the PMSTS protein and ,43 kDa for the HMGR protein (Figure 2B). The HMGR protein band is much more intense compared to the PMSTS band. This is not surprising since the HMGR protein is expressed from the mvaA gene which is an endogenous L. lactis gene while the PMSTS gene is a plant heterologous gene. Therefore, it was expected that the L. lactis host would be able to express the mvaA gene more efficiently since it was not limited by factors such as codon bias which would affect the PMSTS protein synthesis levels. As per the pNZ:PMSTS plasmid, there were also some host protein contaminations in the initial his-tag purified fraction which were subsequently not observed after the desalting step. measurements at 340 nm over a time period of 3 min. The HMG- CoA was added last to initiate the reaction just before spectrophotometer readings were taken. Purified his-tag protein from pNZ:PMSTS clones were used as a negative control. Terpene Synthase Enzymatic Assay To assess the function of the recombinant PMSTS protein, enzymatic assays were performed using the his-tag purified and desalted PMSTS protein. The PMSTS protein was found to produce mainly b-sesquiphellandrene which consisted of 85.4% of the total sesquiterpenes produced (Figure 3) as shown by the high peak at retention time (RT) 7.22 min. Other minor products produced are shown in Table 1 while the mass spectra of the b- sesquiphellandrene peak in comparison with the highest hit from the NIST 2008 and Wiley Registry of Mass Spectral Data, 8th edition library is shown in Figure 4. All peaks were identified by comparison of the mass spectra with the NIST and Wiley libraries only. However, future work to confirm and complete the chemical characterisation of the b-sesquiphellandrene peak identified in this study through NMR and X-ray diffraction studies is suggested. y g y gg b-sesquiphellandrene has been previously detected from other plant terpene synthases such as ginger (Zingiber officinale) [21,22] and grapevine (Vitis vinifera) [23]. However, in grapevine, b- sesquiphellandrene was produced as a secondary sesquiterpene by an a-zingiberene synthase, VvPNaZin. This sesquiterpene synthase produced 79.5% a-zingiberene, 17.5% b-sesquiphellan- drene and 3% b-bisabolene. In fact, to our knowledge, there has not been any sesquiterpene synthase isolated or identified which produced b-sesquiphellandrene as its major product. Whether this is the genuine product of the PMSTS isolated from P. minor, caused by the single amino acid substitution (K266E) introduced during the cloning process or an effect of the lactococcal host expressing the recombinant protein is yet to be resolved. It should also be noted that b-sesquiphellandrene was not detected in the essential oil of the P. minor plant itself, although all the other minor products detected from the in vitro assay of PMSTS was detected in the plant [24]. This further supports the hypothesis that the mutation or host may have affected the major product produced by PMSTS although it could also be argued that the extraction or detection method used by Baharom et al. [24] was not sensitive towards b-sesquiphellandrene detection. As can be seen from their study, analysis using GC-MS and GCxGC-TOF MS produced significant differences in the products detected in the essential oil GC-MS Analysis Products from the enzymatic assays were analyzed using the GC-MS Turbomass Clarus 600 (Perkin Elmer, MA, USA) and the GC column used was Perkin Elmer Elite 5 MS (30 m60.25 mm ID). The GC oven conditions were from an initial temperature of 40uC (1 min hold) to 250uC (3 min hold) with a 35uC ramping temperature increase per min. The column flow was held constant at 1 mL/min. The injection port was maintained at 250uC, the transfer line at 220uC while the MS source temperature was maintained at 200uC. The MS, operated at 70 eV was set to scan from m/z 35 to 400. Products were identified by comparing the spectra to those in the National Institute of Standards and Technology (NIST) library 2008 and the Wiley Registry of Mass Spectral Data, 8th Edition. The cut-off match score for the mass spectra of the products compared to the NIST and Wiley libraries used were .800. In vivo Expression p Overnight culture of L. lactis NZ9000 harbouring pNZ:PMSTS, pNZ:PMSTS:mvaA or empty pNZ8048 (as negative control) were inoculated into fresh GM17 medium at 5% (v/v) and grown to an OD600 of 0.4. The cultures were split into sub-samples consisting of uninduced samples and samples induced with nisin at 40 ng/ mL. During induction, 100 mL of each culture was transferred into 150 mL serum bottles, spiked with chlorohexane as internal control and capped with a crimp top aluminium cap. To measure the sesquiterpenes produced, the headspace of the serum bottle was subjected to SPME sampling at 60uC for 15 min hourly from 1–4 h post-induction. The PDMS fibre was then subjected to GC- MS analysis. Production of sesquiterpenes were measured based on peak area of GC-MS peaks, normalized against chlorohexane, the internal standard and quantified as nerolidol equivalents using nerolidol standards (Sigma, MO, USA) since standards for the sesquiterpenes produced by the recombinant clones in this study were commercially unavailable [20]. Five biological replicates were done for each experiment. Plasmids Construction Two plasmids were constructed in this study, pNZ:PMSTS and pNZ:PMSTS:mvaA. The pNZ:PMSTS plasmid harbours the heterologous sesquiterpene synthase gene, PMSTS from P. minor incorporated into the pNZ8048 plasmid (Figure 1). Two base substitutions, 796A.G and 1077A.G, were introduced into the PMSTS gene during amplification resulting in an amino acid change from lysine to glutamate (K266E) for the former while the latter caused a silent mutation (alanine remains as the original amino acid). An mvaA gene, which codes for the 3-hydroxy-3- methylglutaryl coenzyme-A reductase (HMGR), was cloned downstream of the PMSTS gene to yield the recombinant plasmid pNZ:PMSTS:mvaA. This construct was used for the HMGR overexpression study. Both PMSTS and mvaA genes possess a his- tag sequence for purification purposes. For PMSTS, the his-tag sequence is at the N-terminus while for mvaA, it is located at the C- terminus. In addition, an RBS site was incorporated up-stream of the mvaA gene to allow translation of this second gene using the same pNis promoter. December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org 3 Overexpressing HMGR for Sesquiterpene Production of P minor However it was previously reported that a single point the N-terminal doma Figure 1. Schematic diagram of pNZ:PMSTS (A) and pNZ:PMSTS:mvaA (B) plasmids. doi:10.1371/journal.pone.0052444.g001 Ove Figure 1. Schematic diagram of pNZ:PMSTS (A) and pNZ:PMSTS:mvaA (B) plasmids. doi:10.1371/journal.pone.0052444.g001 Figure 1. Schematic diagram of pNZ:PMSTS (A) and pNZ:PMSTS:mvaA (B) plasmids. doi:10.1371/journal.pone.0052444.g001 of P. minor. However, it was previously reported that a single point mutation in the active site of an aristolochene synthase was sufficient to convert it into an (E)-b-farnesene synthase instead [25]. In the current study, the K266E mutation is ten amino acids upstream of the predicted substrate binding pocket based on sequence analysis. While the C-terminal domain of terpene synthases generally contain the active site, mutational analysis of of P. minor. However, it was previously reported that a single point mutation in the active site of an aristolochene synthase was sufficient to convert it into an (E)-b-farnesene synthase instead [25]. In the current study, the K266E mutation is ten amino acids upstream of the predicted substrate binding pocket based on sequence analysis. While the C-terminal domain of terpene synthases generally contain the active site, mutational analysis of the N-terminal domain indicates that the the domain facilitates proper folding of the active site [26]. December 2012 \| Volume 7 \| Issue 12 \| e52444 Plasmids Construction Since a substitution from lysine to glutamate changes the amino acid charge from positive to negative, the effect on protein folding may be significant and may change the catalytic activity of the enzyme. While many essential oils which contain b-sesquiphellandrene have been shown to possess anti-microbial and anti-oxidant December 2012 \| Volume 7 \| Issue 12 \| e52444 December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 4 Overexpressing HMGR for Sesquiterpene Production activities [22,27], the effect may be caused by other components of the essential oil especially in cases where b-sesquiphellandrene is found only in minute amounts. Denyer et al. [21], however, showed that among a bisabolene from the e Figure 2. His-tag purified PMSTS and HMGR proteins (A) pNZ:PMSTS and (B) pNZ:PMSTS:m Plus Protein Ladder (Fermentas, Canada); Lane 1: His-tag purified protein in Elution Buffer with 300 m after desalting (eluents 1 and 2 respectively). doi:10.1371/journal.pone.0052444.g002 Figure 2. His-tag purified PMSTS and HMGR proteins (A) pNZ:PMSTS and (B) pNZ:PMSTS:mvaA plasmids. Lane M: PageRulerTM Prestained Plus Protein Ladder (Fermentas, Canada); Lane 1: His-tag purified protein in Elution Buffer with 300 mM imidazole; Lanes 2 and 3: Protein from Lane 1 after desalting (eluents 1 and 2 respectively). doi:10.1371/journal.pone.0052444.g002 Figure 2. His-tag purified PMSTS and HMGR proteins (A) pNZ:PMSTS and (B) pNZ:PMSTS:mvaA plasmids. Plus Protein Ladder (Fermentas, Canada); Lane 1: His-tag purified protein in Elution Buffer with 300 mM imidazole; La after desalting (eluents 1 and 2 respectively). doi:10.1371/journal.pone.0052444.g002 Figure 2. His-tag purified PMSTS and HMGR proteins (A) pNZ:PMSTS and (B) pNZ:PMSTS:mvaA plasmids. Lane M: PageRulerTM Prestained Plus Protein Ladder (Fermentas, Canada); Lane 1: His-tag purified protein in Elution Buffer with 300 mM imidazole; Lanes 2 and 3: Protein from Lane 1 after desalting (eluents 1 and 2 respectively). doi:10.1371/journal.pone.0052444.g002 activities [22,27], the effect may be caused by other components of the essential oil especially in cases where b-sesquiphellandrene is found only in minute amounts. Denyer et al. [21], however, showed that among a-zingiberene, b-sesquiphellandrene and b- bisabolene from the essential oils of ginger, b-sesquiphellandrene December 2012 \| Volume 7 \| Issue 12 \| e52444 December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Overexpressing HMGR for Sesquiterpene Production Figure 3. GC-MS chromatogram showing sesquiterpenes produced from purified PMSTS protein. HMGR Enzymatic Assay A HMGR assay was performed to determine the functionality of the endogenous HMGR protein which was produced by overexpressing the mvaA gene downstream of the PMSTS gene in the plasmid pNZ:PMSTS:mvaA. While the HMGR protein was successfully produced as shown in Figure 2B, the functionality of the enzyme remained unknown. Therefore, the purified protein from pNZ:PMSTS:mvaA was used to perform the HMGR assay. Essentially, the purified protein represents the HMGR protein expressed from the plasmid together with the PMSTS protein without the HMGR protein expressed from the host genome. The plasmid-expressed HMGR, if functional, would be responsible for the reduction of the substrate HMG-CoA to mevalonate which is Plasmids Construction Summary of retention times, match score and percentages of sesquiterpenes produced are shown in the Table 1. doi:10.1371/journal.pone.0052444.g003 Figure 3. GC-MS chromatogram showing sesquiterpenes produced from purified PMSTS protein. Summary of retention times, match score and percentages of sesquiterpenes produced are shown in the Table 1. doi:10.1371/journal.pone.0052444.g003 had the highest antirhinoviral activity with an IC50 of 0.44 mM in the plaque reduction test [21]. had the highest antirhinoviral activity with an IC50 of 0.44 mM in the plaque reduction test [21]. coupled to the oxidation of NADPH to NADP+. Figure 5 showed a decrease in absorbance at 340 nm can be observed from the oxidation of NADPH to NADP+, proving that the HMGR enzyme expressed from the plasmid was indeed functional. Negative control was also performed with his-tag purified protein from the pNZ:PMSTS which did not contain the HMGR protein. Overexpressing HMGR for Sesquiterpene Production Table 1. Sesquiterpenes produced by the recombinant PMSTS protein in in vitro enzymatic assay as detected by GC- MS. Sesquiterpenes RT (min) Match score Percentage (%) a-bergamotene 6.708 946 0.8 (Z)-beta-farnesene 6.769 938 3.2 (E)-beta-farnesene 7.006 901 2.0 a-zingiberene 7.041 943 2.6 b-sesquiphellandrene 7.225 913 85.4 (E)-nerolidol 7.356 958 6.0 doi:10.1371/journal.pone.0052444.t001 standard for b-sesquiphellandrene is not commercially available. Such measure was similarly practised by Pitera et al. [20] to quantify amorphadiene concentrations using caryophyllene equivalents. As shown in Table 2, the clone harbouring pNZ:PMSTS:mvaA produced the highest level of 33.7 nM b-sesquiphellandrene after 1 h of induction. The overall production trend showed a gradual decline following the maximal peak production. This production pattern was in line with previous reports observing that heterologous isoprenoids production in vivo followed an exponen- tial growth and ceased at the stationary phase due to the production of the FDP substrate during growth for primary metabolism [30]. For the clone harbouring pNZ:PMSTS, overall production was slightly lower than pNZ:PMSTS:mvaA, with the highest production being 30.0 nM sesquiphellandrene at 2 h post- induction, meeting the same amount of sesquiphellandrene production by pNZ:PMSTS:mvaA at that time. The biggest difference in production between the two clones was achieved at 1 h post-induction with pNZ:PMSTS:mvaA producing about 1.60 folds more sesquiphellandrene than pNZ:PMSTS. However, the overall production of pNZ:PMSTS:mvaA throughout the 4 h was only 1.25 folds more than those produced by pNZ:PMSTS. peak was undetected in the uninduced culture (Figure 6B). The other minor sesquiterpene products, however, were not detected from the recombinant L. lactis cultures probably due to their low concentrations. The clones harbouring the pNZ:PMSTS:mvaA plasmid were expected to produce more b-sesquiphellandrene compared to the clones harbouring the pNZ:PMSTS plasmid based on the assumption that overexpression of the HMGR enzyme encoded by the mvaA gene would enhance production of the downstream products. Apart from having multiple copies of the mvaA gene, since mvaA was cloned into the high copy-number plasmid pNZ8048, it was hoped that the HMGR would also be able to by-pass endogenous host regulation at least at the transcriptional level since transcription would be controlled by the plamid promoter, PNis. This metabolic engineering strategy is commonly used to increase isoprenoid production in yeasts and fungi [28,29] ever since it was established that HMGR is the rate- limiting enzyme in the eukaryotic pathway. In vivo Production of b-sesquiphellandrene Recombinant L. lactis strains harbouring the pNZ:PMSTS and pNZ:PMSTS:mvaA plasmids were analysed to assess its capability to produce P. minor sesquiterpenes in vivo. Upon induction with 40 ng/mL nisin, it was observed that the recombinant L. lactis strains were capable of producing b-sesquiphellandrene, the major product of the recombinant PMSTS protein without any exogenous FPP substrate supplementation. The b-sesquiphellan- drene peak was observed at RT 7.24 min (Figure 6A) and this Figure 4. Mass spectra of the b-sesquiphellandrene. Top panel shows mass spectra of the sample compound while bottom panel shows mass spectra of the highest hit in the National Institute of Standards and Technology (NIST) library 2008 and the Wiley Registry of Mass Spectral Data, 8th Edition mass spectra library. doi:10.1371/journal.pone.0052444.g004 Figure 4. Mass spectra of the b-sesquiphellandrene. Top panel shows mass spectra of the sample compound while bottom panel shows mass spectra of the highest hit in the National Institute of Standards and Technology (NIST) library 2008 and the Wiley Registry of Mass Spectral Data, 8th Edition mass spectra library. doi:10.1371/journal.pone.0052444.g004 December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org Overexpressing HMGR for Sesquiterpene Production Overexpressing HMGR for Sesquiterpene Production Considering the lack of reported work on the prokaryotic mevalonate pathway, the current study attempted to analyse the overexpression effect of HMGR on isoprenoid production in a prokaryotic system. b- sesquiphellandrene concentrations produced by clones harbouring the pNZ:PMSTS plasmid were compared to clones harbouring the pNZ:PMSTS:mvaA plasmid. The concentration of b-sesquiphellan- drene produced was measured as nerolidol equivalents since the Previous studies have also shown the ineffectiveness of the overexpression of HMGR alone to increase production of isoprenoids significantly, especially if the product of interest is further downstream of the pathway such as in the production of triterpenes [31]. Even in the production of sesquiterpenes, Jackson et al. [32] observed that the overexpression of a truncated HMGR along with co-expression of an epi-cedrol synthase in yeast produced significantly less epi-cedrol compared to yeast expressing the epi-cedrol synthase alone. Only with synergistic overexpression of the truncated HMGR and an upc2-1 mutation of the yeast strain was the production of epi-cedrol increased significantly about 4- fold. Rico et al. [33] also found that the overexpression of HMGR alone doubled the production of the monoterpene linalool, just slightly higher than the amount observed in the current study. Regardless of the effectiveness of the overexpression of the HMGR in isoprenoid production, it has been well established that HMGR is indeed the rate-limiting enzyme in eukaryotes and studies have also shown that HMGR is the most effective gene to overexpress Figure 5. HMGR assay using his-tag purified protein expressed from pNZ:PMSTS:mvaA. Protein expressed from his-tag purified pNZ:PMSTS was used as the negative control. doi:10.1371/journal.pone.0052444.g005 PLOS ONE \| l 7 D b 2012 \| V l 7 \| I 12 \| 52444 Figure 5. HMGR assay using his-tag purified protein expressed from pNZ:PMSTS:mvaA. Protein expressed from his-tag purified pNZ:PMSTS was used as the negative control. doi:10.1371/journal.pone.0052444.g005 December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org Overexpressing HMGR for Sesquiterpene Production for prenyl alcohol production in S. cerevisiae in comparison with other genes of the mevalonate pathway [34]. However, studies on the prokaryotic mevalonate pathway are severely lacking with only one study done thus far on the mevalonate pathway of S aureus the mvaK1-mvaD-mvaK2 operon instead which is slightly further downstream of the mevalonate pathway. Therefore, in the current study, the reason the overexpression of HMGR only yielded a slight increase of sesquiterpene production in L lactis harbouring Figure 6. References Wilding EM, Brown JR, Bryant AP, Chalker AF, Holmes DJ, et al. (2000) Identification, evolution, and essentiality of the mevalonate pathway of isopentenyl diphosphate biosynthesis in gram-positive cocci. J Bacteriol 182(15): 4319–4327. 24. Baharum SN, Bunawan H, Ghani MA, Mustapha WAW, Noor NM (2010) Analysis of the chemical composition of the essential oil of Polygonum minus Huds. using two-dimensional gas chromatography-time-of-flight mass spectrometry (GC-TOF MS). Molecules 15: 7006–7015. 8. Kanehisa M, Goto S (2000) KEGG: Kyoto Encyclopedia of genes and genomes. Nucleic Acids Res 28: 27–30. 9. Withers ST, Keasling JD (2007) Biosynthesis and engineering of isoprenoid small molecules. Appl Microbiol Biotechnol 73: 980–990. 25. Deligeorgopoulou A, Allemann RK (2003) Evidence for differential folding of farnesyl pyrophosphate in the active site of aristolchene synthase: a single-point mutation converts aristolchene synthase into a (E)-b-farnesene synthase. Biochem 42: 7741–7747. 10. Song AA, Abdullah JO, Abdullah MP, Shafee N, Rahim RA (2012) Functional expression of an orchid fragrance gene in Lactococcus lactis. Int J Mol Sci 13(2): 1582–1597. 11. Qader SW, Abdullah MA, Chua LS, Najin N, Zain MM, et al. (2011) Antioxidant, total phenolic content and cytotoxicity evaluation of selected Malaysian plants. Molecules 16(4): 3433–3443. 26. Ko¨llner TG, Schnee C, Gershenzon J, Degenhardt J (2004) The variability of sesquiterpenes cultivars is controlled by allelic emitted Zea mays variation of two terpene synthase genes encoding stereoselective multiple product enzymes. Plant Cell 16: 1115–1131. 12. Wasman SQ, Mahmood AA, Salehhuddin H, Zahra AA, Salmah I (2010) Cytoprotective activities of Polygonum minus aqueous leaf extract on ethanol- induced gastric ulcer in rats. J Med Plants Res 4(24): 2658–2665. 27. Lee KH, Kim BS, Keum KS, Yu HH, Chang BS, et al. (2011) Essential oil of curcuma longa inhibits Streptococcus mutans biofilm formation. J Food Sci 76(9): H226–230. g J ( ) 13. Friesen JA, Rodwell VW (2004) The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases. Genome Biol 5: 248. 28. Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, et al. (1998) Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway. Appl Environ Microbiol 64(7): 2676– 2680. 14. Kuipers OP, de Ruyter PG, Kleerebezem M, de Vos WM (1998) Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64: 15– 21. 29. Wang GY, Keasling JD (2002) Amplification of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa. Metab Eng 4: 193–201. 15. Overexpressing HMGR for Sesquiterpene Production Overexpressing HMGR for Sesquiterpene Production Overexpressing HMGR for Sesquiterpene Production Table 2. In vivo production of b-sesquiphellandrene comparing clones harbouring the pNZ:PMSTS:mvaA and the pNZ:PMSTS plasmids. Production of b-sesquiphellandrene (nM) Clones 1 h 2 h 3 h 4 h Total pNZ:PMSTS:mvaA 33.7 29.1 24.5 21.7 109.0 pNZ:PMSTS 20.9 30.0 21.3 15.0 87.2 Data shown is the mean 6 standard deviation (SD) of five biological replicates. doi:10.1371/journal.pone.0052444.t002 a complex process which needs further studies to be understood and we are currently working on enhancing our understanding of the mevalonate pathway of L. lactis by transcriptomic analysis of the lactococcal mevalonate pathway comparing wild-type and recombinant lactococcal strains developed in this study and previous studies. Acknowledgments We thank Kees Leenhouts of the University of Groningen, Netherlands for the generous gift of L. lactis NZ9000 and the plasmid pNZ8048. Conclusions The current study specifically describes the use of L. lactis as a microbial cell factory for P. minor b-sesquiphellandrene. However, on a wider scope, this study represents the use of L. lactis as an alternative heterologous host for isoprenoid pro- duction. It also describes the first study where the prokaryotic mevalonate pathway has been metabolically engineered for increased isoprenoid production by overexpression of HMGR. there are other metabolic bottle-necks further downstream of the mevalonate pathway. Apart from that, while ideally it is postulated that a single optimised heterologous system can be used for overproduction of a variety of isoprenoids, we found that this may not always be true. Our previous study [10] involving the cloning and expression of a sesquiterpene synthase from orchid (Vanda Mimi Palmer) did not yield any plant sesquiterpenes in vivo although in vitro enzymatic studies showed that the recombinant protein was functional and the protein expression levels were three times more than the expression of PMSTS in the current study (data not shown). This shows that the regulation of the prokaryotic mevalonate pathway is Author Contributions Conceived and designed the experiments: AAS ARR RO NMN. Performed the experiments: AAS EFT. Analyzed the data: AAS ARR JOA MPA NS. Contributed reagents/materials/analysis tools: ARR NMN. Wrote the paper: AAS ARR JOA MPA NS. References 18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. 1. de Vos WM, Hugenholtz J (2004) Engineering metabolic highways in Lactococcus and other lactic acid bacteria. Trends Biotechnol 22(2): 72–79. 2. Anuradha K, Foo HL, Mariana NS, Loh TC, Yusoff K, et al. (2010) Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus). J Appl Microbiol 109: 1632–1642. 19. Wilding EM, Kim DY, Brayant AP, Gwynn MN, Lunsford RD, et al. (2000) Essentiality, expression and characterization of the class II 3-hydroxy-3- methylglutaryl coenzyme A reductase of Staphylococcus aureus. J Bacteriol 182(18): 5147–5152. 20. Pitera DJ, Paddon CJ, Newman JD, Keasling JD (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng 9: 193–207. 3. Hernandez I, Molenaar D, Beekwilder J, Bouwmeester H, van Hylckama Vlieg JET (2007) Expression of plant flavor genes in Lactococcus lactis. Appl Environ Microbiol 73(5): 1544–1552. ( ) 4. Pichersky E, Gershenzon J (2002) The formation and function of plant volatiles: Perfumes for pollinator attraction and defense. Curr Opin Plant Biol 5: 237–243. 21. Denyer CV, Jackson P, Loakes DM, Ellis MR, Young DA (1994) Isolation of antirhinoviral sesquiterpenes from ginger (Zingiber officinale). J Nat Prod 57(5): 658–662. 4. Pichersky E, Gershenzon J (2002) The formation and function of plant volatiles: Perfumes for pollinator attraction and defense. Curr Opin Plant Biol 5: 237–243. 5. Kirby J, Keasling JD (2008) Metabolic engineering of microorganisms for isoprenoid production. Nat Prod Rep 2008, 25: 656–661. p p 2 2 5. Kirby J, Keasling JD (2008) Metabolic engineering of microorganisms for isoprenoid production. Nat Prod Rep 2008, 25: 656–661. 22. El-Baroty GS, Abd El-Baky HH, Farag RS, Saleh MA (2010) Characterization of antioxidant and antimicrobial compounds of cinnamon and ginger essential oils. Afr J Biochem Res 4(6): 167–174. 6. Rodrı´guez-Concepcio´n M, Boranat A (2002) Elucidation of the methylerythhri- tol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130: 1079–1089. 23. Martin DM, Aubourg S, Schouwey MB, Daviet L, Schalk M, et al. (2010) Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinefera) terpene synthase gene family based on genome assembly, FLcDNA cloning and enzyme assays. BMC Plant Biol 10: 226. 7. Overexpressing HMGR for Sesquiterpene Production GC-MS chromatogram showing the in vivo production of b-sesquiphellandrene by clones harbouring the pNZ:PMSTS plasmid. The example shown here is for 40 ng/mL nisin induction at 2 h post-induction. B-sesquiphellandrene was detected at 7.24 min in the headspace of the induced culture (indicated by arrow) (A), which was undetected in the uninduced culture (B). Clones harbouring the pNZ:PMSTS:mvaA plasmid showed a similar GC-MS profile (data not shown). doi:10.1371/journal.pone.0052444.g006 Overexpressing HMGR for Sesquiterpene Production Figure 6. GC-MS chromatogram showing the in vivo production of b-sesquiphellandrene by clones harbouring the pNZ:PMSTS plasmid. The example shown here is for 40 ng/mL nisin induction at 2 h post-induction. B-sesquiphellandrene was detected at 7.24 min in the headspace of the induced culture (indicated by arrow) (A), which was undetected in the uninduced culture (B). Clones harbouring the pNZ:PMSTS:mvaA plasmid showed a similar GC-MS profile (data not shown). doi:10.1371/journal.pone.0052444.g006 the mvaK1-mvaD-mvaK2 operon instead which is slightly further downstream of the mevalonate pathway. Therefore, in the current study, the reason the overexpression of HMGR only yielded a slight increase of sesquiterpene production in L. lactis harbouring the pNZ:PMSTS:mvaA plasmid could be due to the fact that the HMGR in L. lactis is similarly not the rate-limiting enzyme and for prenyl alcohol production in S. cerevisiae in comparison with other genes of the mevalonate pathway [34]. However, studies on the prokaryotic mevalonate pathway are severely lacking with only one study done thus far on the mevalonate pathway of S. aureus [35]. It was suggested that unlike the eukaryotic mevalonate pathway, the rate-limiting enzyme in S. aureus is not HMGR but December 2012 \| Volume 7 \| Issue 12 \| e52444 December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org 8 33. Rico J, Pardo E, Orejas M (2010) Enhanced production of plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase catalytic domain in Saccharomyces cerevisiae. Appl Environ Microbiol 76(19): 6449– 6454. 34. Ohto C, Muramatsu M, Obata S, Sakuradani E, Shimizu S (2009) Over- expression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols. Appl Microbiol Biotechnol 82(5): 837–845. 35. Balibar CJ, Shen X, Tao J (2009) The mevalonate pathway of Staphylococcus aureus. J Bacteriol 191(3): 851–861. 32. Jackson BE, Hart-Wells EA, Matsuda SPT (2003) Metabolic engineering to produce sesquiterpenes in yeast. Org Lett 5(10): 1629–1632. Overexpressing HMGR for Sesquiterpene Production 32. Jackson BE, Hart-Wells EA, Matsuda SPT (2003) Metabolic engineering to produce sesquiterpenes in yeast. Org Lett 5(10): 1629–1632. 33. Rico J, Pardo E, Orejas M (2010) Enhanced production of plant monoterpene by overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase catalytic domain in Saccharomyces cerevisiae. Appl Environ Microbiol 76(19): 6449– 6454. 34. Ohto C, Muramatsu M, Obata S, Sakuradani E, Shimizu S (2009) Over- expression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols. Appl Microbiol Biotechnol 82(5): 837–845. 35. Balibar CJ, Shen X, Tao J (2009) The mevalonate pathway of Staphylococcus aureus. J Bacteriol 191(3): 851–861. References Terzaghi BE, Sandine WE (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29: 807–813. 16. Holo H, Nes IF (1989) High-frequency transformation by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55: 3119–3123. 30. Martin VJJ, Yasuo Y, Keasling JD (2001) The in vivo synthesis of plant sesquiterpenes by Escherichia coli. Biotechnol Bioeng 75: 497–503. 17. Chen XY, Chen Y, Heinstein P, Davisson VJ (1995) Cloning, expression and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis. Arch Biochem Biophys 324(2): 255–266. 31. Kirby J, Romanini DW, Paradise EM, Keasling JD (2008) Engineering triterpene production in Saccharomyces cerevisiae-beta-amyrin-synthase from Artemisia annua. FEBS J 275(8): 1852–1859. 9 PLOS ONE \| www.plosone.org December 2012 \| Volume 7 \| Issue 12 \| e52444 December 2012 \| Volume 7 \| Issue 12 \| e52444 December 2012 \| Volume 7 \| Issue 12 \| e52444 Overexpressing HMGR for Sesquiterpene Production PLOS ONE \| www.plosone.org 10 December 2012 \| Volume 7 \| Issue 12 \| e52444 PLOS ONE \| www.plosone.org 10
https://openalex.org/W4301825178	https://chaodaescola.com.br/index.php/rce/article/download/111/108	Portuguese	null	Agadá	Revista Chão da Escola	2,016	cc-by	1,579	Agadá: na alma, na luta e na arte Fabíola Maciel Corrêa João Paulo De Souza Da Silva Fabíola Maciel Corrêa RESUMO - Palavras-chaves: Arte, Relações Étnico-Raciais, Seleção Cultural. 25 INTRODUÇÃO - - - - - ainda persiste em nossas escolas um pensamento - - tas vezes somente importância aos grupos europeus SELEÇÃO CULTURAL - apenas a linguagem das artes visuais, como objeto - O ensino de arte1 no Brasil apresenta um caráter eurocêntrico e estadunidense, quando consideramos a arte contemporânea. De modo que a arte de matriz africana muitas vezes recebe um tratamento que a confundida com folclore, artesanato ou mesmo como “arte primitiva”. que ocorrem processos de transformação do conhe- cimento que incluem, de diferentes formas, a partici- pação da escola e dos professores em um movimento que se desloca do currículo formal – os saberes a se- rem ensinados – para o currículo real – os saberes ensinados e aprendidos (NERI, 2005, p.3). - - Entretanto, a necessidade da presença da cul- - - - O sistema escolar pode, por sua lógica própria, servir à perpetuação dos privilégios culturais sem que os privilegiados tenham de se servir dele. Conferindo às desigualdades culturais uma sanção formalmente conforme aos ideais democráticos, ele fornece a me- - DIEU, 2008, p. 59). Neste artigo pretendemos observar as pos- - cola nas aulas de arte, considerando os pressupos- - - - - - - junto dos conteúdos cognitivos e simbólicos (saberes, competências, representações, tendências, valores) transmitidos (de modo explícito ou implícito) nas práticas pedagógicas e nas situações de escolariza- ção, isto é, tudo aquilo que poderíamos chamar de dimensão cognitiva e cultural da educação escolar (FORQUIN, 2000, p. 48). - - - - - - O currículo é muitas coisas ao mesmo tempo: ideias pedagógicas, estruturação de conteúdos de uma for- termos concretos, estímulo de habilidades nos alu- nos, etc. (SACRISTÁN, 2000, p. 173) - dendo-o como tudo que é suposto de ser ensinado ou aprendido, segundo uma ordem determinada de programação e sob responsabilidade de uma instituição de educa- ção formal, nos limites de um ciclo de estudos. Por extensão, o termo me parece fazer referência ao con- 27 formação na área, aprofundando-se nos estudos sobre relações étnico- raciais e cultura afro-brasileira, garantindo abordagens pedagógicas que promovam o reconhecimento da contribuição do negro para a cultura brasileira, desconstruindo noções de hierarquia racial. formação na área, aprofundando-se nos estudos sobre relações étnico- raciais e cultura afro-brasileira, garantindo abordagens pedagógicas que promovam o reconhecimento da contribuição do negro para a cultura brasileira, desconstruindo noções de hierarquia racial. SELEÇÃO CULTURAL discentes e docentes envolvidos, considerando a so- - - - - - DEMOCRACIA RACIAL: UM MITO A SER COMBATIDO status quo - (...) orienta-se o ensino da área de modo que acolha a diversidade do repertório cultural que o aluno traz para a escola, trabalhe com os produtos da comu- nidade em que a escola está inserida e também se introduzam conteúdos das diversas culturas e épo- cas a partir de critérios de seleção adequados à par- ticipação do estudante na sociedade como cidadão informado. Os conteúdos da área de Arte estão orga- nizados de tal maneira que possam atender apren- dizagens cada vez mais complexas no domínio do conhecimento artístico e estético, seja no exercício do próprio processo criador pelo fazer, seja no con- tato com obras de arte e com outras manifestações presentes nas culturas ou na natureza. O estudo, a análise e a apreciação da arte podem contribuir tanto para o processo pessoal de criação dos alunos como também para sua experiência estética e co- culturas humanas (BRASIL, 1998, p. 40). - - - - - - - - mais bonitos, mais inteligentes, mais honestos, mais e dominar as outras raças, principalmente a negra - rada como a menos honesta, menos inteligente e, - - - Considerando-se que não são muitos os cursos de pedagogia ou licenciatura que preveem conteúdos relacionados à África e à história dos negros no Brasil, é importante que o professor no processo de ação- O racismo, diferentemente do preconceito, é uma tendência ideológica de crer que as características intelectuais e morais de um dado grupo estão diretamente relacionadas ao fenótipo (conjunto pessoa). As diversas características negativas (gente amaldiçoada, suja, violenta, cabelo duro e ruim e etc.) atribuídas à população negra no decorrer da história e mantidas até os dias atuais são compreendidas como racismo. - - - - - - - - - sileiro sua identidade como um povo portador de di- - - LEI 10.639/2003 E O ENSINO DE HISTÓRIA E CULTURA AFRO-BRASILEIRA 29 - - - - - - - - strictu sensu - - institui a obrigatoriedade do ensino da história e - - - - Art.26-A - Nos estabelecimentos de ensino funda- obrigatório o ensino sobre História e Cultura Afro -brasileira. Parágrafo Primeiro – O conteúdo programático a EDUCAÇÃO E RELAÇÕES RACIAIS que se refere o caput deste artigo incluirá o estu- do da África e dos Africanos, a luta dos negros no Brasil, a cultura negra brasileira e o negro na for- mação da sociedade nacional, resgatando a contri- buição do povo negro nas áreas social, econômica e política, pertinentes à História do Brasil. Parágrafo segundo – Os conteúdos referentes à História e Cultura Afro-brasileira serão ministra- dos no âmbito de todo currículo escolar, em espe- cial, nas áreas Educação Artística e de Literatura e Histórias Brasileiras. Art. 79-B – O calendário escolar incluirá o dia 20 de novembro como “Dia Nacional da Consciência Negra”. que se refere o caput deste artigo incluirá o estu- do da África e dos Africanos, a luta dos negros no Brasil, a cultura negra brasileira e o negro na for- mação da sociedade nacional, resgatando a contri- buição do povo negro nas áreas social, econômica e política, pertinentes à História do Brasil. - - - - Parágrafo segundo – Os conteúdos referentes à História e Cultura Afro-brasileira serão ministra- dos no âmbito de todo currículo escolar, em espe- cial, nas áreas Educação Artística e de Literatura e Histórias Brasileiras. - racial no interior da escola e da sala de aula? Art. 79-B – O calendário escolar incluirá o dia 20 de novembro como “Dia Nacional da Consciência Negra”. A falta de preparo pode ser fruto de uma educação eurocêntrica, que nos faz reproduzir, consciente e inconscientemente, os preconceitos introjetados em nós ao longo da história. Além disso, reforça o mito da democracia racial que permeia nossa sociedade ao ignorar essa diversidade nas relações sociais de alunos entre si e de alunos com professores no espaço escolar. as expressões do que eram até então eram con- sideradas minorias, relacionadas à “raça” e gê- nero, ganham espaço e importância. Assim como as manifestações da Cultura Visual que ocorrem fora dos espaços sacralizados da arte (museus, espaços expositivos oficiais, livros, entre outros), como as manifestações em ruas e em outros es- paços públicos e, em alguns casos, sem identifi- cação de autoria. Essas podem quebrar a regra da homogeneização e interagir com o público na construção e na desconstrução de conceitos. (PE- DROSO, 2012, p. 74). - - - - - - - - - históricas, sociais e antropológicas, oriundas da realidade brasileira, e busca combater o racismo e - - - - 31 MUSEU NA ESCOLA: UMA POSSIBILIDADE DE ENSINO DE ARTE - - - papel da escola combater todos os tipos de precon- - Dentre os trabalhos, encontramos obras - - - - - - - pela mantenedora, podem sentir-se mais seguros A NEGRITUDE EM SALA DE AULA - despreparados e inseguros, os professores de Arte passam a apoiar-se cada vez mais nos livros didá- ticos de Educação Artística, produzidos[...] pela 1970”(FUSARI;FERRAZ, 1993, p. 38). - - perior em arte, a partir de um acervo gerido pela - so de escolha, validando-as simbolicamente como - - - - - - - - - - opera como tal na realidade das escolas, em espe- - 33 33 - - - - - - Erbo Stenzel, em 1944, ainda estudando no - negra carregando uma - gerindo o movimento de da mulher negra e as po- - - ainda era aluno da Escola Nacional de um homem negro nos parece, um caja- do ou um rudimen- tar instrumento de Rubem Valentim inicia seu trabalho de pintor - - - - 35 35 - - - admiradores de seus trabalhos em madeira, pedra, - - to utilizou as horas de esconderijo para retomar - Espedito Rocha se apresenta como um perso- - - - - - - - - 37 - morou na Europa de 1989 a 1994, onde ganhou o - de flashs set mulher negra empoderada de sua negritude, pro- CONSIDERAÇÕES FINAIS - determinada obra de arte ou um artista sintetizasse a arte de todo um continente e sua vasta diversidade, - continente em permanente guerra, assolado por - - tomando os pressupostos legais vigentes em nossos - - - - - - - - nas a Europa teria passado por um processo de - 38 - tui obrigatoriedade do ensino de arte e cultura de - - - - - - - - Diante da diversidade cultural que permeia o mundo contemporâneo, é necessário repensar um ensino da Arte que propicie ao estudante o desenvolvimento do pensamento estético voltado a essa diversidade, de relação às formas artísticas, mas também no sentido de compreender as relações da produção artística e cultural, como produto das relações sociais e da indi- vidualidade do artista (CURITIBA, 2006, P. 87) 39
https://openalex.org/W2159203480	https://europepmc.org/articles/pmc4238313?pdf=render	English	null	Effect of negative emotions evoked by light, noise and taste on trigeminal thermal sensitivity	The Journal of headache and pain	2,014	cc-by	8,940	Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Open Access Effect of negative emotions evoked by light, noise and taste on trigeminal thermal sensitivity Guangju Yang1, Lene Baad-Hansen2, Kelun Wang3, Qiu-Fei Xie1* and Peter Svensson2,4 © 2014 Yang et al.; licensee Springer. 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 reproductio in any medium, provided the original work is properly credited. © 2014 Yang et al.; licensee Springer. 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. Abstract Background: Patients with migraine often have impaired somatosensory function and experience headache attacks triggered by exogenous stimulus, such as light, sound or taste. This study aimed to assess the influence of three controlled conditioning stimuli (visual, auditory and gustatory stimuli and combined stimuli) on affective state and thermal sensitivity in healthy human participants. Methods: All participants attended four experimental sessions with visual, auditory and gustatory conditioning stimuli and combination of all stimuli, in a randomized sequence. In each session, the somatosensory sensitivity was tested in the perioral region with use of thermal stimuli with and without the conditioning stimuli. Positive and Negative Affect States (PANAS) were assessed before and after the tests. Subject based ratings of the conditioning and test stimuli in addition to skin temperature and heart rate as indicators of arousal responses were collected in real time during the tests. Results: The three conditioning stimuli all induced significant increases in negative PANAS scores (paired t-test, P ≤0.016). Compared with baseline, the increases were in a near dose-dependent manner during visual and auditory conditioning stimulation. No significant effects of any single conditioning stimuli were observed on trigeminal thermal sensitivity (P ≥0.051) or arousal parameters (P ≥0.057). The effects of combined conditioning stimuli on subjective ratings (P ≤0.038) and negative affect (P = 0.011) were stronger than those of single stimuli. Conclusions: All three conditioning stimuli provided a simple way to evoke a negative affective state without physical arousal or influence on trigeminal thermal sensitivity. Multisensory conditioning had stronger effects but also failed to modulate thermal sensitivity, suggesting that so-called exogenous trigger stimuli e.g. bright light, noise, unpleasant taste in patients with migraine may require a predisposed or sensitized nervous system. Keywords: Quantitative sensory testing; Affective state; Thermal sensitivity; Light; Noise; Taste For a biological system, perception often requires information emanating from sensors of multiple modalities [5]. For instance, human perception of food flavor and texture during consumption involves taste, intraoral somatosensory function (thermal and mechanical), vision, olfaction, and auditory signals that contribute to the overall appreciation of food. Different sensory modalities may interact in a non-linear way as multi-modal sensory integration (MSI) [6]. Moreover, observations that the somatosensory system may modulate activity in auditory brain regions become clinically relevant [7]. The perception of migraine headache is uniquely exacerbated during exposure to ambient light as compared with the pain level perceived in the dark [8]. Study procedure Th i Another possible effect of cross-modality sensory regulation could be simply a distraction effect reducing the perceived stimulus intensity by diverting attention away from pain processing [16-18]. However, one study indicated that distraction tasks that demand greater attention processing do not produce greater reductions in pain [19]. The twenty participants each attended four experimental sessions. In each session the participants received one of four conditions: A. visual stimulation with bright light alone, B. auditory stimulation with recorded chainsaw noise alone, C. gustatory stimulation with bitter taste alone, and D. multisensory stimulation with gustatory stimulation combined with bright light and chainsaw noise. The four conditions were applied in a randomized sequence produced by Microsoft Excel. There was an interval of about one week between sessions A, B and C. Session D was performed on the same day as session C after a 30 min rest period. A total of six thermal sensory tests were performed over perioral region in each participant in the A. visual or B. auditory sessions: I base- line 1, II with conditioning stimulus 1; III baseline 2, IV with conditioning stimulus 2; V baseline 3, VI with condi- tioning stimulus 3. Conditioning stimuli 1–3 (visual, audi- tory depending on group) were of different intensity (see below). In the C/D gustatory and multisensory session, four thermal sensory tests were performed over perioral region in each participant: I baseline 1, II with gustatory conditioning stimulus; III baseline 2, IV with multisensory conditioning stimulus (see below). There was a 2 min adaption period before each test and an interval of 5 min between tests [24,25]. Each session lasted approximately 60 min for each participant (Figure 1 shows the time line of 1 thermal test including the conditioning stimulus). It is obvious that real life experiences mostly rely on the presence of combined stimuli coming from different modalities. For example, music is often used to enhance the emotion impact of movies. The enhancing effect of combined presentation of different modalities is, how- ever, intuitive and understudied [11]. Quantitative sensory testing (QST) provides a reliable and accurate tool for examination of somatosensory func- tion [20,21]. One study, which evaluated the effect of emo- tion on sensory-discriminative parameters, indicated that changes in pain sensitivity are more often observed for pain threshold measures than for pain tolerance measures [22]. Participants Young healthy participants in this study were recruited from university students through the webpage [23] and advertisements at Aarhus University, Denmark. Inclusion criteria were: age between 20 to 30 years old, physically and mentally healthy. Exclusion criteria were: Optical, auditory, gustatory functional disorders; infection in the tested area (perioral region); ongoing orofacial pain, or reported chronic pain in last 6 months; serious systemic diseases; medicine intake with effect on the central nervous system in the last 2 weeks; former experience with similar experiments. Twenty-five people responded to the advertisements. Finally, 20 participants aged between 20 to 30 years old (26.6 ± 3.2), 10 females and 10 males met the criteria and were included in this study. The study adhered to the Helsinki declaration II and was approved by the local ethics committee (NO. 1-10-72-31-13) and all participants gave written informed consent. Background Migraine is an inherited, episodic disorder involving changes in responsivity of the sensory systems. For example, migraine patients complain of headache attacks with increased sensitivity to e.g. light or sound. Some patients mention that otherwise pleasant odors are unpleasant during the attacks. The disorder is considered as a disturbance in the brain of the subcortical aminergic sensory modulatory systems [1]. Overall, these observations could indicate that the migrainous brain does not habituate to signals in a normal way [2-4]. * Correspondence: xieqiuf@163.com 1Department of Prosthodontics and Center for Oral Functional Diagnosis, Treatment and Research, Peking University School and hospital of Stomatology, Zhongguancun Nandajie 22, 100081 Beijing, China Full list of author information is available at the end of the article Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 2 of 12 Page 2 of 12 Page 2 of 12 Methods Participants There are several theories to explain MSI. One of them is the motivational priming model, which sug- gested the affective state as a mediator [9]. The pain sensitivity may be enhanced by evoked unpleasant affective states, while attenuated by pleasant affective states and high arousal augments both effects [9]. Visual stimuli used in previous studies examining emotional processes have been either Pictures of Facial Affect or the International Affective Picture System (IAPS) [10,11]. Music may also evoke comparable emo- tional responses across different musical categories [11,12]. However, the individual background, such as culture and education, is important for the results of such tests [13]. Therefore, for example bright light and a chainsaw noise, to which responses are less ethnic, cultural, or education-dependent, could be useful in some studies. Sucrose has been reported to reduce pain in children, while the effect of unpleasant bitter gusta- tory stimulation on pain has not been systematically studied [14,15]. Study procedure Th i The present study aimed to assess the effect of, and possible interactions among different unpleasant con- ditioning stimulus modalities, i.e. visual (light), auditory (noise), gustatory (bitter taste) on affective state, physio- logical arousal and thermal somatosensory sensitivity in the trigeminal system of young healthy human parti- cipants. The following hypotheses were tested: a. the thermal sensitivity of the participants is increased during exposure to conditioning stimuli compared with no con- ditioning stimuli; b. the unpleasant stimuli evoke negative affective states and physiological arousal compared with baseline; c. combination of conditioning stimuli into a multi-sensory conditioning stimulus exerts larger changes in outcome parameters compared with single conditioning stimuli. The tests were performed in a quiet room with tem perature at 20–22°C. All participants were required to refrain from ingestion of all food, beverages, and oral care products for a minimum of 2 hour before arrival. The participants were free from head colds and allergies on the test days. Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 3 of 12 Figure 1 One thermal somatosensory test consists of: before stimuli (0.5 min), stimulus habituation period (2 min), thermal test (~3.5 min), recovery period (5 min). ‘HR’ = Heart Rate record, ‘T’ = face skin temperature record. Cold Detection Threshold (CDT), Warmth Detection Threshold (WDT), Cold Pain Threshold (CPT), Heat Pain Threshold (HPT). Figure 1 One thermal somatosensory test consists of: before stimuli (0.5 min), stimulus habituation period (2 min), thermal test (~3.5 min), recovery period (5 min). ‘HR’ = Heart Rate record, ‘T’ = face skin temperature record. Cold Detection Threshold (CDT), Warmth Detection Threshold (WDT), Cold Pain Threshold (CPT), Heat Pain Threshold (HPT). Figure 1 One thermal somatosensory test consists of: before stimuli (0.5 min), stimulus habituation period (2 min), thermal test (~3.5 min), recovery period (5 min). ‘HR’ = Heart Rate record, ‘T’ = face skin temperature record. Cold Detection Threshold (CDT), Warmth Detection Threshold (WDT), Cold Pain Threshold (CPT), Heat Pain Threshold (HPT). All the conditioning stimuli were given in repeated trials, 3 trials for the visual or auditory tests, and 1 trial for the gustatory or combination tests. The stimuli were started 2 min before each thermal test, and ended after the emo- tion evaluations; thus, each episode of stimulus lasted about 7 min (Figure 1). Psychometrical and physiological measures h d f h d Psychometrical and physiological measures The perceived intensity of the conditioning stimuli was assessed using a numerical scale from “0 = no stimulus could be detected” to “100 = the strongest stimulus im- aginable”. The intensity rating was measured three times for each test, 3 seconds after the stimulus and at the beginning and ending of the thermal test. The mean value was calculated for statistics analysis. To determine whether the conditioning stimuli induced affective changes, participants rated their emotional reactions using the Positive and Negative Affect Schedule (PANAS), which composes of a list of 20 adjectives used to describe 10 positive emotions (the global Positive Affect Score) and 10 negative emotions (the global Negative Affect Score) Auditory stimuli P i i Participants were instructed to remain awake with eyes covered by eyeshades and were exposed to recorded sounds of an electric chainsaw at three different intensities (60 dB, 75 dB, 90 dB) in randomized sequence produced by Microsoft Excel [6,28]. The noise was generated by a computer through loud-speakers and measured by Sound Pressure Lever Meter (RadioShack 33–2055, USA). A con- trol test was performed before each test without sound stimulus to eliminate the effect of timing. Visual stimuli Visual stimuli were created using three levels of bright light shined directly at the participants’ eyes in a randomized se- quence produced by Microsoft Excel: (i) 1000 lux bright light; (ii) 3000 lux bright light; (iii) 9000 lux bright light [26,27]. The bright light was generated using dental chair lamp with an area of 14 × 9 cm (ESTETICA E80, KaVo Germany) and calibrated by a Luxmeter (LX1010B+, Malmbergs, Sweden). There was a control test before each test without visual stimuli to eliminate timing effect, in which the participants wore eyeshades and closed their eyes to make sure they did not see any light. Thermal sensitivity measures Participants were positioned in a comfortable dental chair. The measures were performed in the right perioral region with the thermode placed on the right angle of the mouth. The thermal tests were performed using a Medoc Pathway with ATS thermode (30 mm × 30 mm, square surface, MEDOC, Israel). Care was taken throughout the session to keep the test sites during the repeated testing the same. Cold and warmth detection thresholds (CDT, WDT) were measured first. Then cold pain and heat pain thresholds (CPT, HPT) were determined [20,21]. The mean threshold temperature of three consecutive measurements was cal- culated. All thresholds were obtained with ramped stimuli (1°C/s) that were terminated when the participants pressed a button. For thermal detection thresholds the ramp back to baseline was 1°C/s, while for thermal pain thresholds this ramp was chosen at 5°C/s. The baseline temperature was 32°C. Cut-off temperatures were set at 0°C and 50°C, respectively [20,21]. A standardized verbal ins truction was read to the participants and a training test was performed before the actual tests to make sure that the participants understood the thermal test program [20]. Gustatory stimulus Participants were instructed to rinse with distilled water at least four times over a 1 min period prior to testing. Gelatin was the vehicle for gustatory stimuli. The partic- ipants were asked to hold 20 ml gelatin (2% bovine skin gelatin, Sigma-Aldrich, St. Louis, USA) in their mouth with either bitter (0.1% quinine, Sigma-Aldrich, adult toxic dose, 2.5 – 4 g) or neutral taste (2% gelatin) [29]. The gustatory stimuli were presented at room temperature (~21°C) with an at least four times distilled water rinse after each test for 5 min until the taste has been washed out [30]. Multisensory stimuli The three conditioning stimulus modalities (visual 9000 lux light, auditory 90 dB noise and gustatory 0.1% quinine gelatin) were presented simultaneously to each participant with a control test (no visual, no auditory & neutral taste). Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 4 of 12 after each thermal sensory test [31,32]. Respondents were required to indicate the extent to which they felt the emo- tions included on the schedule “during the thermal test” on a five-point scale (where 1 = very slightly or not at all, to 5 = extremely) [31,32]. stronger stimuli: significant intensity effect on subjective rating “relative changes”, F = 88.04, P <0.001 for visual test (Figure 2-A); F = 88.25, P <0.001 for auditory test (Figure 2-B). Post-hoc test for multiple contrasts indicated significant increases with increased intensity of light and noise (Figure 2-A,B). There were no gender differences in visual, auditory, gustatory, and multisensory tests (un- paired t-test, t ≤0.266, P >0.791). Heart rate (HR) and skin temperature of contralateral orofacial region were measured using a Patient Monitor (NPB-4000, US) and an infrared thermometer (THER- MOFOCUS, 01500 serien, Italy) continuously. HR and skin temperature were recorded seven times in each test (Figure 1) and the mean was used for further analysis [28]. p , , ) Most of the affective scores to different conditioning stimuli were normally distributed (Shapiro-Wilk, P >0.05). The conditioning stimuli induced statistically significantly higher negative affective states compared with baseline: vis- ual test (baseline: 11.6 ± 2.1, mean ± SD, stimulus: 13.6 ± 4.5; paired t-test, t = −3.742, P <0.001); auditory test (base- line: 12.0 ± 3.0, stimulus: 14.6 ± 4.5; t = −5.182, P <0.001); gustatory test (baseline: 13.6 ± 4.5, stimulus: 16.8 ± 4.1; t = −2.656, P =0.016); multisensory test (baseline: 13.2 ± 4.4, stimulus: 19.6 ± 6.2; t = −6.719, P <0.001) (Figure 3). The negative affective score differences between the condi- tion with stimuli and corresponding baseline (as “delta value” or “changes” in the text and figures) were higher when the participants were exposed to more intense stim- uli: significant intensity effects on negative affective scores for visual test (F = 7.484, P =0.006, Figure 4-A), auditory test (F = 11.335, P <0.001, Figure 4-B). Physiological measures h h d k The heart rate and skin temperature data for different conditioning stimuli were normally distributed (Shapiro- Wilk, P ≥0.069). The conditioning stimuli did not change the two indices of physical arousal, heart rate and skin temperature, in comparison with the corresponding base- line values: pared t-test P value for differences (heart rate, temperature) in visual test (0.408, 0.162), auditory test (0.059, 0.574), gustatory test (0.195, 0.639) and multi- sensory test (0.137, 0.057). Psychometrical measures All the subjective intensity rating scores to different condi- tioning stimuli were normally distributed (Shapiro-Wilk, P ≥0.066). Compared with baseline, subjective stimulus in- tensity rating during conditioning stimuli tests was signifi- cantly higher in visual (paired t-test, P <0.001), auditory (P <0.001), gustatory (P <0.001), and multisensory test (P <0.001) (Table 1). The participants’ subjective rating differences between the condition with stimulus and corresponding baseline (“relative changes” in the text and figures) was higher when participants were exposed to Multisensory stimuli The Post-hoc test for multiple comparisons indicated a statistically significant increase in negative affective scores with increasing inten- sities in the light and noise tests (Figure 4-A,B). Besides, there were statistically significant gender differences, with males having higher negative affective scores when exposed to the same stimuli compared to females: in auditory test (unpaired t-test gender differences, t = −3.308, P = 0.004 for 75 dB noise; t = −3.519, P =0.002 for 90 dB noise, Figure 4-B) and gustatory test (t = −2.694, P = 0.015, Figure 4-C). As could be expected, all the stimuli failed to induce positive affective changes of the participants com- pared with baseline: visual test (paired t-test, P = 0.117), auditory test (P = 0.091), gustatory test (P = 0.169), and multisensory test (P = 0.080). Statistical analysis All statistical calculations were performed using SPSS 17.0 software for windows (IBM, inc., USA). The assumption of normal distribution of all data was investigated by the Shapiro-Wilk method. Paired t-test was used to compare the difference between baselines and stimuli tests. Differ- ences between genders were evaluated by unpaired t-test. The changes under conditioning stimuli compared to base- line tests were calculated as delta values: △value = value (stimuli) – value (baseline), which was used in results and figures as “relative changes”. Differences between delta values within one session (dose dependent effect) were an- alyzed using two-way repeated measures ANOVA with the stimulus intensity as within-participant factor and gender as between-participant factor. To evaluate the multisensory effect, the delta values (“changes”) in multisensory stimula- tion test, gustatory test, 9000 lux light visual test, and 90 dB noise auditory test were compared using two-way repeated ANOVA with conditioning stimuli as within- participant factor, gender as between-participant factor. Post-hoc comparisons were estimated using Bonferroni post-hoc test with correction for multiple comparisons. P <0.05 was taken as an indication of a statistically signifi- cant difference. Participants There was no significant age difference between female (25.4 ± 2.9 years, mean ± SD) and male (27.7 ± 3.2 years) participants (unpaired t-test age: P = 0.110). Influence of conditioning stimuli on affective state (Shapiro-Wilk, P >0.05). Compared with baseline, the sensi- tivity to thermal stimuli did not change when the partici- pants were exposed to conditioning stimuli: visual test (paired t-test P value for CDT, WDT, CPT, HPT were 0.074, 0.641, 0.165, 0.095 respectively; Figure 5-A), auditory test (paired t-test P value for CDT, WDT, CPT, HPT were 0.055, 0.922, 0.515, 0.418 respectively; Figure 5-B), gustatory test (paired t-test P value for CDT, WDT, CPT, HPT were 0.347, 0.207, 0.202, 0.729 respectively; Figure 5-C), multi- sensory stimulation (paired t-test P value for CDT, WDT, CPT, HPT were 0.526, 0.051, 0.737, 0.331 respectively; Figure 5-D). g The experimental visual stimuli used in previous studies to examine the affective processes were composed of emo- tional videos [33,34] or photos [35,36]. Viewing faces with sad or happy expressions specifically evoked the expressed feelings in the viewer, defined as “emotional contagion” [35]. This emotional contagion was considered as a “pre- wired” decoding instrument, which was considered a fast, repeatable and stable process [35]. These visual stimuli activate an emotion perception process characterized by focusing attention to external events, and accurately under- standing the information conducted, which need the full co-operation of the investigated individuals [11]. Addition- ally, some studies concluded that the experimentally induced changes in the affective state were obtained only after repeated stimuli presentation accompanied with specified verbal inductions [37]. Furthermore, in another Electroencephalography (EEG) study, emotional reactions could not be evoked by presenting emotional pictures alone [11]. In migraineurs, bright light often exacerbate the headache, which could be accounted for a pathophysio- logical hypothesis of a disturbance of the subcortical sen- sory modulation system [1,8]. Although the trigeminal autonomic reflex acts as a feed-forward system to facilitate the acute attack, the fundamental problem was thought to be in the brain [1]. The possible effect of bright light to experimentally evoke an affective state and somatosensory sensitivity changes has not been assessed before. The hypothesis of the present study was that bright light could evoke a negative affective state and change the somatosen- sory sensitivity. In the present experimental study, bright Thermal somatosensory sensitivity Page 6 of 12 Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 6 of 12 Figure 3 Negative affective state differences between tests with conditioning stimuli (grizzle bars) and corresponding baselines (white bars). The results are presented as means ± SDs of the negative affective scores form Positive and Negative Affect Schedule. The conditioning stimuli induced statistically significantly higher negative affective states compared with baseline. **= P <0.001, = P <0.05. Thermal somatosensory sensitivity Most of the QST data in different conditioning stimuli were normally distributed only after logarithmic transformation Yang et al. The Journal of Headache and Pain 2014, 15:71 Page 5 of 12 http://www.thejournalofheadacheandpain.com/content/15/1/71 Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 5 of 12 Table 1 Subjective intensity rating to conditioning stimuli (−/100) Conditions Baseline (mean ± SD) With stimuli (mean ± SD) P Visual 0 ± 0 59.0 ± 24.1 < 0.001 Auditory 18.8 ± 15.0 65.6 ± 24.1 < 0.001 Gustatory 6.6 ± 4.4 40.3 ± 17.3 < 0.001 Multisensory 7.5 ± 4.9 47.3 ± 22.8 < 0.001 Subjective rating to conditioning stimuli was assessed by numerical scale, in which “0 = no stimulus could be detected” to “100 = the strongest stimulus imaginable”. The comparisons between the conditions with stimuli and corresponding baseline were investigated by paired t-test. Table 1 Subjective intensity rating to conditioning stimuli (−/100) ive rating to conditioning stimuli was assessed by numerical scale, in which “0 = no stimulus could be detected” to “100 = the stronges able”. The comparisons between the conditions with stimuli and corresponding baseline were investigated by paired t-test. Figure 2 Subjective rating (changes) to different conditioning stimuli. The differences between test with conditioning stimuli and corresponding baseline were calculated as delta values: △value = value (conditioned) – value (baseline), which was used in figures (A visual and B auditory test) as “relative changes”. Compared with baseline, subjective stimulus intensity rating during gustatory and multisensory tests was significantly higher. The participants’ subjective rating changes were higher when they were exposed to stronger stimuli in visual and auditory tests. (C) gustatory test, (D) multisensory test. *= P <0.001. Fi 2 S bj i i ( h ) diff di i i i li Th diff b i h di i i i li d Figure 2 Subjective rating (changes) to different conditioning stimuli. The differences between test with conditioning stimuli and corresponding baseline were calculated as delta values: △value = value (conditioned) – value (baseline), which was used in figures (A visual and B auditory test) as “relative changes”. Compared with baseline, subjective stimulus intensity rating during gustatory and multisensory tests was significantly higher. The participants’ subjective rating changes were higher when they were exposed to stronger stimuli in visual and auditory tests. (C) gustatory test, (D) multisensory test. = P <0.001. Discussions This study investigated for the first time the effects of three unpleasant conditioning stimuli (bright light, chain- saw noise and bitter taste) on negative affective state, physiological arousal and skin thermal sensitivity. The three conditioning stimuli, as expected, all induced statis- tically significant increases in negative affective scores compared with baseline. Additionally, a dose dependent phenomenon was demonstrated in the visual and auditory tests (only one intensity gustatory stimulus was applied in this study). There were no significant changes in positive affective scores, physiological arousal (heart rate or skin temperature) between tests with and without conditioning stimuli. The skin thermal sensitivity (via, CDT, WDT, CPT, HPT) in the perioral region did not change signifi- cantly during conditioning stimuli tests compared with baseline. Combining the individual conditioning stimuli into a multisensory conditioning stimulus led to a stren gthened effect compared with single conditioning stimuli for several outcome parameters, i.e. subjective ratings, negative affective scores and warmth detection threshold (WDT). Figure 3 Negative affective state differences between tests with conditioning stimuli (grizzle bars) and corresponding baselines (white bars). The results are presented as means ± SDs of the negative affective scores form Positive and Negative Affect Schedule. The conditioning stimuli induced statistically significantly higher negative affective states compared with baseline. *= P <0.001, = P <0.05. Multisensory stimulation effect When the participants were exposed to multisensory (combined) conditioning stimuli, they subjectively rated higher “delta values” to the same bitter taste compared with in the condition of taste stimulus alone (whole group: F = 5.017, P = 0.038; male: F = 8.980, P = 0.015; female: F = 1.304, P >0.05 Figure 6-A). The female participants presented higher “delta values” for the negative affective scores during the multisensory stimuli condition than dur- ing noise stimulation alone (noise: 2.0 ± 2.4, mean ± SD; taste: 1.5 ± 6.3; combination: 6.8 ± 5.8; Post-hoc test between combination test and noise test, P = 0.011, Figure 6-C). The participants presented a statistically sig- nificant decrease in WDT (“delta” WDT decreasing) dur- ing multisensory conditioning stimuli compared with light stimulus alone (male: Post-hoc test, P = 0.019, Figure 6-G, Table 2) or taste alone (whole group: P =0.004; male: P = 0.003; female: P = 0.438, Figure 6-G, Table 2). Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 7 of 12 Page 7 of 12 Figure 4 Effects of conditioning stimuli on negative affective (changes) between genders. The negative affective score differences between the condition with stimuli and corresponding baseline (as “changes” in the figure) were higher when the participants were exposed to more intense stimuli in visual (A) and auditory (B) tests. Besides, there were statistically significant gender differences in auditory test (B) and gustatory test (C). (D) indicates multisensory test. *= P <0.001, = P <0.01, = P <0.05. Figure 4 Effects of conditioning stimuli on negative affective (changes) between genders. The negative affective score differences between the condition with stimuli and corresponding baseline (as “changes” in the figure) were higher when the participants were exposed to more intense stimuli in visual (A) and auditory (B) tests. Besides, there were statistically significant gender differences in auditory test (B) and gustatory test (C). (D) indicates multisensory test. = P <0.001, = P <0.01, = P <0.05. light successfully evoked a negative affective state in a dose-dependent manner according to the intensity of the light (Figure 4-A). Bright light may be a simple and effecient way to induce a negative affective state in human studies. However, bright light did not significantly change somatosensory sensitivity to thermal stimuli. This indicates that the light regulation of thermal somatosensory sensi- tivity may not exist in healthy humans without prior sensitization or predisposition. Multisensory stimulation effect and termination of changes in affective state [13]. Music may affect trained musicians and non-musicians differently and different individuals have different musical preferences and experiences, which may be important in studies of influence of music on different outcome parameters [13]. In contrast, to most people a chainsaw noise as used in the present study can be considered an unpleasant auditory stimulus. The participants in this study rated moderate to strong unpleasantness to the noise (Figure 2-B). The hypothesis of the present study was that an unpleasant auditory stimulus could increase negative affect and change somatosensoty sensitivity. The chainsaw noise successfully evoked a negative affective state in a dose-dependent manner as the intensity of the auditory stimulus increased (Figure 4-B). Again, chainsaw noise may be a simple and effective way to induce a negative affective state in human studies. However, the chainsaw noise did not significantly Several studies have demonstrated that music is a power- ful mediator of changes in affective state [38,39]. Func- tional neuro-imaging and lesion studies have shown that music-evoked affective state changes can modulate activity in virtually all limbic and paralimbic brain structures, such as amygdala, parahippocampal gyrus and hippocampus [12,40-42]. These structures are crucially involved in the initiation, generation, detection, maintenance, regulation Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 8 of 12 Figure 5 Thermal sensitivity differences between tests with conditioning stimuli (grizzle bars) and corresponding baselines (white bars). The results are presented as means ± SDs of the threshold values from visual test (A), auditory test (B), gustatory test (C) and multisensory test (D). Compared with baseline (white bars), the sensitivity to thermal stimulus did not change when the participants were exposed to conditioning stimuli (grizzle bars). CDT = cold detection threshold, WDT = warmth detection threshold, CPT = cold pain threshold, HPT = heat pain threshold. Figure 5 Thermal sensitivity differences between tests with conditioning stimuli (grizzle bars) and corresponding baselines (white bars). The results are presented as means ± SDs of the threshold values from visual test (A), auditory test (B), gustatory test (C) and multisensory test (D). Compared with baseline (white bars), the sensitivity to thermal stimulus did not change when the participants were exposed to conditioning stimuli (grizzle bars). CDT = cold detection threshold, WDT = warmth detection threshold, CPT = cold pain threshold, HPT = heat pain threshold. negative affective state. Multisensory stimulation effect These contradictory results may possibly been explained by the “preference theory”, since the subjective rating to the bitter taste in this study (40.3 ± 17.3, mean ± SD, in a 0–100 numerical scale) was much higher than the study by Horjales-Aaujo (29.7 ± 19.7) [14,45]. Despite the negative affective state, there were no changes in somatosensory sensitivity in healthy volunteers. change somatosensory sensitivity to thermal stimuli. This indicates that auditory stimulus regulation of thermal som- atosensory is not significant in healthy volunteers. How- ever, in some pathological conditions, such as tension-type and cervicogenic headache, phonophobia is a common symptom [43]. Again, this may require a sensitized or predisposed somatosensory system. It has been reported that sucrose reduces pain during the Cold Pressor Test (CPT) in 5- to 10-year-old children, and this kind of analgesia is dependent on the sweet taste pref- erence [14]. However, sucrose was not an effective analgesia in adult women in the same study. The age-related decline in the analgesic efficacy of sucrose mirrors the age-related decline in the hedonic value of sweet tastes [44]. In a recent study, the taste of quinine gelatin (0.1%) failed to produce robust affective state changes [45]. However, in the present study the same stimulus evoked a significant change in Multisensory conditioning stimulus effects There were several statistically significant effects of the multisensory conditioning stimulus, which indicated that the multisensory stimulation enhanced the effect of indi- vidual stimuli with regard to subjective rating changes, affective score changes and induced slight thermal sensi- tivity changes. This is in line with a neurophysiological study, which demonstrates a strong emotion enhancement effect by simultaneous presentation of congruent emotional Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 9 of 12 Page 9 of 12 Figure 6 Multisensory effects between genders. To diminish the effect of timing, the delta values between test with stimuli and corresponding baseline, defined as “changes” in the figure was used. Multisensory conditioning stimulus led to a strengthened effect compared with single conditioning stimuli for several outcome parameters, i.e. subjective ratings to gustatory stimulus (A), negative affective scores (C) and warmth detection threshold (G). CDT = cold detection threshold, WDT = warmth detection threshold, CPT = cold pain threshold, HPT = heat pain threshold. Positive affective changes (B), heat rate changes (D), skin temperature changes (E), cold detection threshold changes (F), cold pain threshold changes (H), and heat pain threshold changes (I) in different tests were also presented. = P <0.01, = P <0.05. Figure 6 Multisensory effects between genders. To diminish the effect of timing, the delta values between test with stimuli and corresponding baseline, defined as “changes” in the figure was used. Multisensory conditioning stimulus led to a strengthened effect compared with single conditioning stimuli for several outcome parameters, i.e. subjective ratings to gustatory stimulus (A), negative affective scores (C) and warmth detection threshold (G). CDT = cold detection threshold, WDT = warmth detection threshold, CPT = cold pain threshold, HPT = heat pain threshold. Positive affective changes (B), heat rate changes (D), skin temperature changes (E), cold detection threshold changes (F), cold pain threshold changes (H), and heat pain threshold changes (I) in different tests were also presented. *= P <0.01, = P <0.05. pictures and music, regarding subjective ratings, peripheral and central physiological measures [11]. The findings also fit with a study, which shows that when either positive (joy- ful) or negative (fearful) music is played simultaneously with an emotionally neutral film clip, it evokes stronger signal changes in the amygdala and in areas of the ventro- lateral frontal cortex, compared with when only music or only film clips are presented [46]. Multisensory conditioning stimulus effects The activation increase may be due to the cross modal integration of three sensory stimuli, and this stronger activation possibly suggests en- hanced activation in a distributed neuronal network, which needs to be confirmed in functional imaging studies [6]. Physiological arousal changes It has been proposed that most affective changes are asso- ciated with undifferentiated physiological activity changes Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Page 10 of 12 Table 2 Impact of gender on warmth detection threshold (WDT, °C) changes with different stimuli Gender Visual (1) Auditory (2) Gustatory (3) Multisensory (4) F P 1 vs .4 2 vs .4 3 vs .4 Female Mean −0.08 −0.12 −0.02 −0.15 0.098 N.S 0.784 0.938 0.438 SD 0.76 0.64 0.35 0.60 Male Mean 0.17 −0.03 0.27 −0.33 6.036 0.003 0.019 N.S 0.003 SD 0.29 0.21 0.50 0.33 Sum Mean 0.04 −0.08 0.13 −0.24 2.241 N.S 0.058 0.387 0.004 SD 0.57 0.46 0.45 0.48 To evaluate the multisensory effect, the delta values (“changes”) in 9000 lux light visual test, 90 dB noise auditory test, gustatory test, and multisensory stimulation test were compared using two-way repeated ANOVA with conditioning stimuli as within-participant factor, gender as between-participant factor. The male participants presented a statistically significant decrease in “delta” WDT during multisensory conditioning stimuli compared with light stimulus alone (P = 0.019) or taste alone (P = 0.003). Italics values indicate statistically significant P values (P < 0.05). Table 2 Impact of gender on warmth detection threshold (WDT, °C) changes with different stimul Gender Visual (1) Auditory (2) Gustatory (3) Multisensory (4) F P 1 vs der on warmth detection threshold (WDT, °C) changes with different stimuli To evaluate the multisensory effect, the delta values (“changes”) in 9000 lux light visual test, 90 dB noise auditory test, gustatory test, and multisensory stimulation test were compared using two-way repeated ANOVA with conditioning stimuli as within-participant factor, gender as between-participant factor. The male participants presented a statistically significant decrease in “delta” WDT during multisensory conditioning stimuli compared with light stimulus alone (P = 0.019) or taste alone (P = 0.003). Italics values indicate statistically significant P values (P < 0.05). response is inhibited, when the appetitive system is primed by a positive affective state [53]. According to the motiv- ational priming theory, participants exposed to negative affective loading, whose aversive systems are activated, should get their perception facilitated [9,22]. However, somatosensory sensitivity to thermal stimuli was not chan- ged during induction of negative affective states in the present study. Physiological arousal changes Another perspective on somatosensory and affective processing is provided by the four-region neuro- biological model, which indicates a complex relationship between acute pain and simple emotion [54]. For instance, fear and pain sites overlap in the anterior midcingulate cortex (aMCC) and this region is involved in avoidance behaviors; posterior midcingulate cortex (pMCC) has no consistent emotion activations, yet has robust nociceptive responses; subgenual anterior cingulate cortex (sACC) acti- vation during noxious stimulation of the skin and viscera in a person-specific manner; the cingulate cortex is not uniformly involved in emotion, and not all pain-activation sites are associated with affect or emotion [54]. [47]. Thus it seems reasonable to examine physiological arousal measurements related to affective reactions. Skin temperature and heart rate are the most frequently used physiological expressions of affective state [47]. However, in the present study, these two parameters did not change significantly in response to changes in negative affective state by the different conditioning stimuli. This is in line with another study, where it was demonstrated that the verbally reported affective experiences are not always consistent with the physiological parameters, especially for unpleasant stimuli [47]. On the other hand, it may be because of the limited number of participants or that the applied stimulus was inadequate for induction of a signifi- cant physiological change in arousal. [47]. Thus it seems reasonable to examine physiological arousal measurements related to affective reactions. Skin temperature and heart rate are the most frequently used physiological expressions of affective state [47]. However, in the present study, these two parameters did not change significantly in response to changes in negative affective state by the different conditioning stimuli. This is in line with another study, where it was demonstrated that the verbally reported affective experiences are not always consistent with the physiological parameters, especially for unpleasant stimuli [47]. On the other hand, it may be because of the limited number of participants or that the applied stimulus was inadequate for induction of a signifi- cant physiological change in arousal. Abbreviations MSI M l d Abbreviations MSI: Multi-modal sensory integration; IAPS: Pictures of facial affect or the international affective picture system; QST: Quantitative sensory testing; CDT: Cold detection threshold; WDT: Warmth detection threshold; CPT: Cold pain threshold; HPT: Heat pain threshold; PANAS: Positive and negative affect schedule; HR: Heart rate; EEG: Electroencephalography; aMCC: Anterior MidCingulate cortex; pMCC: Posterior MidCingulate cortex; sACC: Subgenual anterior cingulate cortex. Interactions between somatosensory sensitivity and conditioning stimuli The perception of migraine headache, which is mediated by nociceptive signals, is exacerbated by exposure to en- vironment stimuli, i.e. light or noise, which are mediated by optical and acoustic signals. Prolonged neuronal activa- tion during a migraine attack is thought to induce periph- eral and central sensitization along the trigeminovascular pathway, which stands to explain the throbbing headache [48], accompanying scalp and neck-muscle tenderness [49], and whole-body cutaneous allodynia [50]. Several theories exist, which predict the relationship between conditioning stimuli and somatosensory sensitivity. To develop a more comprehensive model of somatosensory modulation by conditioning stimuli, it may be useful to consider a theory of “motivational priming” [9]. It is pro- posed that affective state processing evoked by stimuli could be viewed as two opposing motivational systems. One system is appetitive and produces approach behav- iors, and the other system is aversive and promotes avoid- ance behaviors [9]. Considerable evidence indicates that prior activation of these systems modulates defensive be- haviors, which facilitates somatosensory capability [51]. These defensive behaviors increase in magnitude when the aversive system is primed by a negative affective state, i.e. anticipation of shock [52]. Conversely, this defensive Conclusions The three conditioning stimuli induced significant increases in subjective rating and negative effective scores accompanied with no physiological arousal changes. These stimuli did not, however, modulate skin thermal sensitivity. The multisensory conditioning stimuli induced stronger effects on subjective rating and negative affect than individual conditioning stimuli alone. The often observed trigger or exacerbation of migraine by visual, auditory or gustatory stimuli may require a pre- disposed or sensitized nervous system. Abbreviations MSI: Multi-modal sensory integration; IAPS: Pictures of facial affect or the international affective picture system; QST: Quantitative sensory testing; CDT: Cold detection threshold; WDT: Warmth detection threshold; CPT: Cold pain threshold; HPT: Heat pain threshold; PANAS: Positive and negative affect schedule; HR: Heart rate; EEG: Electroencephalography; aMCC: Anterior MidCingulate cortex; pMCC: Posterior MidCingulate cortex; sACC: Subgenual anterior cingulate cortex. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Page 11 of 12 Page 11 of 12 Yang et al. The Journal of Headache and Pain 2014, 15:71 http://www.thejournalofheadacheandpain.com/content/15/1/71 Authors’ contributions d h 16. Bushnell EW, Baxt C (1999) Children’s haptic and cross-modal recognition with familiar and unfamiliar objects. J Exp Psychol Hum Percept Perform 25(6):1867–1881, doi:10.1037/0096-1523.25.6 YG recruited the participants, carried out the study, performed the statistical analysis, and drafted the manuscript. SP and BHL participated in the design of the study, statistical analysis, and revision of the draft. XQ and WK helped to draft and revise the manuscript. All authors read and approved the final manuscript. 17. Longe SE, Wise R, Bantick S, Lloyd D, Johansen-Berg H, McGlone F, Tracey I (2001) Counter-stimulatory effects on pain perception and processing are significantly altered by attention: an fMRI study. 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https://openalex.org/W3120162216	https://europepmc.org/articles/pmc7796421?pdf=render	English	null	Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo	Materials	2,021	cc-by	9,952	Citation: Pham, H.T.; Ono, M.; Hara, E.S.; Nguyen, H.T.T.; Dang, A.T.; Do, H.T.; Komori, T.; Tosa, I.; Hazehara-Kunitomo, Y.; Yoshioka, Y.; et al. Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo. Materials 2021, 14, 208. https:// doi.org/10.3390/ma14010208 Received: 1 December 2020 Accepted: 21 December 2020 Published: 4 January 2021 Citation: Pham, H.T.; Ono, M.; Hara, E.S.; Nguyen, H.T.T.; Dang, A.T.; Do, H.T.; Komori, T.; Tosa, I.; Hazehara-Kunitomo, Y.; Yoshioka, Y.; et al. Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo. Materials 2021, 14, 208. https:// doi.org/10.3390/ma14010208 Received: 1 December 2020 Accepted: 21 December 2020 Published: 4 January 2021 Abstract: Aging tissues present a progressive decline in homeostasis and regenerative capacities, which has been associated with degenerative changes in tissue-speciﬁc stem cells and stem cell niches. We hypothesized that amino acids could regulate the stem cell phenotype and differentiation ability of human bone marrow-derived mesenchymal stromal cells (hBMSCs). Thus, we performed a screening of 22 standard amino acids and found that D-tryptophan (10 µM) increased the number of cells positive for the early stem cell marker SSEA-4, and the gene expression levels of OCT-4, NANOG, and SOX-2 in hBMSCs. Comparison between D- and L-tryptophan isomers showed that the latter presents a stronger effect in inducing the mRNA levels of Oct-4 and Nanog, and in increasing the osteogenic differentiation of hBMSCs. On the other hand, L-tryptophan suppressed adipogenesis. The migration and colony-forming ability of hBMSCs were also enhanced by L-tryptophan treatment. In vivo experiments delivering L-tryptophan (50 mg/kg/day) by intraperitoneal injections for three weeks conﬁrmed that L-tryptophan signiﬁcantly increased the percentage of cells positive for SSEA-4, mRNA levels of Nanog and Oct-4, and the migration and colony-forming ability of mouse BMSCs. L-kynurenine, a major metabolite of L-tryptophan, also induced similar effects of L-tryptophan in enhancing stemness and osteogenic differentiation of BMSCs in vitro and in vivo, possibly indicating the involvement of the kynurenine pathway as the downstream signaling of L-tryptophan. Finally, since BMSCs migrate to the wound healing site to promote bone healing, surgical defects of 1 mm in diameter were created in mouse femur to evaluate bone formation after two weeks of L-tryptophan or L-kynurenine injection. Both L-tryptophan and L-kynurenine accelerated bone healing compared to the PBS-injected control group. Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo Hai Thanh Pham 1,2 , Mitsuaki Ono 3,, Emilio Satoshi Hara 4, , Ha Thi Thu Nguyen 1,2,3, Anh Tuan Dang 1,2,3, Hang Thuy Do 1,2,3, Taishi Komori 1, Ikue Tosa 1, Yuri Hazehara-Kunitomo 1,3, Yuya Yoshioka 1, Yasutaka Oida 1, Kentaro Akiyama 1 and Takuo Kuboki 1 1 Department of Oral Rehabilitation and Regenerative Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama 700-8558, Japan; pth_uytki@yahoo.com (H.T.P.); thuharhm@gmail.com (H.T.T.N.); ppjj5xuj@s.okayama-u.ac.jp (A.T.D.); pa62884x@s.okayama-u.ac.jp (H.T.D.); de19016@s.okayama-u.ac.jp (T.K.); de421035@s.okayama-u.ac.jp (I.T.); de19034@s.okayama-u.ac.jp (Y.H.-K.); de17054@s.okayama-u.ac.jp (Y.Y.); gmd20103@s.okayama-u.ac.jp (Y.O.); akentaro@md.okayama-u.ac.jp (K.A.); kuboki@md.okayama-u.ac.jp (T.K.) 1 Department of Oral Rehabilitation and Regenerative Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama 700-8558, Japan; pth_uytki@yahoo.com (H.T.P.); thuharhm@gmail.com (H.T.T.N.); ppjj5xuj@s.okayama-u.ac.jp (A.T.D.); pa62884x@s.okayama-u.ac.jp (H.T.D.); de19016@s.okayama-u.ac.jp (T.K.); de421035@s.okayama-u.ac.jp (I.T.); de19034@s.okayama-u.ac.jp (Y.H.-K.); de17054@s.okayama-u.ac.jp (Y.Y.); gmd20103@s.okayama-u.ac.jp (Y.O.); akentaro@md.okayama-u.ac.jp (K.A.); kuboki@md.okayama-u.ac.jp (T.K.) 2 Faculty of Dentistry, Hai Phong University of Medical and Pharmacy, Haiphong 04211, Vietnam 3 Department of Molecular Biology and Biochemistry, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama 700-8558, Japan y y p 4 Department of Biomaterials, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama 700-8558, Japan * Correspondence: mitsuaki@md.okayama-u.ac.jp (M.O.); gmd421209@s.okayama-u.ac.jp (E.S.H.); Tel.: +81-86-235-7127 (M.O.); +81-86-235-6667 (E.S.H.); Fax: +81-86-222-7768 (M.O.); +81-86-235-6669 (E.S.H.) materials materials Citation: Pham, H.T.; Ono, M.; Hara, E.S.; Nguyen, H.T.T.; Dang, A.T.; Do, H.T.; Komori, T.; Tosa, I.; Hazehara-Kunitomo, Y.; Yoshioka, Y.; et al. Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo. Materials 2021, 14, 208. https:// doi.org/10.3390/ma14010208 Received: 1 December 2020 Accepted: 21 December 2020 Published: 4 January 2021 1. Introduction Aging has been associated with a decline in the function of multiple organs due to degenerative changes in tissue-speciﬁc stem cells or stem cell niches, as well as decreased bone metabolism and osteoporosis, which are high-risk factors for bone fractures [1,2]. The current and unprecedented super-aging of the population requires urgent adaptation in the health care system worldwide. In this context, preventive approaches focusing on antiaging therapies or the treatment of age-related diseases or dysfunctions have attracted researchers to search for novel management and therapeutic approaches, including stem cell therapy. Therefore, a deeper understanding of the molecular mechanisms involved in the age-dependent deterioration of stem cell function is of critical importance for the development of new methods or materials for prevention or treatment of aging-related diseases or dysfunctions. y Amino acids are organic compounds containing amine (–NH2) and carboxylic acid (–COOH) functional groups. There are more than 500 amino acids in nature [3,4]; however, only 22 of them can be found in the human body, which are termed as 22 common amino acids, and include nine essential (need to be obtained from the diet) and 13 non-essential (synthesized in the body) amino acids [5]. Amino acids pass through the cell membrane via the amino acid transporter [6], and can be used for protein synthesis, and/or be degraded into one of seven common metabolic intermediates that can be further converted into glucose or oxidized by the citric acid cycle [7]. Additionally, amino acids play fundamental roles in processes such as neurotransmitter transport and gene transcription [8]. p p g p Amino acids are widely known as notable antiaging agents due to their abilities to promote tissue repair and cell renewal. For instance, arginine derived from nitric hoxide was proven to be essential to healthy skin [9], and glutamine regulates the body’s pH, thereby inﬂuencing the regeneration of cells and the synthesis of bone-protective antioxidants [10]. A recent report also showed that large amounts of methionine are required for the maintenance of stemness and differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) [11]. Furthermore, considerable evidence suggests that the regulation of stem cell renewal and differentiation was partially modulated by the modiﬁcation of metabolic and nutrient-sensing pathway [12]. In summary, L-tryptophan enhanced the stemness and osteoblastic differentiation of BMSCs and may be used as an essential factor to maintain the stem cell properties and accelerate bone healing and/or prevent bone loss. Citation: Pham, H.T.; Ono, M.; Hara, E.S.; Nguyen, H.T.T.; Dang, A.T.; Do, H.T.; Komori, T.; Tosa, I.; Hazehara-Kunitomo, Y.; Yoshioka, Y.; et al. Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo. Materials 2021, 14, 208. https:// doi.org/10.3390/ma14010208 Received: 1 December 2020 Accepted: 21 December 2020 Published: 4 January 2021 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal afﬁliations. Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Keywords: amino acid; mesenchymal stromal cells; stemness; tryptophan; kynurenine; osteogenesis; adipogenesis; screening; injury/fracture healing; anabolics https://www.mdpi.com/journal/materials Materials 2021, 14, 208. https://doi.org/10.3390/ma14010208 2 of 15 Materials 2021, 14, 208 1. Introduction On the other hand, the lack of dietary amino acids causes several symptoms or dis- orders such as weight loss, muscle mass decrease, decline in infection protection, and especially in changing of bone tissue homeostasis [13]. It is proven that dietary amino acids inﬂuence positively bone health by reducing bone hip fracture, promoting postfracture recovery, and increasing bone strength [14]. The impact of certain amino acids or their metabolites on typical bone diseases, age-related osteoporosis has also been reported. Arginine, for instance, was shown to support bone formation by inducing collagen and fa- cilitating the growth of osteoblast [1,15]. In addition, a recent report has also demonstrated the importance of the kynurenine pathway during osteogenic differentiating of human mesenchymal stromal cells (hMSCs) [16]. A deeper understanding of whether amino acids can regulate both the stemness and differentiation ability towards osteogenesis and adipogenesis of bone marrow-derived stromal cells (hBMSCs) is important for future potential applications of amino acid sup- plementation as preventive approaches for aging-related dysfunctions of stem cell niches, particularly in the bone marrow. Therefore, we performed a screening of 22 common amino acids using human BMSCs (hBMSCs) and found that L-tryptophan could enhance stem cell markers, as well as osteogenic differentiation of BMSCs. Since L-kynurenine is the main and initial metabolite of L-tryptophan, we also investigated its effect on the cellular behavior of BMSCs in vitro and in vivo. The null hypothesis of this study was that treatment of L-tryptophan and L-kynurenine does not induce any changes in the stem cell phenotype of BMSCs. 3 of 15 Materials 2021, 14, 208 2.2. Differentiation Assays Osteogenesis: hBMSCs or mBMSCs were cultured up to 100% conﬂuence, and there- after induced to differentiate toward the osteogenic lineage by mineralization medium consisted of the respective human or mouse basal medium supplemented with 10 nM dexamethasone (Sigma-Aldrich), 2 mM β-glycerophosphate (Sigma-Aldrich), and 10 nM L-ascorbic acid phosphate, for 14 days. Cultures were then analyzed for gene expression of osteocalcin (OCN) and osteopontin (OPN) or stained for the calciﬁed matrix with 1% Alizarin Red S (Sigma-Aldrich) staining solution [19]. Adipogenesis: hBMSCs or mBMSCs were cultured up to 100% conﬂuence and then incubated in the respective human or mouse basal medium supplemented with 10 µg/mL insulin (Sigma), 0.5 mM 1-methyl-3-isobutylxanthine (Sigma), 60 µM indomethacin (Sigma), 0.5 µM Hydrocortisone (Sigma) for 14 days. After induction, cultures were analyzed for gene expression of Peroxisome proliferator-activated receptor gamma (PPAR-γ) and Lipoprotein lipase (LPL), or stained for oil droplets with 0.5% Oil Red O (Sigma) stain- ing solution [20]. 2.1. BMSC Culture Human BMSCs were purchased from Lonza (# PT-2501, Basel, Switzeland) and cul- tured under guidance of a previous protocol [17]. Brieﬂy, hBMSCs were cultured with basal medium, consisting of alpha-modiﬁed minimum essential medium (α-MEM, Invitrogen, Carlsbad, CA, USA), 15% fetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine (Invit- rogen), 100 units/mL penicillin (Sigma-Aldrich, Saint Louis, MO, USA) and 100 mg/mL streptomycin (Sigma), at 37 ◦C under 5% CO2 in air. p y g mBMSCs were obtained following a previous reported protocol with minor modiﬁca- tions [17,18]. mBMSCs were isolated from the femur by ﬂushing the bone marrow. The cells were collected by centrifugation and cultured in α-MEM containing 20% FBS, 1% antibiotics (penicillin 100 units/mL and streptomycin 100 mg/mL), 2 mM L-glutamine (In- vitrogen), 0.1 mM L-ascorbic acid phosphate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 55 µM β-mercaptoethanol (Life Technologies, Grand Island, NY, USA). All experiments were repeated at least three times. For pretreatment experiments with amino acid, BMSCs were cultured up to 60% conﬂuence and further incubated with 10 µM of each amino acid for two days, and then used for subsequent analysis. BMSCs from fourth to eighth passages were used in this study. An orphan ligand library (Enzo Life Sciences, Tokyo, Japan) containing 22 common D-isoform amino acids was used for the screening (Table S1). 2.3. Flow-Cytometry (FCM) Cultured hBMSCs or mBMSCs were dissociated with accutase (Inovative Cell Tech- nologies, CA, USA) and ﬁltered through a 70 µm cell strainer, washed, resuspended in phosphate-buffered saline (PBS) containing 1% FBS, and incubated with antihuman SSEA- 4 (eBioscience, San Diego, CA, USA) for 30 min on ice. Cells were washed again and subjected to FCM analysis by Accuri C6 (BD Biosciences, Franklin Lakes, NJ, USA) [21]. 2.7. Cell Viability A total of 1 × 104 hBMSCs were seeded onto 96 well plates and treated with amino acids for 48 h. Cell viability was assessed by a colorimetric assay based on the bio-reduction of a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt; MTS] by viable cells (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA), according to manufac- turer’s instructions. 2.8. Migration Assay Migration assay was performed according to the Boyden chamber method, using cell culture inserts with a light-opaque, polyethylene terephthalate, 8-µm, microporous membrane (BD Falcon HTS FluoroBlok inserts, BD Biosciences). BMSCs were dissociated with accutase, resuspended in basal medium and counted. Cells were then washed with PBS, centrifuged, and resuspended with serum-starved (1% FBS) medium. Cells were counted one more time, and 5000 cells contained in 250 µL of serum-starved medium were seeded in the upper chamber (cell insert), while in the lower chamber 750 µL of basal culture medium (15% FBS). After 24 h of incubation, cells were ﬁxed with 4% PFA, stained with 20 µg/mL of Alexa Fluor 546-conjugated Phalloidin (Invitrogen) overnight, washed with PBS, and observed under a ﬂuorescence microscope (BZ-X700, Keyence, Osaka, Japan). The total number of migrated cells observed at the bottom of the chamber was counted as an average of four different pictures taken per chamber [26]. 2.6. Immunocytochemistry A total of 1 × 104 hBMSCs were seeded onto 96-well plates and cultured in the presence of amino acids for 48 h in basal medium. The cells were subsequently ﬁxed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with PBS containing 0.25% Triton X-100 for 10 min, blocked with 5% normal goat serum (Invitrogen), and then incubated with primary antibody anti-CD146 (Abcam, Cambridge, MA, USA), anti-Ki-67 (Abcam) or the respective IgG (Abcam) overnight at 4 ◦C. Cells were then incubated with secondary antibody Alexa Fluor 488-conjugated IgG (Invitrogen) for 1 h at room temperature, and observed under a ﬂuorescence microscope (BZ-X700, KEYENCE, Osaka, Japan). Cell nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, Invitrogen) [25]. 2.5.2. Second Screening In the second screening, three candidate molecules (D-methionine, D-proline, D-tryptophan) were selected from the ﬁrst screening and used to treat hBMSCs for two days at dose of 10 µM. The total cellular RNA was extracted from the cells for analysis of the mRNA expression levels of the stem cell markers NANOG, SOX-2, and OCT-4 [24]. 2.5.1. First Screening For library screening, 22 D-isoform common amino acids from an orphan ligand library were selected and used to treat hBMSCs in culture for two days at a dose of 10 µM. The cells were then harvested and analyzed by FCM for the expression of the early stem cell surface marker, SSEA-4 [23]. 2.5. Screening Process 2.5. Screening Process 2.5.1. First Screening 2.4. Real-Time Reverse-Transcription Polymerase Chain Reaction (Real-Time RT-PCR) Total cellular RNA from cells was extracted by Purelink RNA mini kit (Invitrogen) according to the manufacturer’s instructions and puriﬁed by removing genomic DNA with RNase-Free DNase set (Purelink DNase, Invitrogen). The relative levels of mRNA of target genes were normalized to that of the reference gene ribosomal protein S29 [22]. Primer sequences are shown in Supplementary Table S2. All experiments were repeated at least three times, independently. Materials 2021, 14, 208 4 of 15 2.12. Histological Analysis Femurs were ﬁxed in 4% PFA for 2–3 days, kept in 70% ethanol before being decalciﬁed with Morse’s solution (22.5% formic acid-10% sodium citrate solution). Samples were then washed and dehydrated through a graded ethanol series and xylene before parafﬁn embedding. Five-micrometer sections were stained by Hematoxylin and Eosin (H&E) and observed under a microscope (Biozero BZ-X700) [21]. 2.9. Animal Experiments Five-week-old C57BL/6 mice were used in the experiments, according to the Guide- lines for Animal Research of Okayama University Dental School, under the approval of the Okayama University Ethical Committee (OKU-2013125). For analysis of the ef- fect of L-tryptophan or L-kynurenine in vivo, mice were injected intraperitoneally with 50 mg/kg/day of L-tryptophan (Sigma-Aldrich) or L-kynurenine sulfate (Sigma-Aldrich) for consecutive three weeks, and then femurs were dissected from mice for mBMSC isola- tion or further micro-CT and histological analysis. Materials 2021, 14, 208 5 of 15 For the surgical experiment, mice were anesthetized by inhalation of isoﬂurane (Isoﬂu: Dainippon Sumitomo Pharma Co., Osaka, Japan). The left lower limb was shaved and aseptically cleaned with 70% ethanol. An incision of approximately 15 mm in the frontal skin of the mid-femur was performed to expose the muscle. The muscle was then elevated and the periosteum was separated to expose the femur surface. A drill was used to make a surgical defect of 1 mm in diameter in the anterior portion of the diaphysis, 5 mm above the knee joint [27]. The diaphysis was irrigated with saline during the surgery. Thereafter, the muscles were replaced in the original position and the incision line was sutured. 2.11. Micro-Computed Tomographic Analysis Mouse femur defects were scanned by micro-computed tomography (micro-CT) at a resolution of 6.4 µm, and the bone volume in defect site was analyzed with Skyscan 1174 version 2 software (Nrecon, CTAn, CTvol, and CTvox), as described previously [29]. 2.10. Colony-Forming Assay Colony-forming unit-ﬁbroblast (CFU-F) assay was performed under two different protocols. In the ﬁrst one, 2 × 106 of mouse bone marrow cells collected from mouse femur were cultured in 6 cm2 culture dishes with or without L-tryptophan or L-kynurenine (10 µM) for three weeks. In the second protocol, mice were injected L-tryptophan or L- kynurenine or PBS continuously for three weeks, and then a total of 2 × 106 cells collected from the mice femurs were seeded onto 6 cm2 culture dishes and cultured for three weeks. After the 3-week culture period, the cells were then washed with PBS and stained with 0.1% toluidine blue contained in 1% PFA overnight. On the following day, dishes were washed to remove excess dye, and only stained clusters containing more than 50 cells were counted as colonies [28]. 2.13. Statistical Analysis Statistical analyses were performed with unpaired Student’s t-test or one-way ANOVA with Tukey correction tests. Prism GraphPad sofware version 5.0 (San Diego, CA, USA) was used for the analyses. A statistically signiﬁcant difference was considered as p < 0.05. 3.1. Screening Process 3.1. Screening Process 3.1. Screening Process In the ﬁrst screening, three candidates (D-methionine, D-proline, D-tryptophan) dif- ferentially increased the percentage of hBMSCs positive for SSEA-4 after 48 h of treatment (Figure 1A). Notably, however, in the second screening, gene expression analysis showed a higher increase in the mRNA levels of the stem cell markers NANOG, SOX-2, and OCT-4 upon D-tryptophan treatment, compared to D-methionine or D-proline (Figure 1B). Therefore, D-tryptophan was selected for the subsequent steps. 6 of 15 6 of 16 Materials 2021, 14, 208 Materials 2021 14 x FO Figure 1. Screening process. (A) First screening by flow cytometry. Three candidate amino acids were selected (red columns): D-methionine (D-Met), D-proline (D-Pro), and D-tryptophan (D-Tr The experiment was performed once. (B) Second screening by real-time RT-PCR. D-tryptophan (D-Trp) strongly enhanced the mRNA levels of stem cell markers, NANOG, OCT-4, and SOX-2. Results are representative of two independent experiments. (C, D) hBMSCs were treated with fo different concentrations (2, 10, 50, and 100 μM) of D-Trp for 48 h, and 10 μM concentration in- duced the highest increase in gene expression levels of NANOG, OCT-4 and SOX2 (C) and the number of SSEA 4 positive cells (D) Results are representative of at least two independent expe Figure 1. Screening process. (A) First screening by ﬂow cytometry. Three candidate amino acids were selected (red columns): D-methionine (D-Met), D-proline (D-Pro), and D-tryptophan (D-Trp). The experiment was performed once. (B) Second screening by real-time RT-PCR. D-tryptophan (D-Trp) strongly enhanced the mRNA levels of stem cell markers, NANOG, OCT-4, and SOX-2. Results are representative of two independent experiments. (C,D) hBMSCs were treated with four different concentrations (2, 10, 50, and 100 µM) of D-Trp for 48 h, and 10 µM concentration induced the highest increase in gene expression levels of NANOG, OCT-4 and SOX2 (C) and the number of SSEA-4 positive cells (D). Results are representative of at least two independent experiments. ( p < 0.01, one-way ANOVA/Tukey). Figure 1. Screening process. (A) First screening by flow cytometry. Three candidate amino acid were selected (red columns): D-methionine (D-Met), D-proline (D-Pro), and D-tryptophan (D-T The experiment was performed once. (B) Second screening by real-time RT-PCR. D-tryptophan (D-Trp) strongly enhanced the mRNA levels of stem cell markers, NANOG, OCT-4, and SOX-2 Results are representative of two independent experiments. 3.1. Screening Process (C, D) hBMSCs were treated with f different concentrations (2, 10, 50, and 100 μM) of D-Trp for 48 h, and 10 μM concentration in- duced the highest increase in gene expression levels of NANOG, OCT-4 and SOX2 (C) and the number of SSEA-4 positive cells (D). Results are representative of at least two independent exp Figure 1. Screening process. (A) First screening by ﬂow cytometry. Three candidate amino acids were selected (red columns): D-methionine (D-Met), D-proline (D-Pro), and D-tryptophan (D-Trp). The experiment was performed once. (B) Second screening by real-time RT-PCR. D-tryptophan (D-Trp) strongly enhanced the mRNA levels of stem cell markers, NANOG, OCT-4, and SOX-2. Results are representative of two independent experiments. (C,D) hBMSCs were treated with four different concentrations (2, 10, 50, and 100 µM) of D-Trp for 48 h, and 10 µM concentration induced the highest increase in gene expression levels of NANOG, OCT-4 and SOX2 (C) and the number of SSEA-4 positive cells (D). Results are representative of at least two independent experiments. ( p < 0.01, one-way ANOVA/Tukey). ments. (** p < 0.01, one-way ANOVA/Tukey). In order to determine the optimal effective dosage of D-Tryptophan for hBMSCs, we analyzed four concentrations of D-tryptophan (2, 10, 50, and 100 μM). As shown in Figure 1C, 10 μM of D-tryptophan yielded the highest increase in mRNA levels of NANOG, OCT- 4, and SOX2 in hBMSCs. This result was also confirmed by FCM analysis, which showed the highest increase in the number of cells positive for SSEA-4 upon treatment with 10 μM of D-tryptophan (Figure 1D). Therefore, we selected 10 μM as the optimal concentration In order to determine the optimal effective dosage of D-Tryptophan for hBMSCs, we analyzed four concentrations of D-tryptophan (2, 10, 50, and 100 µM). As shown in Figure 1C, 10 µM of D-tryptophan yielded the highest increase in mRNA levels of NANOG, OCT-4, and SOX2 in hBMSCs. This result was also conﬁrmed by FCM analysis, which showed the highest increase in the number of cells positive for SSEA-4 upon treatment with 10 µM of D-tryptophan (Figure 1D). Therefore, we selected 10 µM as the optimal concentration for our cells. 7 of 15 Materials 2021, 14, 208 Tryptophan is an essential amino acid that needs to be taken from the diet. It exists in two mirror forms, i.e., L- and D- isomers. 3.1. Screening Process Therefore, we compared the effect of the two isomers on the stem cell phenotype of hBMSCs. As shown in Table 1, L-tryptophan showed similar effects compared to D-isomer in increasing the number of SSEA-4 positive cells and the gene expression levels of NANOG, SOX2, and OCT-4 in hBMSCs. Additionally, immunoﬂuorescence staining demonstrated an increase in the expression of CD146, which is one major marker of BMSCs, after L-tryptophan treatment (Figure S1). Based on these results, and taking into consideration that L-tryptophan is the most common isomer in natural food and the isomer used for protein synthesis in the body [30], L-tryptophan was then selected for the subsequent experiments, at a concentration of 10 µM. Table 1. Comparison effect of L- and D-tryptophan. Both amino acids induced an increase in the percentage of SSEA-4 positive cells (FCM analysis) and mRNA levels of stem cell markers as NANOG, SOX2, OCT-4, compared to the control group. The value is the ratio between treated and control groups. The results are representative of at least three independent experiments. * p < 0.05, p < 0.01, * p < 0.001, ns: non-signiﬁcant, unpaired t-test. Stem Cell Marker Amino Acid D-Trp (10 µM) L-Trp (10 µM) SSEA-4+ cells 1.39 ± 0.03 (**) 1.21 ± 0.08 () NANOG mRNA levels 2.25 ± 0.59 (*) 3.57 ±0.96 () OCT-4 mRNA levels 1.23 ± 0.43 (NS) 1.78 ± 0.86 (NS) SOX2 mRNA levels 1.45 ± 0.34 () 2.74 ± 1.07 () 3.2. L-Tryptophan Enhances Migration, Colony Formation and Osteogenic Differentiation of hBMSCs In Vitro 3.2. L-Tryptophan Enhances Migration, Colony Formation and Osteogenic Differentiation of hBMSCs In Vitro 3.2. L-Tryptophan Enhances Migration, Colony Formation and Osteogenic Differentiation of hBMSCs In Vitro We then investigated the effect of L-tryptophan on other cellular activities of BMSCs, including colony formation, cell migration, and differentiation towards osteogenic and adipogenic lineages. Interestingly, treatment with L-tryptophan signiﬁcantly enhanced the formation of mBMSC colonies (Figure 2A). Next, we examined the effect of L-tryptophan on cell migration and proliferation. As shown in Figure 2B, L-tryptophan-treated hBMSCs exhibited a higher ability for migration than the control group; however, L-tryptophan treatment induced no signiﬁcant changes in the number of cells positive for the cell prolif- eration marker Ki-67 (Figure S2A,B), or in the cell proliferation (viability) analyzed by MTS assay (Figure S2C). 3.1. Screening Process y g Regarding the differentiation ability of hBMSCs, L-tryptophan enhanced osteogen- esis as demonstrated by stronger staining of the mineralized matrix with Alizarin Red S (Figure 2C); as well as by an increase in mRNA levels of osteogenic markers OPN and OCN (Figure 2D). Interestingly, L-tryptophan suppressed adipogenesis as can be seen in Oil Red O staining of cell cultures, and decreased mRNA levels of adipogenic markers PPARγ and LPL (Figure 2E,F). 3.3. L-Tryptophan Enhances Migration, Colony Formation and Osteogenic Differentiation of mBMSCs In Vivo Next, in order to analyze the effect of L-tryptophan in vivo, we injected 10 mg/kg/day or 50 kg/mg/day of L-tryptophan in mice intraperitoneally for consecutive three weeks. Mice femurs were then dissected for isolation of mBMSCs in culture dishes for subsequent assays. After 21 days of in vitro culture, mBMSCs were submitted to FCM analysis, which showed that the concentration of 50 mg/kg/day induced a signiﬁcant increase in the number of SSEA-4 positive cells, whereas 10 mg/kg/day induced no signiﬁcant changes (Figure 3A). Consistently, L-tryptophan (50 kg/mg/day) increased the mRNA levels of Nanog and Oct-4 (Figure 3B) and the colony-forming ability of mBMSCs, compared to the PBS-injected control group (Figure 3C). We also examined the differentiation ability of mBMSCs toward osteogenesis and adipogenesis. Consistent with the in vitro results with Materials 2021, 14, 208 8 of 15 hBMSCs, L-tryptophan enhanced the deposition of the mineralized matrix as shown by Alizarin Red S staining (Figure 3D), as well as the mRNA levels of Opn and Ocn (Figure 3E). On the other hand, there was no signiﬁcant difference in the adipogenic differentiation of mBMSCs after L-tryptophan injection, as analyzed by Oil Red O staining and mRNA levels of Pparγ and Lpl (Figure 3F,G). Taken together, these results indicate that L-tryptophan can promote an increase in stemness and osteogenic ability of BMSCs in vitro and in vivo. hBMSCs, L-tryptophan enhanced the deposition of the mineralized matrix as shown by Alizarin Red S staining (Figure 3D), as well as the mRNA levels of Opn and Ocn (Figure 3E). On the other hand, there was no signiﬁcant difference in the adipogenic differentiation of mBMSCs after L-tryptophan injection, as analyzed by Oil Red O staining and mRNA levels of Pparγ and Lpl (Figure 3F,G). Taken together, these results indicate that L-tryptophan can promote an increase in stemness and osteogenic ability of BMSCs in vitro and in vivo. promote an increase in stemness and osteogenic ability of BMSCs in vitro and in vivo. Figure 2. Effect of L-tryptophan on the stemness-related function and differentiation capability of hBMSCs in vitro. (A) CFU-F assay was performed using primary mBMSCs. The colony-forming potential was increased by the treatment of L-Trp. Data represent the means ± SD (n = 3). p < 0.01, unpaired t-test. (B) L-Trp induced higher migration ability compared to the control group. The images are representatives of three independent experiments. 3.3. L-Tryptophan Enhances Migration, Colony Formation and Osteogenic Differentiation of mBMSCs In Vivo Figure 3. Effect of L-tryptophan on the stem cell phenotype of mBMSCs and trabecular bone in vivo. (A of L-tryptophan on the stem cell phenotype of mBMSCs and trabecular bone in vivo. (A) FCM analysis of the percentage of mBMSCs positive for SSEA-4. mBMSCs from L-Trp (50 mg/kg)-treated mice showed a signiﬁcantly higher number of SSEA-4 positive cells than the control group. * p < 0.05, one-way ANOVA/Turkey. (B) Gene expression levels of Nanog and Oct-4 in mBMSCs from 50 mg/kg L-Trp-treated mice was signiﬁcantly higher than mBMSCs from the PBS-injected control group. Data represent the mean ± SD (n = 3). * p < 0.05, *** p < 0.001, unpaired t-test. (C) Treatment with L-Trp increased the colony-forming ability of mBMSCs. Data represent the mean ± SD (n = 3). * p < 0.05, unpaired t-test. (D,E) Differentiation ability. L-Trp induced stronger deposition of the calciﬁed matrix as detected by Alizarin Red S staining (D) as well as an increase in the mRNA levels of Opn and Ocn on mBMSCs (E). No substantial change in Oil Red O staining (F) or gene expression levels of Ppar-γ or Lpl (G) could be observed upon L-Trp treatment. * p < 0.05, * p < 0.001, unpaired test. Images and graphs are representative of at least three independent experiments. 3.3. L-Tryptophan Enhances Migration, Colony Formation and Osteogenic Differentiation of mBMSCs In Vivo Graph shows the average number of migrated cells counted in four different pictures taken at each chamber. p < 0.01, unpaired t-test. Pre-treatment of L-Trp induced osteogenic differentiation of hBMSCs (C,D). L-Trp decreased the adipogenic ability of hBMSCs (E,F). * p < 0.05, p < 0.01, unpaired t-test. Images and graphs are representatives of at least three independent experiments. Figure 2. Effect of L-tryptophan on the stemness-related function and differentiation capability of hBMSCs in vitro. (A) CFU-F assay was performed using primary mBMSCs. The colony-forming potential was increased by the treatment of L-Trp. Data represent the means ± SD (n = 3). p < 0.01, unpaired t-test. (B) L-Trp induced higher migration ability compared to the control group. The images are representatives of three independent experiments. Graph shows the average number of migrated cells counted in four different pictures taken at each chamber. ** p < 0.01, unpaired t-test. Pre-treatment of L-Trp induced osteogenic differentiation of hBMSCs (C,D). L-Trp decreased the adipogenic ability of hBMSCs (E,F). * p < 0.05, ** p < 0.01, unpaired t-test. Images and graphs are representatives of at least three independent experiments. 9 of 15 Materials 2021, 14, 208 Figure 3. Effect of L-tryptophan on the stem cell phenotype of mBMSCs and trabecular bone in vivo. (A) FCM analysis of the percentage of mBMSCs positive for SSEA-4. mBMSCs from L-Trp (50 mg/kg)-treated mice showed a signiﬁcantly higher number of SSEA-4 positive cells than the control group. * p < 0.05, one-way ANOVA/Turkey. (B) Gene expression evels of Nanog and Oct-4 in mBMSCs from 50 mg/kg L-Trp-treated mice was signiﬁcantly higher than mBMSCs from the PBS-injected control group. Data represent the mean ± SD (n = 3). * p < 0.05, *** p < 0.001, unpaired t-test. (C) Treatment with L-Trp increased the colony-forming ability of mBMSCs. Data represent the mean ± SD (n = 3). * p < 0.05, unpaired -test. (D,E) Differentiation ability. L-Trp induced stronger deposition of the calciﬁed matrix as detected by Alizarin Red S staining (D) as well as an increase in the mRNA levels of Opn and Ocn on mBMSCs (E). No substantial change in Oil Red O staining (F) or gene expression levels of Ppar-γ or Lpl (G) could be observed upon L-Trp treatment. * p < 0.05, * p < 0.001, unpaired test. Images and graphs are representative of at least three independent experiments. 3.4. L-Kynurenine, A Main Metabolite of L-Tryptophan, Enhances the Stem Cell Phenotype of hBMSCs Previous investigations reported that 99% of L-tryptophan is catalyzed by the en- zymes tryptophan 2,3 dioxygenase (TDO) and Indoleamine 2, 3-dioxygenase (IDO1) and subsequently metabolized through the L-kynurenine pathway [30,31]. Therefore, since L-kynurenine is the main metabolite of L-tryptophan, we evaluated the effects of L- Materials 2021, 14, 208 10 of 15 10 of 15 kynurenine on the stem cell phenotype and differentiation ability of hBMSCs. As expected, in vitro analyses showed that L-kynurenine induced similar effects of those promoted by L-tryptophan, including an increase in the number of cells positive for SSEA-4 (Figure 4A), in gene expression levels of NANOG, SOX2, and OCT-4 (Figure 4B), as well as in the ability of mBMSCs to form colonies (Figure 4C) and to migrate (Figure 4D). Figure 4E shows that L-kynurenine also enhanced osteogenesis of hBMSCs as demonstrated by stronger staining of the calciﬁed matrix with Alizarin Red S, and the increase in OPN and OCN mRNA levels. On the other hand, no signiﬁcant changes in adipogenic differentiation of hBMSCs were observed upon L-kynurenine treatment, as analyzed by Oil Red O staining of oil droplets, and gene expression levels of PPARγ and LPL (Figure 4H). kynurenine on the stem cell phenotype and differentiation ability of hBMSCs. As expected, in vitro analyses showed that L-kynurenine induced similar effects of those promoted by L-tryptophan, including an increase in the number of cells positive for SSEA-4 (Figure 4A), in gene expression levels of NANOG, SOX2, and OCT-4 (Figure 4B), as well as in the ability of mBMSCs to form colonies (Figure 4C) and to migrate (Figure 4D). Figure 4E shows that L-kynurenine also enhanced osteogenesis of hBMSCs as demonstrated by stronger staining of the calciﬁed matrix with Alizarin Red S, and the increase in OPN and OCN mRNA levels. On the other hand, no signiﬁcant changes in adipogenic differentiation of hBMSCs were observed upon L-kynurenine treatment, as analyzed by Oil Red O staining of oil droplets, and gene expression levels of PPARγ and LPL (Figure 4H). Figure 4. Effect of L-kynurenine on the stemness, function and differentiation capability of human bone marrow-derived mesenchymal stromal cells in vitro. (A) Flow cytometry showed the cells cultured with L-Kyn had a higher percentage positive with SSEA-4+ cells compared to control samples. (B) L-Kyn increased the gene expression levels of NANOG, OCT-4, SOX2. p < 0.01, *** p < 0.001, unpaired t-test. 3.4. L-Kynurenine, A Main Metabolite of L-Tryptophan, Enhances the Stem Cell Phenotype of hBMSCs Both L-Tryptophan and L-Kynurenine Accelerate Bone Regeneration in Mouse Femur Surgical Defect 3.5. Both L-Tryptophan and L-Kynurenine Accelerate Bone Regeneration in Mouse Femur Surgical Defect We have previously demonstrated that during the inﬂammatory phase of wound healing, the inﬂammatory cytokine, tumor necrosis factor-α (TNF-α), enhanced the stem cell phenotype of MSCs [20]. Additionally, we also showed that MSCs intensively migrate to the wound site during the inﬂammatory phase of healing (initial two to three days post-trauma) to accelerate bone healing [20,32]. Based on these previous data, as well as on the fact that L-tryptophan and L-kynurenine enhanced the stemness and migration ability of BMSCs, we hypothesized that in vivo treatment with the two amino acids could also enhance bone healing. Therefore, we used a mouse model of femur surgical defect and administrated L-tryptophan or L-kynurenine (50 mg/kg/day) or PBS by intraperitoneal injections one week before the surgical defect was made, and subsequently for two weeks after the surgery (Figure 5A). As demonstrated by micro-CT analysis and quantitative mea- surements, we observed an enhanced bone regeneration both in L-tryptophan (Figure 5B) and L-kynurenine-treated groups (Figure 5D), compared to their respective PBS-injected control group. Moreover, histological sections further conﬁrmed that L-tryptophan and L-kynurenine accelerated new bone formation (Figure 5C,E). Figure 5. Effect of tryptophan or kynurenine on bone regeneration in mice femur surgical defect. (A) Schema showing the study design. Mice were then administered the two amino acids for 2 more weeks. (B,D) Three-dimensional micro-CT images of mouse femur defects after intraperitoneal injection with PBS or L-Trp (50 mg/kg) or L-Kyn (50 mg/kg). The bone healing ability after three weeks of L-Trp or L-Kyn-treated group was accelerated compared to the respective PBS-injected control group. The analysis was performed with Skyscan software. The images are representative of four different samples. The white arrow indicates the defect. (C,E) Histological analysis of mouse femurs in the defect area. Note the amount of regenerated bone volume in L-Trp (C) and L-Kyn-treated (E) groups. Data represent the mean ± SD (n = 4). * p < 0.05, ** p < 0.01, unpaired t-test. Figure 5. Effect of tryptophan or kynurenine on bone regeneration in mice femur surgical defect. (A) Schema showing the study design. Mice were then administered the two amino acids for 2 more weeks. (B,D) Three-dimensional micro-CT images of mouse femur defects after intraperitoneal injection with PBS or L-Trp (50 mg/kg) or L-Kyn (50 mg/kg). 3.4. L-Kynurenine, A Main Metabolite of L-Tryptophan, Enhances the Stem Cell Phenotype of hBMSCs Data are representative of at least three independent experiments. (C) A higher number of colonies was observed in L-Kyn-treated group. Data represent the mean ± SD (n = 3). * p < 0.05, unpaired t-test. (D) hBMSCs treated with L-Kyn presented higher migration ability. Images are representative of three independent experiments. Scale bar = 100 µM. Graph shows the average number of migrated cells counted in four different pictures taken in each chamber. ** p < 0.01, unpaired t-test. (E,F) L-Kyn-treated hBMSCs presented stronger deposition of the calciﬁed matrix as detected by Alizarin Red S staining (E) as well as an increase in the mRNA levels of Opn and Ocn (F). No change in adipogenesis of hBMSC was observed after treatment with L-Kyn (G,H). * p < 0.05, * p < 0.001, unpaired t-test. Images and graphs are representative of at least three independent experiments. Figure 4. Effect of L-kynurenine on the stemness, function and differentiation capability of human bone marrow-derived mesenchymal stromal cells in vitro. (A) Flow cytometry showed the cells cultured with L-Kyn had a higher percentage positive with SSEA-4+ cells compared to control samples. (B) L-Kyn increased the gene expression levels of NANOG, OCT-4, SOX2. p < 0.01, *** p < 0.001, unpaired t-test. Data are representative of at least three independent experiments. (C) A higher number of colonies was observed in L-Kyn-treated group. Data represent the mean ± SD (n = 3). * p < 0.05, unpaired t-test. (D) hBMSCs treated with L-Kyn presented higher migration ability. Images are representative of three independent experiments. Scale bar = 100 µM. Graph shows the average number of migrated cells counted in four different pictures taken in each chamber. ** p < 0.01, unpaired t-test. (E,F) L-Kyn-treated hBMSCs presented stronger deposition of the calciﬁed matrix as detected by Alizarin Red S staining (E) as well as an increase in the mRNA levels of Opn and Ocn (F). No change in adipogenesis of hBMSC was observed after treatment with L-Kyn (G,H). * p < 0.05, *** p < 0.001, unpaired t-test. Images and graphs are representative of at least three independent experiments. Taken together, these results suggest that L-kynurenine is the major pathway associ- ated with the effect of L-tryptophan on promoting the increase in stemness and osteogenic ability of BMSCs. 11 of 15 11 of 15 Materials 2021, 14, 208 3.5. 4. Discussion The involvement of amino acids in aging and bone homeostasis, as well as in the maintenance and differentiation of iPS cells as outlined in recent reports, strongly supports the notion that amino acids are essential for the regulation of maintenance and differ- entiation of adult stem cells. In our screening of over 22 amino acids, tryptophan was shown to differentially enhance the expression of stem cell markers in hBMSCs in vitro, and also maintain the stemness of BMSCs in vivo. Additionally, we demonstrated that L-tryptophan can enhance osteogenesis of BMSCs and bone regeneration in vivo. These results are in accordance with previous reports showing that the tryptophan-free diet decreased body weight and delayed femoral bone growth and bone mineral density in rats [33]. Therefore, tryptophan has crucial roles in the maintenance of stemness of BMSCs and bone homeostasis. The in vitro and in vivo results showing enhanced stemness of BMSCs and enhanced bone formation upon L-tryptophan stimulation may seem contradictory. However, since BMSCs consist of a heterogenic population, the effect of L-tryptophan could be both on stem/progenitor cells and osteoblastic cells. Moreover, the enhanced stem cell phenotype of BMSCs by the amino acid treatment could, in a latter step, promote a stronger differen- tiation of the cells. However, it is not clear from the present results whether the effect of L-tryptophan on inducing no signiﬁcant changes or an inhibition of the adipogenic differ- entiation of BMSCs was naturally due to a direct effect of the amino acid in suppressing the expression of master genes of adipogenic differentiation (e.g., PPARγ) or an indirect effect due to activation of the master genes of osteoblastic differentiation (e.g., RUNX2), which, consequently, would activate the osteoblast-adipocyte switch towards the osteoblastic lineage. Future studies investigating the possible regulation of this molecular switch by amino acids may provide a deeper understanding of the mechanisms of L-tryptophan associated inhibition of adipogenic differentiation. L-tryptophan passes through the cell membrane via the L-amino acid transporter system, which is the major route for providing the living cells with essential amino acids [6]. Tryptophan can be then metabolized into two main pathways (serotonin or kynurenine pathways). It has been reported that serotonin has either an inhibitory or stimulatory effect on bone growth, depending on whether it originated from the gut or the central nervous system (acting as a neurotransmitter), respectively [34–36]. 3.4. L-Kynurenine, A Main Metabolite of L-Tryptophan, Enhances the Stem Cell Phenotype of hBMSCs The bone healing ability after three weeks of L-Trp or L-Kyn-treated group was accelerated compared to the respective PBS-injected control group. The analysis was performed with Skyscan software. The images are representative of four different samples. The white arrow indicates the defect. (C,E) Histological analysis of mouse femurs in the defect area. Note the amount of regenerated bone volume in L-Trp (C) and L-Kyn-treated (E) groups. Data represent the mean ± SD (n = 4). * p < 0.05, ** p < 0.01, unpaired t-test. Materials 2021, 14, 208 12 of 15 12 of 15 4. Discussion Importantly, only 1% of tryptophan is metabolized into the serotonin pathway, while the majority of 99% of tryptophan is degraded through the kynurenine pathway [31,37] by the TDO and IDO1 enzymes. In addition, in in vitro assays of osteogenic differentiation of hBMSCs, no changes in serotonin levels in the supernatant of hBMSCs culture medium could be detected, while the kynurenine pathway was shown to be strongly activated [16]. y p y g y In this context, we considered L-kynurenine as the major metabolite of tryptophan and then proceeded with the next experiments investigating the effects of L-kynurenine on the stemness of hBMSCs. As expected, we found that L-kynurenine, similar to L-tryptophan, enhanced the stem cell phenotype of BMSCs in vitro and in vivo. These ﬁndings are also in agreement with those of a recent study using cancer cells, that reported that L- kynurenine acts as an endogenous ligand of the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) and can subsequently activate the transcription level of the master pluripotency factor Oct-4 [8]. Additionally, in accordance with previous reports, we also demonstrated that L- kynurenine enhances osteogenesis and bone formation in vivo [16]. A previous clinical epidemiological study also found high serum levels of kynurenine to be associated with lowerer BMD and risk of hip fracture in elderly individuals [38,39]. Importantly, the deletion of IDO-1, the major enzyme degrading tryptophan to kynurenine, markedly reduced the osteoblast number and bone volume in the knockout mice [16], indicating, therefore, the potential role of kynurenine as the major downstream pathway of tryptophan for the maintenance of bone homeostasis. Materials 2021, 14, 208 13 of 15 13 of 15 Regarding the process of stem cell aging, recent evidence indicated that the main- tenance of stem cell function and differentiation is modulated by mTORC1 pathway or by the modiﬁcation of the nutrient-sensing pathways, such as AMPK [12]. In an attempt to ﬁnd other cellular pathways that could be associated with the increase in stemness of hBMSCs, we performed a pathway array and an RNA array for stem cell signaling genes; however, we could identify no molecules involved in the upregulation of stem cell markers in hBMSCs (data not shown). Future studies using different approaches or analyzing changes at a single cell level may provide a deeper understanding of the intracellular mechanisms of amino acid-regulation of stemness. 5. Conclusions In summary, our data provide evidence of the novel effect of L-tryptophan and L- kynurenine in increasing the stem cell phenotype of BMCSs, which could further implicate in the maintenance of bone homeostasis. Further understanding of the molecular signaling involved in these effects may also elucidate the processes underlying stem cell aging. Finally, L-tryptophan and/or L-kynurenine could be potential targets for the development of novel materials and therapeutics for the aging-related decline in stem cell properties of tissue-speciﬁc stem/progenitor cells, and bone loss, including osteoporosis. Supplementary Materials: The following are available online at https://www.mdpi.com/1996-194 4/14/1/208/s1, Figure S1: Effect of L-Tryptophan on the stem cell phenotype of hBMSCs, Figure S2: Effect of L-Tryptophan on cell cycle and cell viability, Table S1: List of D-isomer amino acids included in the orphan ligand library that was used in the screening, Table S2: List of primer sequences used for quantitative analysis of gene expression levels by real-time RT-PCR. Author Contributions: Conceptualization, M.O. and E.S.H.; Data curation, H.T.P.; Formal analysis, H.T.P.; Funding acquisition, M.O., E.S.H., Y.H.-K. and T.K (Takuo Kuboki); Investigation, H.T.T.N., A.T.D., H.T.D., T.K. (Taishi Komori), I.T., Y.H.-K., Y.Y., Y.O. and K.A.; Methodology, M.O.; Project administration, M.O. and E.S.H.; Supervision, K.A. and T.K. (Takuo Kuboki). Validation, H.T.T.N., A.T.D., H.T.D. and I.T.; Writing—original draft, H.T.P., M.O., E.S.H., H.T.T.N., A.T.D., H.T.D., T.K. (Taishi Komori), I.T., Y.H.-K., Y.Y. and Y.O.; Writing—review & editing, M.O., E.S.H., K.A. and T.K. (Takuo Kuboki). All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI grant numbers (JP19K19092, JP20K21679, JP20H04534). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Okayama University (OKU- 2013125, April 2013). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Okayama University (OKU- 2013125, April 2013). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Acknowledgments: This work was supported by Central Research Laboratory of Okayama Univer- sity Medical School (Okayama, Japan). Conﬂicts of Interest: The authors declare that there is no conﬂict of interest. J.M.; Tymoczko, J.L.; Stryer, L.; Stryer, L. Biochemistry, 5th ed.; W.H. Freeman: New York, NY, USA, 2002. agner, I.; Musso, H. New naturally-occurring amino-acids. Angew. Chem. Int. Ed. 1983, 22, 816–828. [CrossR de, D.R. Handbook of Chemistry and Physics, 72nd ed.; CRC Press: Boca Raton, FL, USA, 1991. 6. Christensen, H.N. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol. Rev. 1990, 70, 43–77. [CrossRef] [PubMed] References A practical guide to culturing mouse and human bone marrow stromal cells. Curr. Protoc. Immunol. 2013, 22F, 1–13. 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https://openalex.org/W4297787607	https://eprints.lancs.ac.uk/id/eprint/168744/1/fulltext.pdf	English	null	The Economic Burden of Overweight and Obesity in Saudi Arabia	The World Bank eBooks	2,022	cc-by	4,638	PLOS ONE RESEARCH ARTICLE The economic burden of overweight and obesity in Saudi Arabia Jesse D. MalkinID1, Drishti Baid2, Reem F. Alsukait3,4, Taghred Alghaith5, Mohammed Alluhidan5,6, Hana Alabdulkarim7, Abdulaziz Altowaijri8, Ziyad S. AlmalkiID9, Christopher H. Herbst4, Eric Andrew Finkelstein10, Sameh El-SahartyID11, Nahar Alazemi5 Jesse D. MalkinID1, Drishti Baid2, Reem F. Alsukait3,4, Taghred Alghaith5, , , , g g , Mohammed Alluhidan5,6, Hana Alabdulkarim7, Abdulaziz Altowaijri8, Ziyad S. AlmalkiID9, Christopher H. Herbst4, Eric Andrew Finkelstein10, Sameh El-SahartyID11, Nahar Alazemi5 1 World Bank Group Consultant, Colorado Springs, Colo., United States of America, 2 Sol Price School of Public Policy, University of Southern California, Los Angeles, Calif., United States of America, 3 Community Health Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia, 4 Health, Nutrition and Population Global Practice, World Bank, Riyadh, Saudi Arabia, 5 Saudi Health Council, Riyadh, Saudi Arabia, 6 Lancaster University, Lancaster, United Kingdom, 7 Drug Policy and Economic Centre, Ministry of National Guards Health Affairs, Riyadh, Saudi Arabia, 8 Program for Health Assurance and Purchasing, Ministry of Health, Riyadh, Saudi Arabia, 9 Department of Clinical Pharmacy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudi Arabia, 10 Duke-NUS Medical School, Singapore, Singapore, 11 Health, Nutrition and Population Global Practice, World Bank, Kuwait City, Kuwait a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * jesse.malkin@gmail.com Settings and design The cost of overweight and obesity in Saudi Arabia was estimated from a societal perspec- tive using an epidemiologic approach. Copyright: © 2022 Malkin 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. Statistical analysis used Data Availability Statement: The minimum data set is held in the figshare public respository. https://figshare.com/articles/dataset/The_ economic_burden_of_overweight_and_obesity_ in_Saudi_Arabia/18677513. Data Availability Statement: The minimum data set is held in the figshare public respository. https://figshare.com/articles/dataset/The_ economic_burden_of_overweight_and_obesity_ in_Saudi_Arabia/18677513. Overweight/obesity-attributable costs were calculated for six major noncommunicable dis- eases; sensitivity analyses were conducted for key model parameters. Overweight/obesity-attributable costs were calculated for six major noncommunicable dis- eases; sensitivity analyses were conducted for key model parameters. https://figshare.com/articles/dataset/The_ economic_burden_of_overweight_and_obesity_ in_Saudi_Arabia/18677513. p g economic_burden_of_overweight_and_obesity_ in_Saudi_Arabia/18677513. Methods and materials Data were obtained from previously published studies and secondary databases. OPEN ACCESS Citation: Malkin JD, Baid D, Alsukait RF, Alghaith T, Alluhidan M, Alabdulkarim H, et al. (2022) The economic burden of overweight and obesity in Saudi Arabia. PLoS ONE 17(3): e0264993. https:// doi.org/10.1371/journal.pone.0264993 The prevalence of overweight and obesity in Saudi Arabia has been rising. Although the health burden of excess weight is well established, little is known about the economic burden. Aims Editor: Venkata Naga Srikanth Garikipati, The Ohio State University College of Medicine, UNITED STATES To assess the economic burden—both direct medical costs and the value of absenteeism and presenteeism—resulting from overweight and obesity in Saudi Arabia. * jesse.malkin@gmail.com PLOS ONE PLOS ONE Introduction The prevalence of overweight and obesity is high among the population of Saudi Arabia. According to the 2019 Kingdom of Saudi Arabia World Health Survey—the latest nationally representative survey that includes anthropometric measurements of adults—the prevalence of overweight [Body Mass Index (BMI) 25.0 kg/m2 to <30 kg/m2] is 38 percent and the preva- lence of obesity (BMI 30 kg/m2) is 20 percent [1]. The median age of the Saudi population is 31.8 years [1]; obese Saudis who are young are at increased risk for obesity-attributable dis- eases that are more likely to occur in mid- and late life. Obesity is a well-established risk factor for all-cause mortality and noncommunicable dis- eases (NCDs) such as hypertension, dyslipidemia, type 2 diabetes, stroke, cardiovascular dis- eases, osteoarthritis, several cancers and other conditions. Even people who are overweight but not obese are at increased risk for several of these conditions [2, 3]. This increase in risk trans- lates into substantially higher health care utilization and medical costs among those with excess weight. Much of the increase in health care utilization and costs results from treating the medical conditions such as type 2 diabetes that are caused by excess weight. However, cost increases also result from direct treatments for obesity, including weight loss surgeries and medications, which are increasingly available in Saudi Arabia. In addition to direct medical costs, excess weight leads to an increase in indirect costs, including increased absenteeism (workdays missed due to illness or injury), presenteeism (reduced productivity while working), reduced labor force participation, and premature mortality. Other, less tangible costs include the mone- tary value of pain and suffering and opportunity costs resulting from lower economic output. Much has been written about the economic costs of overweight and obesity in North Amer- ica [4, 5], Europe [6–11], Brazil [12], Australia [13], and China [14]. These studies used a range of approaches, including regression analyses, epidemiological methods, and simulations. By contrast, we identified just one study that attempted to quantify the economic burden of over- weight and obesity in Saudi Arabia [15]. This study included more than 50 countries and con- tained many assumptions required for comparability across countries. Given the paucity of research on the costs of overweight and obesity in Saudi Arabia, the objective of this study was to provide estimates of direct and indirect costs using the best avail- able data from within the country. Introduction This limitation required us to use an epidemiological approach and to limit the analysis to costs resulting from six major NCDs. Using these diseases we quantified (1) direct medical costs, including the cost of hospitalizations, outpatient visits, emergency department visits, general practitioner visits, and prescription drugs; and (2) indi- rect costs arising from absenteeism and presenteeism only. Conclusions Even when limited to six diseases and a subset of total indirect costs, results indicate that overweight and obesity are a significant economic burden in Saudi Arabia. Future studies should identify strategies to reduce the health and economic burden resulting from excess weight in Saudi Arabia. Competing interests: The authors have declared that no competing interests exist. Results Funding: Funding for this research was provided by the World Bank under its advisory services program (P172148) to the Saudi Health Council, Saudi Arabia. The sponsors—the Saudi Health Council and the World Bank—participated in the preparation of this paper. However, the findings, The impact of overweight and obesity for these diseases is found to directly cost a total of $3.8 billion, equal to 4.3 percent of total health expenditures in Saudi Arabia in 2019. Esti- mated overweight and obesity–attributable absenteeism and presenteeism costs a total of $15.5 billion, equal to 0.9 percent of GDP in 2019. 1 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 PLOS ONE The economic burden of overweight and obesity in Saudi Arabia interpretations, and conclusions expressed in this work are those of the authors and do not necessarily reflect the views of the Saudi Health Council or the World Bank, their Boards of Directors, or the governments they represent. Subjects and methods We used a population attributable fraction (PAF) approach—an epidemiologic method widely used to assess the public health impact of exposures in populations—to estimate costs. Direct 2 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 PLOS ONE The economic burden of overweight and obesity in Saudi Arabia medical, absenteeism, and presenteeism costs resulting from excess weight in Saudi Arabia in 2019 were quantified for the six NCDs for which data were available: coronary heart disease, stroke, type 2 diabetes, breast cancer in women, colon cancer, and asthma. We excluded NCDs for which data were not available such as major depressive disorder, anxiety disorders, Alzhei- mer’s, epilepsy, sleep disorders, and several cancers. We began by obtaining risk ratios—the proportionate increase in risk as a result of over- weight or obesity, respectively—for each of the six NCDs. We used sex-specific risk ratio esti- mates from a previous published study [2], whose results were based upon a meta-analysis of the literature. Our approach assumes that (a) the relative risk from excess weight for these six diseases is the same for Saudi Arabians as it is for those from other countries and (b) the rela- tive risk from excess weight for these diseases has not changed over time. Next, we calculated the sex-specific PAFs for overweight and obesity, respectively, using the following formulas: PAF1ð%Þ ¼ p1ðRR1 1Þ 1 þ p1ðRR1 1Þ þ p2ðRR2 1Þ PAF1ð%Þ ¼ p1ðRR1 1Þ 1 þ p1ðRR1 1Þ þ p2ðRR2 1Þ PAF2ð%Þ ¼ p2ðRR2 1Þ 1 þ p1ðRR1 1Þ þ p2ðRR2 1Þ PAF2ð%Þ ¼ p2ðRR2 1Þ 1 þ p1ðRR1 1Þ þ p2ðRR2 1Þ where p1 and p2 represent the prevalence of overweight and obesity in the population of inter- est (that is, Saudi Arabia’s population) respectively; RR1 represents the unadjusted relative risk of a particular NCD of interest for overweight relative to normal weight (18.5 kg/m2 BMI < 25 kg/m2) individuals; and RR2 represents the corresponding figure for obesity relative to normal weight individuals. We obtained sex-specific prevalence estimates for overweight and obesity from the 2019 Kingdom of Saudi Arabia World Health Survey [1]. We obtained estimates of the total direct cost, absenteeism cost, and presenteeism cost of the six diseases in Saudi Arabia for the year 2019 from a previous published study [16]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 Results Estimated costs due to overweight and obesity are presented in Table 1. The estimated annual direct medical cost of excess weight (overweight and obesity combined) is $3.8 billion, equal to 4.3 percent of health expenditures in Saudi Arabia and 0.1 percent of GDP in 2019. Estimated costs of overweight/obesity-attributable absenteeism and presenteeism are $1.6 billion (0.1 percent of GDP in 2019) and $13.8 billion (0.8 percent of GDP in 2019), respectively. Type 2 diabetes is by far the largest cost driver, accounting for 88 percent of overweight/obesity-attrib- utable direct medical costs and 83 percent of overweight/obesity-attributable absenteeism and presenteeism costs. The results of our sensitivity analyses are shown in Table 2. When we replace our base case per-unit cost estimates for type 2 diabetes with lower estimates from the literature [20], esti- mated medical costs decline to $3.2 billion, which is 3.7 percent of estimated health expendi- tures in 2019. When we replace our base case per-unit cost estimates for type 2 diabetes with higher estimates from the literature [21], estimated direct medical costs attributable to over- weight and obesity balloon to $15.9 billion, equal to 18.3 percent of estimated health expendi- tures in 2019. When we replace our base case estimate of type 2 diabetes prevalence with upper bound estimates from the literature [22], estimated direct medical costs due to overweight and obesity rise to $6.2 billion (7.1 percent of health expenditures in 2019), estimated overweight/ obesity-attributable absenteeism costs rise slightly to $2.3 billion (0.1 percent of GDP in 2019) and estimated overweight/obesity-attributable presenteeism costs rise to $22.5 billion (1.3 per- cent of GDP in 2019). Subjects and methods We obtained sex-specific prevalence rates for the year 2019 from the Global Burden of Disease database [17]. To estimate direct medical costs of overweight and obesity, we multiplied the sex-specific prevalence rate by total cost then divided the product by the all-sex prevalence rate to obtain sex-specific total cost estimates. We then multiplied the sex-specific direct medical cost esti- mates by PAFs to obtain sex-specific estimates of overweight/obesity-attributable direct medi- cal costs. We summed the sex-specific overweight/obesity-attributable direct medical cost estimates for each disease (i.e., direct medical costs for men plus direct medical costs for women) to obtain overweight/obesity-attributable cost estimates for both sexes in the aggregate. To estimate absenteeism and presenteeism costs of overweight and obesity, we first calcu- lated a weighted average of PAFs for males and females, where the average was weighted by the percentage of the workforce that is male (84.2 percent) or female (15.8 percent), respec- tively. The breakdown of men and women in Saudi Arabia’s workforce was obtained from The World Bank’s Databank [18]. Next, we multiplied the weighted PAF by total absenteeism or presenteeism costs, respectively, to obtain estimates of overweight- and obesity-attributable costs for both sexes in the aggregate. gg g All cost estimates are reported in 2019 International dollars. To test the variation of our results to our assumptions, we conducted several sensitivity analyses. We considered the impact of replacing the base case per unit type 2 diabetes mellitus cost estimates (based on a previous published study [19]) with lower and higher per unit cost PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 3 / 8 PLOS ONE The economic burden of overweight and obesity in Saudi Arabia estimates from published studies conducted in Saudi Arabia [20, 21]. We replaced base case type 2 diabetes prevalence data from the IHME’s Global Burden of Disease database [17] with higher prevalence estimates from the International Diabetes Foundation [22]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 Discussion Our estimates, albeit limited to six diseases and estimated with great uncertainty, reveal the substantial economic toll of overweight and obesity in Saudi Arabia. Despite limiting our anal- ysis to a small number of NCDs, our base case estimate of annual direct medical costs due to overweight and obesity is $3.8 billion annually, which represents 4.3 percent of total health spending in 2019. The estimated impact of overweight and obesity on absenteeism and presen- teeism is even higher: We estimate that overweight and obesity raise absenteeism and presen- teeism costs in the aggregate by $15.5 billion annually—equal to 0.9 percent of GDP in 2019. Our analysis, however, is subject to several important limitations: • Due to data constraints, we excluded some NCDs for which overweight and obesity-attribut- able costs may be significant. • The literature provides a wide range of estimates of the per unit cost and prevalence of type 2 diabetes, which is by far the largest driver of overweight/obesity-attributable costs among the diseases we considered. • As noted in the introduction, given the paucity of available information, the objective of this study was to provide estimates of direct and indirect costs of overweight and obesity using the best available data. This limitation required us to use an epidemiological approach and stratifications by gender only. We could not, for example, quantify the extent to which costs differ for additional population subsets, such as stratifications by employment characteris- tics, as this level of detail was not available in the existing data. If such data become available more detailed stratifications are recommended. PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 4 / 8 PLOS ONE The economic burden of overweight and obesity in Saudi Arabia g y a. Overweight only Relative risk of overweight a Population Attributable Fraction b Sex-specific prevalence c Total direct medical costs d, e Sex-specific total direct medical costs d Total costs attributable to overweight d Direct medical Absenteeism Presenteeism Males Females Males Females Males Females Males Females Male Female All All All Coronary heart disease 1.29 1.80 0.10 0.16 2.98% 1.68% 798 511 287 51 46 97 166 404 Stroke 1.23 1.15 0.08 0.04 1.28% 1.56% 1,118 504 614 40 25 65 63 153 Type 2 diabetes 2.40 3.92 0.22 0.23 7.78% 6.46% 4,796 2,620 2,176 576 500 1,077 286 4,074 Breast cancer n.a. 1.08 n.a. Discussion 0.03 0.00% 0.32% 43 0 43 0 1 1 1 1 Colorectal cancer 1.51 1.45 0.16 0.12 0.06% 0.03% 45 29 16 5 2 7 12 15 Asthma 1.20 1.25 0.07 0.07 2.54% 6.46% 352 99 253 7 18 25 130 286 All n.a. n.a. n.a. n.a. n.a. n.a. 7,152 3,763 3,389 679 592 1,271 658 4,932 b. Obese only Relative risk of obesity a Population Attributable Fraction b Sex-specific prevalence c Total direct medical costs d, e Sex-specific total direct medical costs d Total costs attributable to obesity d Direct medical Absenteeism Presenteeism Males Females Males Females Males Females Males Females Male Female All All All Coronary heart disease 1.72 3.10 0.11 0.25 2.98% 1.68% 798 511 287 56 72 128 201 487 Stroke 1.51 1.49 0.08 0.09 1.28% 1.56% 1,118 504 614 40 55 96 70 169 Type 2 diabetes 6.74 12.41 0.41 0.54 7.78% 6.46% 4,796 2,620 2,176 1,074 1,175 2,249 555 7,916 Breast cancer n.a. 1.13 n.a. 0.02 0.00% 0.32% 43 0 43 0 1 1 1 1 Colorectal cancer 1.95 1.66 0.13 0.10 0.06% 0.03% 45 29 16 4 2 5 10 12 Asthma 1.43 1.78 0.07 0.13 2.54% 6.46% 352 99 253 7 33 40 148 325 All n.a. n.a. n.a. n.a. n.a. n.a. 7,152 3,763 3,389 1,182 1,337 2,519 984 8,910 c. Overweight and obesity Total costs attributable to both overweight and obesity e Direct medical Absenteeism Presenteeism Absenteeism + Presenteeism Males Females All All All All Coronary heart disease 107 118 225 367 891 1,257 Stroke 81 80 160 133 322 455 Type 2 diabetes 1,651 1,675 3,326 841 11,990 12,831 Breast cancer n.a. 2 2 2 2 4 Colorectal cancer 8 4 12 22 26 48 Asthma 14 51 64 278 611 888 All 1,861 1,929 3,790 1,642 13,842 15,484 % of health expenditures in 2019 2.1 2.2 4.3 n.a. n.a. n.a. % of GDP in 2019 0.1 0.1 0.2 0.1% 0.8% 0.9% n.a. = not applicable. a Relative risks of comorbidities—the proportionate increase in risk as a result of overweight or obesity—were obtained from Guh, Zhang, Bansback et al. 2009 [2]. We attributed all overweight/ obesity-attributable breast cancer costs to women. Discussion b Population attributable fractions (PAFs) for overweight and obesity are calculated using the formula: PAF1 (%) = [p1(RR1 −1)]/ [1 + p1(RR1 −1) + p2(RR2 −1)] and PAF2 (%) = [p2(RR2 −1)]/ [1 + p1(RR1 −1) + p2(RR2 −1)], respectively, where 1 and 2 represent the overweight and obesity groups, respectively; p = prevalence rate; RR = relative risk. Prevalence estimates for overweight (Male: 43%, Female: 33%) and obesity (Male: 19%, Female: 20%) were obtained from preliminary findings for the 2019 Kingdom of Saudi Arabia World Health Survey [1]. c Global Burden of Disease database [17]. d Milli f 2019 I t ti l d ll LOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 n.a. = not applicable. a Relative risks of comorbidities—the proportionate increase in risk as a result of overweight or obesity—were obtained from Guh, Zhang, Bansback et al. 2009 [2]. We attributed all overweight/ obesity-attributable breast cancer costs to women. b Population attributable fractions (PAFs) for overweight and obesity are calculated using the formula: PAF1 (%) = [p1(RR1 −1)]/ [1 + p1(RR1 −1) + p2(RR2 −1)] and PAF2 (%) = [p2(RR2 −1)]/ [1 + p1(RR1 −1) + p2(RR2 −1)], respectively, where 1 and 2 represent the overweight and obesity groups, respectively; p = prevalence rate; RR = relative risk. Prevalence estimates for overweight (Male: 43%, Female: 33%) and obesity (Male: 19%, Female: 20%) were obtained from preliminary findings for the 2019 Kingdom of Saudi Arabia World Health Survey [1]. c Global Burden of Disease database [17]. d 5 / 8 PLOS ONE The economic burden of overweight and obesity in Saudi Arabia Table 2. Sensitivity analyses. Estimates of costs arising from overweight and obesity a Direct Medical Absenteeism Presenteeism $ % of health expenditures in 2019 $ % of GDP in 2019 $ % of GDP in 2019 Base case 3,790 4.3 1,642 0.1 13,842 0.8 Higher per-unit diabetes costs [20] 15,943 18.3 Lower per-unit diabetes costs [19] 3,222 3.7 Higher diabetes prevalence [21] 6,203 7.1 2,252 0.1 22,541 1.3 An asterisk () indicates no change from the base case. a All cost estimates are presented in millions of 2019 International dollars. https://doi.org/10.1371/journal.pone.0264993.t002 An asterisk () indicates no change from the base case. An asterisk () indicates no change from the base case. a All cost estimates are presented in millions of 2019 International dollars. Discussion • We were unable to capture some kinds of indirect costs such as early retirement and care provided by family and friends. • The PAF approach assumes that, conditional on diagnosis, treatment and costs do not vary by baseline BMI. If for example, individuals with excess weight are more difficult to treat, then our results would be conservative. We took into account some but not all of these uncertainties in our sensitivity analyses. As noted above, we identified only one previous study estimating the cost of overweight and obesity in Saudi Arabia. Cecchini and Vuik (2019) [15] estimate that overweight and obe- sity will increase Saudi Arabia’s annual health expenditures by 12.7 percent on average between the years 2020 and 2050. Compared to studies in other countries, this estimate is an outlier. The rest of the literature spanning Europe, North America, Australia, China, and Brazil indicates that overweight/obesity-attributable direct medical costs are equal to between 2 and 9 percent of total health expenditures [4–14]. Our base case estimate for Saudi Arabia, although limited to 6 diseases, reveals that overweight/obesity-attributable direct medical costs are equal to 4.3 percent of national health expenditures, which is in the range of previous estimates. Cecchini and Vuik (2019) [15]—taking into account the effect of excess weight on absentee- ism, presenteeism, unemployment, and early retirement—estimate that overweight and obe- sity will reduce annual GDP in Saudi Arabia by 4.4 percent on average between 2020 and 2050. This, too, is an outlier compared to prior studies. Other researchers have estimated indirect costs resulting from overweight/obesity in France, Australia, Germany, and Canada ranging from 0.5 percent to 1.6 percent of GDP [5, 6, 8, 10, 13]. Our base case estimate of absenteeism and presenteeism costs arising from overweight and obesity in Saudi Arabia—0.9 percent of GDP—is in between these prior estimates. Conclusion Despite uncertainties, our estimates are broadly consistent with previous estimates of the costs of overweight and obesity in countries other than Saudi Arabia. Our findings indicate that overweight and obesity are a significant economic burden in Saudi Arabia, resulting in esti- mated direct medical costs of 4.3 percent of annual health expenditures and select indirect costs accounting for an estimated 0.9 percent of GDP. This raises the possibility that interven- tions that prevent overweight and obesity, such as Saudi Arabia’s excise tax on sugar-sweet- ened beverages [23], may be both health improving and cost saving. 6 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 PLOS ONE The economic burden of overweight and obesity in Saudi Arabia Author Contributions Conceptualization: Jesse D. Malkin, Reem F. Alsukait, Taghred Alghaith, Christopher H. Herbst, Eric Andrew Finkelstein, Nahar Alazemi. Conceptualization: Jesse D. Malkin, Reem F. Alsukait, Taghred Alghaith, Christopher H. Herbst, Eric Andrew Finkelstein, Nahar Alazemi. Formal analysis: Jesse D. Malkin, Eric Andrew Finkelstein. Formal analysis: Jesse D. Malkin, Eric Andrew Finkelstein. Formal analysis: Jesse D. Malkin, Eric Andrew Finkelstein. Funding acquisition: Christopher H. Herbst. Funding acquisition: Christopher H. Herbst. Funding acquisition: Christopher H. Herbst. Methodology: Jesse D. Malkin, Drishti Baid, Eric Andrew Finkelstein. Project administration: Jesse D. Malkin, Reem F. Alsukait, Christopher H. Herbst, Eric Andrew Finkelstein. Supervision: Taghred Alghaith, Eric Andrew Finkelstein. Writing – original draft: Jesse D. Malkin, Eric Andrew Finkelstein. Writing – review & editing: Jesse D. Malkin, Drishti Baid, Reem F. Alsukait, Taghred Alghaith, Mohammed Alluhidan, Hana Alabdulkarim, Abdulaziz Altowaijri, Ziyad S. Almalki, Christopher H. Herbst, Eric Andrew Finkelstein, Sameh El-Saharty, Nahar Alazemi. Acknowledgments The authors are grateful for the overall support provided by Rekha Menon, World Bank Prac- tice Manager, Health, Nutrition and Population, Middle East and North Africa, and Issam Abousleiman, Country Director, Gulf Cooperation Council countries. PLOS ONE \| https://doi.org/10.1371/journal.pone.0264993 March 8, 2022 References 1. Ministry of Health. 2020. Kingdom of Saudi Arabia World Health Survey 2019 Report. Riyadh: Ministry of Health. 2. Guh DP, Zhang W, Bansback N, Amarsi Z, Birmingham CL, Anis AH. The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis. BMC Public Health. 2009 Mar 25; 9:88. https://doi.org/10.1186/1471-2458-9-88 PMID: 19320986; PMCID: PMC2667420. 3. World Health Organization. 2021. Obesity and Overweight Factsheet, June 9, 2021. https://www.who. int/en/news-room/fact-sheets/detail/obesity-and-overweight. 4. Finkelstein EA, Trogdon JG, Cohen JW, Dietz W. 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https://openalex.org/W3013441161	https://discovery.ucl.ac.uk/id/eprint/10095303/1/s12877-020-01531-w.pdf	English	null	Gait speed, cognition and falls in people living with mild-to-moderate Alzheimer disease: data from NILVAD	BMC geriatrics	2,020	cc-by	9,879	Dyer et al. BMC Geriatrics (2020) 20:117 https://doi.org/10.1186/s12877-020-01531-w Dyer et al. BMC Geriatrics (2020) 20:117 https://doi.org/10.1186/s12877-020-01531-w Open Access Gait speed, cognition and falls in people living with mild-to-moderate Alzheimer disease: data from NILVAD Adam H. Dyer1,2, Brian Lawlor2,3, Sean P. Kennelly1,2 and for the NILVAD Study Group Abstract 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: dyera@tcd.ie 1Department of Age-Related Healthcare, Tallaght University Hospital, Dublin, Ireland 2Department of Medical Gerontology, School of Medicine, Trinity College Dublin, Dublin 2, Ireland Full list of author information is available at the end of the article Abstract Background: Previous evidence suggests that slower gait speed is longitudinally associated with cognitive impairment, dementia and falls in older adults. Despite this, the longitudinal relationship between gait speed, cognition and falls in those with a diagnosis of dementia remains poorly explored. We sought to assess this longitudinal relationship in a cohort of older adults with mild to-moderate Alzheimer Disease (AD). Methods: Analysis of data from NILVAD, an 18-month randomised-controlled trial of Nilvadipine in mild to moderate AD. We examined: (i) the cross-sectional (baseline) association between slow gait speed and cognitive function, (ii) the relationship between baseline slow gait speed and cognitive function at 18 months (Alzheimer Disease Assessment Scale, Cognitive Subsection: ADAS-Cog), (iii) the relationship between baseline cognitive function and incident slow gait speed at 18 months and finally (iv) the relationship of baseline slow gait speed and incident falls over the study period. Results: Overall, one-tenth (10.03%, N = 37/369) of participants with mild-to-moderate AD met criteria for slow gait speed at baseline and a further 14.09% (N = 52/369) developed incident slow gait speed at 18 months. At baseline, there was a significant association between poorer cognition and slow gait speed (OR 1.05, 95% CI 1.01–1.09, p = 0.025). Whilst there was no association between baseline slow gait speed and change in ADAS-Cog score at 18 months, a greater cognitive severity at baseline predicted incident slow gait speed over 18 months (OR 1.04, 1.01–1.08, p = 0.011). Further, slow gait speed at baseline was associated with a significant risk of incident falls over the study period, which persisted after covariate adjustment (IRR 3.48, 2.05–5.92, p < 0.001). Conclusions: Poorer baseline cognition was associated with both baseline and incident slow gait speed. Slow gait speed was associated with a significantly increased risk of falls over the study period. Our study adds further evidence to the complex relationship between gait and cognition in this vulnerable group and highlights increased falls risk in older adults with AD and slow gait speed. Trial registration: Secondary analysis of the NILVAD trial (Clincaltrials.gov NCT02017340; EudraCT number 2012–002764-27). First registered: 20/12/2013. Trial registration: Secondary analysis of the NILVAD trial (Clincaltrials.gov NCT02017340; EudraCT number 2012–002764-27). First registered: 20/12/2013. Keywords: Dementia, Alzheimer disease, Cognition, Falls © The Author(s). Background adults with a diagnosis of AD, particularly those with mild-to-moderate AD [27]. Further, most studies on com- munity dwelling older adults with AD have either oc- curred in cross-section or retrospectively with < 12 months follow up [27]. Gait disorders affect a significant number of community- dwelling older adults and have recently been highlighted as a potential indicator of later cognitive decline. Gait dis- orders are believed to affect up to one third of older adults aged > 70 years and may be divided based on the particu- lar sensorimotor deficit involved: (i) lower level gait disor- ders such as osteoarthritis, (ii) mid-level gait disorders which result from a focal neurological lesion such as that seen in Parkinson’s Disease and (iii) higher level gait disor- ders with no demonstrable deficits in the pyramidal/extra- pyramidal/sensory or cerebellar systems (and may be particularly related to cerebrovascular disease) [1–3]. Based on the above evidence and the known adverse effects experienced by those with slow gait speed, it fol- lows that slow gait speed may be an indicator of which patients with AD are particularly vulnerable of further cognitive decline and adverse outcomes such as falls. In the current study, we aimed to assess the relationships between (i) baseline cognition and baseline slow gait speed in mild-moderate AD, (ii) baseline slow gait speed and cognitive decline at 18 months (iii) baseline cogni- tive impairment and incident slow gait speed at 18 months (decline in gait speed) and (iv) the longitudinal relationship between baseline slow gait speed and inci- dent falls over 18 months in older adults with a diagnosis of mild-to moderate AD. Not only does slow gait speed predict a decline in func- tional status, but has also been linked with adverse out- comes including mortality in older adults [4–9]. Further, there is now a significant body of evidence linking slow gait speed, and in particular a decline in gait speed, with later risk of cognitive decline and dementia. For instance, in a recently published study from the English Longitu- dinal Study of Aging, both baseline gait speed and change in gait speed was associated with dementia risk over a fol- low up of > 10 years [10]. This follows a large body of evi- dence indicating the strong relationship between slow gait speed and later cognitive decline [11–16]. Methods Setting g The current study was embedded within NILVAD, a randomised controlled trial of the antihypertensive Nil- vadipine in mild-to-moderate Alzheimer Disease (AD). The trial’s primary outcome of a therapeutic effect of Nilvadipine in mild/ moderate AD was not achieved. Briefly, participants were recruited from 23 centres in 9 European countries (Clincaltrials.gov NCT02017340; EudraCT number 2012–002764-27). The full trial proto- col, the sub-study protocol in addition to the main trial results have been published elsewhere [28–30]. Ethical approval for the trial was granted from the appropriate National Competent Authorities, Independent Ethics Committees and Institutional Review Boards for all study sites. Whilst the evidence for the relationship between slow gait speed and later dementia risk is mounting, the rela- tionship between gait speed and cognitive trajectories in those with Alzheimer Disease (AD) is less clear. Whilst a handful of studies have evaluated the association be- tween gait speed and dementia in cross-section [17–19], longitudinal analyses are limited. A notable study has demonstrated a decline in gait speed associated with clinical progression in those with Mild Cognitive Impair- ment (MCI) [20]. In the only longitudinal study of gait speed in those with a diagnosis of dementia, baseline gait-speed was associated with a decline in verbal fluency over a 1 year period, but not in overall cognition [21]. Thus, whilst the cross-sectional relationship between gait and cognition in dementia has been demonstrated, the longitudinal relationship between slow gait speed and cognitive function in AD remains unclear. Participants once enrolled were randomized to either Nilvadapine 8 mg or placebo for the 18 month duration of the study. © 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. Dyer et al. BMC Geriatrics (2020) 20:117 Page 2 of 10 Page 2 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 Statistical analysis ( Gait speed was measured as part of the NILVAD frailty sub-study, the protocol of which has been previously pub- lished [28]. Briefly, gait speed was measured both at base- line and 18 months. Four markings were placed on an even floor with adhesive tape over a distance of 6 m total. The markings were spaced as follows (i) first mark at start, (ii) second mark at 1 m, (iii) third mark 4 m from mark 2 and (iv) fourth mark 1 m from mark 3. Participants were asked to stand at mark 1 and then instructed to walk from mark 1 to mark 4 at normal walking speed. The stopwatch was started once the participant’s first foot touched/ crossed mark two and was stopped once the participant’s last foot crossed mark 3. A stopwatch accurate to two decimal places was used. Thus the total speed represented the time for the participant to walk 4 m. If the total time was > 6 s to complete this 4 m walk (< 0.67 ms−1), gait speed was recorded as slow gait speed. This pre-specified binary cut-off was applied as per previous literature and included in the initial sub-study protocol [23, 28]. Statistical analysis STATA V15.0 (Stata Corp, College Station, Texas, USA) was used for all analysis in the current study, with the significance level considered as p < 0.05. Baseline de- scriptive statistics are reported as means with standard deviations where parametric and medians with Inter- quartile Ranges (IQR) where appropriate. Univariate analysis of between group differences employed T-tests, Wilcoxon rank-sum tests and Chi-square tests. Logistic regression was used to assess predictors of slow gait speed at baseline and adjusted Odds Ratios (OR), 95% Confidence Intervals (CI) and p-values presented as ap- propriate. Change in ADAS-Cog Scores was considered the difference between the baseline ADAS-Cog score and ADAS-Cog score at 18 months, with an increasing score indicating greater cognitive decline. In the current analysis, we sought to assess the longi- tudinal relationship between slow gait speed and cogni- tive function in older adults with mild-to-moderate Alzheimer Disease. Inclusion/exclusion criteria The inclusion/exclusion criteria for the NILVAD trial have been previously published [28]. Of note, included participants were those aged > 50 years who met the National Institute of Neurological and Communicative Disorders and Stroke/Alz- heimer’s Disease Criteria: NINCDS-ADRDA for AD. Criteria for inclusion was a diagnosis of mild-to-moderate AD, de- fined as a Mini-Mental State (MMSE) Score between 12 and 26 at trial enrolment and a diagnosis of AD as per the NINCDS-ADRDA criteria. Further, those included had a Mini-Mental State (MMSE) Score between 12 and 26 at trial enrolment, meeting criteria for mild to moderate AD. There is a significant body of evidence reporting that older adults with a diagnosis of AD are at greater risk of falls than their age matched counterparts without demen- tia [22–26]. The increased risk represents the interaction of several risk factors such as autonomic symptoms, phys- ical activity profile, orthostatic hypotension and polyphar- macy [24]. Several studies have reported associations between spatiotemporal gait parameters and falls risk in older adults with dementia [27], however there are also conflicting results. Many studies have taken place in nurs- ing home or long term care facilities and the association has been less-well explored in community dwelling older Relevant exclusion criteria to the current analysis include a comorbid neurological condition such as Parkinson’s Disease Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Page 3 of 10 Page 3 of 10 or Huntington’s Disease. Similarly, participants with a history of significant head trauma, known structural brain abnormal- ities or any other condition known to interfere with cognitive function were excluded. Patients meeting criteria for a sub- stance use disorder were also excluded as were participants with significant cardiovascular disease. For full exclusion cri- teria, readers are directed to the initial trial protocol. also obtained at time of study enrolment and reviewed at follow up visits in order to identify any changes in concomitant medication usage. Medications were classi- fied according to Anatomic Therapeutic Classification (ATC) codes to ensure consistency. For the current ana- lysis, only medications taken for the entire 18-month study duration were considered. Short term and histor- ical medication use were excluded. Cognitive function and dementia severity assessment Cognitive function and dementia severity assessment The Alzheimer’s Disease Assessment Scale, Cognitive Subsection (ADAS-Cog) was used to assess cognition and was the primary cognitive endpoint in the NILVAD trial. For the current study, we considered ADAS-Cog at initial visit to be baseline ADAS-Cog and analysed the change in ADAS-Cog at 18 months as the main cogni- tive outcome. ADAS-Cog sub-scores analysed included the following: word recall task, naming, commands, con- structional praxis, delated word recall, ideational praxis, orientation, word recognition task, spoken language abil- ity, comprehension, word finding difficult in spontan- eous speech, remembering test instructions. To assess the cross-sectional relationship at baseline between cognitive function (independent variable, linear) and slow gait speed (dependent variable; binary), we used multivariate logistic regression adjusting for im- portant confounders known to influence gait speed and/ or cognition. We adjusted for age, gender, Body Mass Index, study group (Nilvadipine vs.placebo), diagnosis duration, symptom duration, years of formal education, total number of medications, antidepressant and benzo- diazepine use, total medical comorbidities, diabetes, hypertension, arthritis, baseline ADAS-Cog and baseline CDR-Sb. Dementia severity at baseline and at 18-months was assessed using the Clinical Dementia Rating scale sum of boxes (CDR-Sb) score. In order to assess whether baseline slow gait speed (inde- pendent variable; categorical) was associated with cognitive outcomes at 18 months (dependent variable; linear), mixed effects linear regression was used with country as a random effect. The association was tested unadjusted in the first Statistical analysis ( Our research questions were as fol- lows: (i) is there an association at baseline between slow gait speed and baseline cognitive function?, (ii) does slow gait speed at baseline predict greater cognitive decline at 18 months in mild-to-moderate AD?, (iii) does cognitive function at baseline predict the development of slow gait speed at 18 months (incident slow gait speed)?, and fi- nally, (iv) does slow gait speed predict falls in those with AD over an 18-month period. Baseline slow gait speed was considered as those below this cut-off at trial initiation and incident slow gait speed defined as those below this cut off at 18 months who did not meet the slow great criteria at baseline. Participant characteristics Of those randomized to either Nilvadipine or placebo at baseline (N = 511), 465 participants had a gait speed meas- urement performed. Of these, 369 had full clinical and 18- month follow up data obtained including repeat gait speed and cognitive assessment at 18 months. This population constituted those included in the current analysis. Mean age of included participants was 72.8 ± 8.06 and 63.14% (N = 233/369) were female. Overall, 50.68% (N = 187/369) were assigned to the treatment group. Median duration of AD symptoms was 3.65 years (IQR: 2.26–5.18) and dur- ation since diagnosis was 0.91 years (IQR: 0.42–2.12). At Change in ADAS-cog scores over 18 months Change in ADAS-cog scores over 18 months Overall, the mean ADAS-Cog in the included cohort in- creased by 8.87 (± 9.18), indicating both a clinically sig- nificant cognitive decline [31] and a statistically significant progression (t = −18.56, p < 0.001). Analyses were then repeated by ADAS-Cog sub-scores for each task, with the number of errors on each task analysed by Poisson regression using the same covariates the above models. Finally, we repeated the above models using the CDR-Sb as the dependent variable in order to assess the relationship between slow gait speed and de- mentia severity at baseline at 18 months. Medical history and regular medications A comprehensive medical history was taken from partic- ipants/carers at time of study enrolment and updated on follow up visits. A list of concomitant medications was Dyer et al. BMC Geriatrics (2020) 20:117 Page 4 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 baseline, mean ADAS-Cog was 32.97 ± 9.81 and mean CDR-Sb was 4.82 ± 2.52. instance (model 1). The association was then tested control- ling for important demographic and AD-related covariates including baseline cognitive function (ADAS-Cog Score), age, gender, study arm, body mass index, years of formal education and diagnosis duration (model 2). Finally we incor- porated total number of medical comorbidities and total number of medications (model 3). Baseline slow gait speed and cognitive decline at 18 months Unadjusted, there was no association between baseline slow gait speed and ADAS-Cog scores at 18 months (β: − 1.49, −4.61 – 1.63, p = 0.35). There was no association on controlling for baseline ADAS-Cog, age, gender, study arm, BMI, education and diagnosis duration (β: −0.63, − 3.69 – 2.42, p = 0.69). Further, following further adjust- ment for important medical comorbidities and concomi- tant medication use, there was no association between baseline gait speed and ADAS-Cog scores at 18 months (β: −0.80, −3.86 – 2.25, p = 0.69) (Table 3). Under the same models, there was no association between slow gait speed at baseline and CDR-Sb score at 18 months either unadjusted (β: 0.23, −0.79 – 1.24, p = 0.66), or under model 2 (β: 0.11, −0.92 – 1.14, p = 0.84) or model 3 (β: 0.16, −0.92 – 1.15, p = 0.83). In all above analyses, the following data were categor- ical: gender, group (Nilvadipine vs. placebo), slow gait speed (present vs absent), antidepressant use (user vs non-user), benzodiazepine use (user vs non-user), dia- betes (present vs absent), arthritis (present vs absent), hypertension (present vs absent). All other variables were continuous as detailed above. Baseline association between slow gait speed and cognition At baseline, one-tenth (10.03%, N = 37/369) of participants had slow gait speed defined as described above. On uni- variate analysis, those with slow gait speed were older, had a higher BMI, fewer years of education, greater number of total medications and comorbidities, poorer baseline cog- nition rated using the ADAS-Cog and greater dementia severity rated using the CDR-Sb (all p < 0.05, see Table 1). On multivariate analysis controlling for all covariates, in- creasing age (OR 1.10, 1.04–1.17, p < 0.001) and poorer baseline cognition on the ADAS-Cog (OR 1.05, 1.01–1.09, p = 0.025) were associated with greater risk of baseline slow gait speed (See Table 2). Similarly, there was a non- significant statistical trend for greater dementia severity using the CDR-Sb as a predictor of slow gait speed under this model (OR 1.19, 0.98–1.45, p = 0.08). On analysis of ADAS-Cog subset-scores, there were no specific associa- tions between gait speed and separate cognitive domain subset-scores at 18 months. For assessment of whether baseline cognition (inde- pendent variable; linear) was associated with incident slow gait speed (dependent variable; categorical), logistic regression was used Again the association was tested un- adjusted followed by adjustment for baseline ADAS-Cog, age, gender, study arm, body mass index, years of formal education, diagnosis duration (model 2) followed by ro- bust adjustment (model 3) for total number of medical conditions, diabetes mellitus, arthritis, total number of medications, antidepressant use and benzodiazepine use based on known impact on gait speed. Finally, in order to assess the relationship between slow gait speed (independent variable; categorical) and falls (dependent variable; count), a Poisson regression was used, again unadjusted in the first instance, followed by adjustment for baseline ADAS-Cog, age, gender, study arm, body mass index, years of formal education, diagno- sis duration (model 2) in addition to total medical comor- bidities and total number of medications (model 3). months ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection; CDR-Sb: Clinical Dementia Rating, Sum of Boxes, BMI: Body Mass Index criteria for incident slow gait speed had higher baseline ADAS-Cog scores, however this was not a statistically sig- nificant or clinically meaningful difference (36.47 +/−9.44 vs 36.08 +/−11.42, t = −0.23, p = 0.82) [31]. Unadjusted, ADAS-Cog score at baseline was associated with a greater likelihood of incident slow gait speed at 18 months (OR: 1.04, 1.01–1.07, p = 0.015). This association persisted after controlling for age, gender, BMI, study group, years of education and diagnosis duration (OR: 1.04, 1.01–1.08, p = 0.009). Further, after robust covariate adjustment for total medical comorbidities, diabetes, arthritis, total num- ber of medications, benzodiazepines and antidepressant use, the association remained significant (OR: 1.04, 1.01– 1.08, p = 0.011) (see Table 4). Further, dementia severity at baseline rated using the CDR-Sb was significant under the same models with associations seen unadjusted (OR: 1.17, 1.05–1.30, p = 0.003) in addition to under models 2 (OR: 1.16, 1.03–1.30, p = 0.018) and 3 (OR: 1.15, 1.01–1.30, p = 0.031). Table 2 Multivariate Analysis of Slow Gait Speed at Baseline. At baseline, increasing age and poorer scores on the ADAS-Cog at baseline was associated with significantly greater likelihood of slow gait speed (dependent variable) at baseline. ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection; CDR-Sb: Clinical Dementia Rating, Sum of Boxes, BMI: Body Mass Index Characteristic OR (95% CI) P-Value Age 1.10 (1.04–1.17) < 0.001* Gender 1.14 (0.47–2.75) 0.773 BMI 1.08 (0.99–1.16) 0.078 Group 1.00 (0.45–2.21) 0.996 Diagnosis Duration 0.88 (0.64–1.20) 0.417 Symptom Duration 0.94 (0.78–1.14) 0.541 Education (Years) 0.93 (0.83–1.04) 0.214 Total Medications 1.07 (0.91–1.25) 0.421 Antidepressant Use 1.53 (0.69–3.41) 0.298 Benzodiazepine Use 0.36 (0.07–1.60) 0.178 Total Comorbidities 1.10 (0.93–1.31) 0.270 Diabetes 0.96 (0.30–3.11) 0.951 Hypertension 1.76 (0.77–3.98) 0.179 Arthritis 0.86 (0.23–3.20) 0.820 Baseline ADAS-Cog 1.05 (1.01–1.09) 0.025* Baseline CDR-Sb 1.19 (0.98–1.45) 0.081 Table 2 Multivariate Analysis of Slow Gait Speed at Baseline. At baseline, increasing age and poorer scores on the ADAS-Cog at baseline was associated with significantly greater likelihood of slow gait speed (dependent variable) at baseline. months Overall, 14.09% (N = 52/369) of participants with mild to moderate AD met the criteria for incident slow gait speed at 18 month follow up (having had normal gait speed at baseline). Overall, those participants meeting Page 5 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 Table 1 Baseline Differences between Those with Mild to Moderate AD with Slow Gait Speed at Baseline and Those Without (Univariate Analysis). ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection; CDR-Sb: Clinical Dementia Rating, Sum of Boxes, BMI: Body Mass Index Characteristic No Slow Gait Speed (N = 332) Slow Gait Speed (N = 37) P-Value Age, mean (SD) 72.25 (8.02) 77.68 (6.75) < 0.001* Gender, female (%) 207 (62.35%) 26 (70.27%) 0.343 BMI, mean (SD) 25.44 (4.23) 27.97 (5.56) 0.005* Group, nilvadipine (%) 169 (50.9%) 18 (48.65%) 0.795 Diagnosis Duration, median (IQR) 0.90 (0.43–2.22) 1.01 (0.48–1.71) 0.687 Symptom Duration, median (IQR) 3.69 (2.20–5.25) 3.45 (2.50–4.28) 0.689 Education, median (IQR) 16 (14–18) 14 (12–16) 0.003* Total Medications, median (IQR) 5 (3–6.5) 5 (4–8) 0.022* Antidepressant Use, N (%) 126 (37.95%) 18 (48.65%) 0.206 Benzodiazepine Use, N (%) 40 (12.05%) 3 (8.11%) 0.479 Total Comorbidities, median (IQR) 4 (2–5) 4 (3–6) 0.009* Diabetes, N (%) 22 (6.63%) 5 (13.51%) 0.297 Hypertension, N (%) 94 (28.40%) 14 (37.84%) 0.232 Arthritis, N (%) 26 (7.83%) 4 (10.81) 0.529 Baseline ADAS-Cog, mean (SD) 32.49 (9.49) 37.29 (11.56) 0.002* Baseline CDR-Sb, mean (SD) 4.67 (2.44) 6.13 (2.84) 0.004* Table 1 Baseline Differences between Those with Mild to Moderate AD with Slow Gait Speed at Baseline and Those Without (Univariate Analysis). ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection; CDR-Sb: Clinical Dementia Rating, Sum of Boxes BMI: Body Mass Index Table 1 Baseline Differences between Those with Mild to Moderate AD with Slow Gait Speed at Baseline and Those Without (Univariate Analysis). ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection; CDR-Sb: Clinical Dementia Rating, Sum of Boxes, BMI: Body Mass Index Table 1 Baseline Differences between Those with Mild to Moderate AD with Slow Gait Speed at Baseline and Those Without (Univariate Analysis). months ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection; CDR-Sb: Clinical Dementia Rating, Sum of Boxes, BMI: Body Mass Index Cognitive Scores (ADAS-Cog) of those at baseline with baseline slow gait speed and normal gait speed in addition to those meeting criteria for incident slow gait speed are detailed below in Table 5. Baseline slow gait speed and incident falls over an 18 month duration Overall, 57 (15.45%) participants experienced at least one fall over the 18 month duration of the trial. A small Dyer et al. BMC Geriatrics (2020) 20:117 Page 6 of 10 Page 6 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Table 3 Analysis of The Relationship Between Baseline Slow Gait Speed and Cognition at 18 Months (ADAS-Cog) in Mild to Moderate AD. Under unadjusted or adjusted models, there was no association between baseline slow gait speed and cognition at 18 months (dependent variable). ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection Table 3 Analysis of The Relationship Between Baseline Slow Gait Speed and Cognition at 18 Months (ADAS-Cog) in Mild to Moderate AD. Under unadjusted or adjusted models, there was no association between baseline slow gait speed and cognition at 18 months (dependent variable). ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection p g g Predictor Model 1 Model 2 Model 3 β -Coef (95% CI) P β -Coef (95% CI) P β Coef (95% CI) P Baseline Slow Gait Speed −1.49 (−4.61, 1.63) 0.35 −0.63 (−3.69, 2.42) 0.69 −0.80 (−3.86, 2.25) 0.606 Baseline ADAS-Cog 0.22 (0.13, 0.31) < 0.001* 0.22 (0.13, 0.32) < 0.001* Age −0.21 (−0.32, −0.10) < 0.001* −0.22 (−0.34, −0.10) < 0.001* Gender (Female) 0.94 (−2.80, 0.92) 0.321 0.97 (−2.83, 0.90) 0.310 Study Arm −0.73 (−2.49, 1.02) 0.414 −0.70 (−2.46, 1.06) 0.437 Body Mass Index −0.25 (−0.46 - -0.04) 0.018* −0.27 (−0.48 - -0.06) 0.011* Education (Years) 0.13 (−0.11–0.37) 0.291 0.14 (−0.10–0.38) 0.244 Diagnosis Duration −0.67 (−1.22 – −0.12) 0.017 −0.68 (−1.22 – −0.12) 0.015 Total Medications 0.17 (−0.18–0.52) 0.345 Total Comorbidities 0.08 (−0.33–0.49) 0.697 minority, 21 (5.69%), experienced recurrent (> 1) falls over the study period. Unadjusted, slow baseline gait speed was associated with increasing likelihood of falls (IRR: 3.18, 1.97–5.11, p < 0.001). Controlling for demo- graphic and baseline AD variables, this association per- sisted (IRR: 3.27, 1.96–5.50, p < 0.001). Finally, after robust adjustment for important comorbidities and med- ications, the association remained significant (IRR: 3.48, 2.05–5.92, p < 0.001). See Table 6 for details. Under all models, slow gait speed was the strongest independent predictor of falls over the 18 month duration. Discussion One of the most interesting findings from the current analysis is the relationship between baseline cognitive function and incident slow gait speed over the duration of the study. Even in a fully adjusted model, baseline cognition was the strongest (apart from age) predictor of incident slow gait speed. This is particularly interesting given that 14% of the participants with normal gait speed at baseline converted to slow gait speed (incident slow gait speed). Thus, this may represent a subpopulation within those with mild-to-moderate AD who may be particularly vulnerable to adverse consequences. At baseline, the only significant predictors of slow gait speed were greater age and poorer cognition in multi- variate analysis. This finding is noteworthy and is largely consistent with previous studies in the literature which have demonstrated a relationship between cognitive function and gait speed in a cross-sectional manner in those with AD [17–19]. The lack of association between baseline slow gait speed and cognitive outcomes is of interest. The only previous study in mild to moderate dementia to assess gait speed and cognition in a longitudinal fashion similarly found no association in longitudinal analysis for general cognition, but found a positive association for particular cognitive sub-domains, namely executive function [21]. One of the reasons for the lack of association in the current study may be that gait was considered as a dichotomous variable based on a pre-specified cut-off. It may be the case that more subtle abnormalities in gait speed may correlate with The association between baseline slow gait speed and incident falls is particularly stark. Baseline slow gait speed was associated with a greater than threefold in- creased risk in incident falls over the 18 month study period. Indeed, baseline slow gait speed was the stron- gest predictor of incident falls under a fully adjusted model. This is particularly pertinent given that more Table 6 Association between Baseline Slow Gait Speed and Falls at 18 Months in Mild to Moderate AD. Under all models, slow gait speed was significantly associated with falls (dependent variable) risk at 18 months. ADAS-Cog = Alzheimer Disease Assessment Scale b Table 6 Association between Baseline Slow Gait Speed and Falls at 18 Months in Mild to Moderate AD. Under all models, slow gait speed was significantly associated with falls (dependent variable) risk at 18 months. Discussion Subsection Baseline Slow Gait Speed Incident Slow Gait S ADAS-Cog (Baseline) 37.29 +/−9.49 36.47 +/−11.56 ADAS-Cog (18 Months) 44.70 +/−16.01 47.28 +/−15.91 Table 6 Association between Baseline Slow Gait Speed and Falls a speed was significantly associated with falls (dependent variable) r – Cognitive Subsection Predictor Model 1 IRR (95% CI) P Baseline Slow Gait Speed 3.18 (1.97–5.11) < 0.001 Baseline ADAS-Cog Age Gender (Female) Study Arm Body Mass Index Education (Years) Diagnosis Duration Total Comorbidities Diabetes Mellitus Arthritis Total Medications Benzodiazepine Use Antidepressant Use Table 5 Cognitive Scores (ADAS-Cog) at baseline and 18 months by Gait Speed Group at baseline and 18 Months in Mild to Moderate AD. Data presented as means with standard deviations. ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection Baseline Slow Gait Speed Incident Slow Gait Speed Normal Gait Speed at Baseline Normal Gait Speed at 18 Months ADAS-Cog (Baseline) 37.29 +/−9.49 36.47 +/−11.56 32.49 +/−9.49 36.08 +/−11.42 ADAS-Cog (18 Months) 44.70 +/−16.01 47.28 +/−15.91 41.53 +/−14.43 40.96 +/−14.22 Table 5 Cognitive Scores (ADAS-Cog) at baseline and 18 months by Gait Speed Group at baseline and 18 Months in Mild to Moderate AD. Data presented as means with standard deviations. ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection Baseline Slow Gait Speed Incident Slow Gait Speed Normal Gait Speed at Baseline Normal Gait Speed at 18 Months ADAS-Cog (Baseline) 37.29 +/−9.49 36.47 +/−11.56 32.49 +/−9.49 36.08 +/−11.42 ADAS-Cog (18 Months) 44 70 +/−16 01 47 28 +/−15 91 41 53 +/−14 43 40 96 +/−14 22 Baseline Slow Gait Speed Incident Slow Gait Speed Normal Gait Speed at Baseline Normal Gait Speed at 18 Months ADAS-Cog (Baseline) 37.29 +/−9.49 36.47 +/−11.56 32.49 +/−9.49 36.08 +/−11.42 ADAS-Cog (18 Months) 44.70 +/−16.01 47.28 +/−15.91 41.53 +/−14.43 40.96 +/−14.22 than threefold increased risk of falls over an 18 month period in those with mild to moderate AD. This is one of the first studies in the literature to assess the longitu- dinal relationship between slow gait speed and cognitive function in those with a diagnosis of AD cognitive function and that such a stringent cut off may mask such an association. Nevertheless, it is possible that whilst slow gait speed may be a predictor of a later diagno- sis of dementia, it may lose its predictive value once a diagnosis of dementia has been established. Discussion Th study in mild to moderate dementia to a and cognition in a longitudinal fashion sim association in longitudinal analysis for ge but found a positive association for part sub-domains, namely executive function [ reasons for the lack of association in th may be that gait was considered as a dicho based on a pre-specified cut-off. It may b more subtle abnormalities in gait speed ma Table 5 Cognitive Scores (ADAS-Cog) at baseli Moderate AD. Data presented as means with st Subsection Baseline Slow Gait Speed ADAS-Cog (Baseline) 37.29 +/−9.49 ADAS-Cog (18 Months) 44.70 +/−16.01 Table 6 Association between Baseline Slow Ga speed was significantly associated with falls (de – Cognitive Subsection Predictor Model 1 IRR (95% CI) Baseline Slow Gait Speed 3.18 (1.97–5.11) Baseline ADAS-Cog Age Gender (Female) Study Arm Body Mass Index Education (Years) Diagnosis Duration Total Comorbidities Diabetes Mellitus Arthritis Total Medications Benzodiazepine Use Antidepressant Use than threefold increased risk of falls over an 18 month period in those with mild to moderate AD. This is one of the first studies in the literature to assess the longitu- dinal relationship between slow gait speed and cognitive function in those with a diagnosis of AD At baseline, the only significant predictors of slow gait speed were greater age and poorer cognition in multi- variate analysis. This finding is noteworthy and is largely consistent with previous studies in the literature which have demonstrated a relationship between cognitive function and gait speed in a cross-sectional manner in those with AD [17–19]. The lack of association between baseline slow gait speed and cognitive outcomes is of interest. The only previous study in mild to moderate dementia to assess gait speed and cognition in a longitudinal fashion similarly found no association in longitudinal analysis for general cognition, but found a positive association for particular cognitive sub-domains, namely executive function [21]. One of the reasons for the lack of association in the current study may be that gait was considered as a dichotomous variable based on a pre-specified cut-off. It may be the case that more subtle abnormalities in gait speed may correlate with Table 5 Cognitive Scores (ADAS-Cog) at baseline and 18 months b Moderate AD. Data presented as means with standard deviations. Discussion In this study of older adults mild-to-moderate AD, greater cognitive impairment at baseline was signifi- cantly associated with both baseline slow gait speed and incident slow gait speed at 18 months. There was no as- sociation between baseline slow gait speed and longitu- dinal cognitive performance at 18 months. This is despite a clinically meaningful cognitive decline experi- enced by the cohort overall (with the mean ADAS-Cog score increasing by a mean of 8.87 points) [32]. Further, slow gait speed at baseline was associated with a more Table 4 Association between Baseline Cognition (ADAS-Cog) and incident Slow Gait Speed at 18 Months in Mild to Moderate AD. Under all models, baseline cognition was significantly associated with incident slow gait speed (dependent). ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection Predictor Model 1 Model 2 Model 3 OR (95% CI) P OR (95% CI) P OR (95% CI) P Baseline ADAS-Cog 1.04 (1.01, 1.07) 0.015* 1.04 (1.01–1.08) 0.009* 1.04 (1.01–1.08) 0.011* Age 1.10 (1.05–1.15) < 0.001* 1.10 (1.05–1.16) < 0.001* Gender (Female) 0.61 (0.32–1.18) 0.143 0.63 (0.32–1.24) 0.178 Study Arm 0.82 (0.44–1.54) 0.534 0.70 (0.37–1.36) 0.294 Body Mass Index 1.09 (1.01, 1.16) 0.023* 1.10 (1.02, 1.18) 0.014* Education (Years) 1.08 (0.99–1.16) 0.076 1.08 (0.99–1.17) 0.064 Diagnosis Duration 1.07 (0.90–1.26) 0.443 1.07 (0.90–1.26) 0.466 Total Comorbidities 0.96 (0.82–1.11) 0.531 Diabetes Mellitus 0.41 (0.11–1.59) 0.195 Arthritis 0.54 (0.14–2.10) 0.373 Total Medications 1.06 (0.94–1.21) 0.330 Benzodiazepine Use 1.49 (0.55–4.01) 0.433 Antidepressant Use 0.99 (0.49–1.98) 0.989 Page 7 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 than threefold increased risk of falls ove period in those with mild to moderate A of the first studies in the literature to ass dinal relationship between slow gait spee function in those with a diagnosis of AD At baseline, the only significant predict speed were greater age and poorer cogn variate analysis. This finding is noteworth consistent with previous studies in the l have demonstrated a relationship betw function and gait speed in a cross-sectio those with AD [17–19]. The lack of association between baseline and cognitive outcomes is of interest. Acknowledgements NILVAD Study Group. Verhey, Department of Psychiatry and Neuropsychology, School of Mental Health and Neuroscience, Alzheimer Center Limburg, Maastricht University, Maas- tricht, the Netherlands; Raffaello Nemni, IRCCS Don Gnocchi Foundation- University of Milan, Italy; Flavio Nobili, Dept. of Neuroscience (DINOGMI), Uni- versity of Genoa, and IRCCS AOU Polyclinic, Hospital San Martino, Genoa, Italy; Massimo Franceschi, Neurology Department, Multimedica, Castellanza, Italy; Giovanni Frisoni, Centro San Giovanni di Dio—IRCCS Fatebenefratelli, Brescia, Italy; Orazio Zanetti, Centro San Giovanni di Dio—IRCCS Brian Lawlor, Mercer’s Institute for Research on Ageing, St. James’s Hospital and Department of Medical Gerontology, Trinity College, Dublin, Ireland; Ricardo Segurado, CSTAR and School of Public Health, Physiotherapy and Sport Science, University College Dublin (UCD), Dublin, Ireland; Sean Kennelly, Department of Age Related Healthcare, Tallaght Hospital, Dublin 24 and Department of Medical Gerontology, Trinity College Dublin; Marcel G. M. Olde Rikkert, Department of Geriatric Medicine, Radboud umc Alzheimer Center, Donders Institute of Medical Neurosciences, Radboudumc, Nijmegen, the Netherlands; Robert Howard, Division of Psychiatry, University College London and King’s College London; Florence Pasquier, CHU Lille, Univ. Lille, DISTALZ Laboratory of Excellence, F-59000 Lille, France; Anne Bo¨rjesson-Han- son, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg; Magda Tso- laki, Papanikolaou General Hospital of Thessaloniki, Greece; Ugo Lucca, La- boratory of Geriatric Neuropsychiatry, IRCCS Istituto di Ricerche Our study has several limitations. Principal amongst these is the fact that gait was assessed as a binary vari- able. Applying such a stringent cut-off in a dichotomous fashion may not produce the same results as analysis re- peated with gait speed as a continuous variable in meters per second for instance. However, in the current study we were limited by the fact that gait speed was recorded as an individual binary variable and not a specific speed in metres per second. This may introduce less accuracy into our results than more detailed recording of gait speeds. Future longitudinal studies should examine the intricacies of the gait-cognition relationship in those with a diagnosis of dementia. A further limitation of the current study is that gait and cognition was only consid- ered over an 18 month period. Such a period may be too short to demonstrate the relationship between gait speed and cognition. Acknowledgements NILVAD Study Group. Brian Lawlor, Mercer’s Institute for Research on Ageing, St. James’s Hospital and Department of Medical Gerontology, Trinity College, Dublin, Ireland; Ricardo Segurado, CSTAR and School of Public Health, Physiotherapy and Sport Science, University College Dublin (UCD), Dublin, Ireland; Sean Kennelly, Department of Age Related Healthcare, Tallaght Hospital, Dublin 24 and Department of Medical Gerontology, Trinity College Dublin; Marcel G. M. Olde Rikkert, Department of Geriatric Medicine, Radboud umc Alzheimer Center, Donders Institute of Medical Neurosciences, Radboudumc, Nijmegen, the Netherlands; Robert Howard, Division of Psychiatry, University College London and King’s College London; Florence Pasquier, CHU Lille, Univ. Lille, DISTALZ Laboratory of Excellence, F-59000 Lille, France; Anne Bo¨rjesson-Han- son, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg; Magda Tso- laki, Papanikolaou General Hospital of Thessaloniki, Greece; Ugo Lucca, La- boratory of Geriatric Neuropsychiatry, IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; D. William Molloy, University Col- lege Cork Centre for Gerontology and Rehabilitation, Cork, Ireland; Robert Coen, Mercer’s Institute for Research on Ageing, St. James’s Hospital, Dublin, Ireland; Matthias W. Riepe, Department of Geriatrics and Old Age Psychiatry, Psychiatry II, Ulm University at BKH Gu¨nzburg, Germany; Ja’nos Ka’lma’n, De- partment of Psychiatry, University of Szeged, Hungary; Rose Anne Kenny, De- partment of Medical Gerontology, Trinity College Dublin (TCD), Dublin, Ireland; Fiona Cregg, Department of Medical Gerontology, Trinity College Dublin (TCD), Dublin, Ireland; Sarah O’Dwyer, Mercer’s Institute for Research on Ageing, St. James’s Hospital, Dublin, Ireland; Cathal Walsh, Health Re- search Institute and MACSI, Department of Mathematics and Statistics, Uni- versity of Limerick, Ireland; Jessica Adams, Department of Old Age Psychiatry, King’s College London; Rita Banzi, Laboratory of Geriatric Neuropsychiatry, IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; Laetitia Breuilh, CHU Lille, Univ. Lille, DISTALZ Laboratory of Excellence, F-59000 Lille, France; Leslie Daly, CSTAR and School of Public Health, Physiotherapy and Sport Science, University College Dublin (UCD), Dublin, Ireland; Suzanne Hen- drix, Pentara Corporation, 2180 Claybourne Avenue, Salt Lake City, Utah; Paul Aisen, Department of Neurology, University of Southern California; Siobhan Gaynor, Molecular Medicine Ireland (MMI), Dublin, Ireland; Ali Sheikhi, Health Research Institute and MACSI, Department of Mathematics and Statistics, Uni- versity of Limerick, Ireland; Diana G. Taekema, Department of Geriatric Medi- cine, Rijnstate Hospital, Arnhem, the Netherlands; Frans R. Discussion ADAS-Cog = Alzheimer Disease Assessment Scale – Cognitive Subsection Predictor Model 1 Model 2 Model 3 IRR (95% CI) P IRR (95% CI) P IRR (95% CI) P Baseline Slow Gait Speed 3.18 (1.97–5.11) < 0.001 3.27 (1.96–5.50) < 0.001* 3.09 (1.82–5.22) < 0.001* Baseline ADAS-Cog 0.95 (0.92–0.97) 0.001* 0.95 (0.92–0.98) 0.001* Age 1.07 (1.04–1.10) < 0.001* 1.07 (1.03–1.10) < 0.001* Gender (Female) 1.90 (1.15–3.11) 0.012* 1.60 (0.97–2.65) 0.068 Study Arm 1.20 (0.78–1.83) 0.409 1.32 (0.85–2.06) 0.218 Body Mass Index 0.98 (0.94, 1.03) 0.489 0.96 (0.92–1.01) 0.149 Education (Years) 1.07 (1.01–1.12) 0.021* 1.10 (1.04–1.15) 0.001* Diagnosis Duration 1.02 (0.88–1.17) 0.816 0.96 (0.84–1.11) 0.612 Total Comorbidities 0.97 (0.88–1.07) 0.527 Diabetes Mellitus 1.73 (0.85–3.51) 0.128 Arthritis 2.19 (1.19–4.04) 0.012* Total Medications 1.03 (0.95–1.12) 0.490 Benzodiazepine Use 1.05 (1.04–3.64) 0.036* Antidepressant Use 0.67 (0.41–1.07) 0.096 Page 8 of 10 Page 8 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 than 15% of the participants with mild to moderate AD experienced at least one fall over the study period. Tar- geted interventions for falls prevention focussed on those with AD and slow gait speed are warranted and slow gait speed may be a way to select those with mild to moderate AD who are particularly vulnerable to ex- periencing falls, given the adverse consequences of falls in such a cohort [23]. with incident slow gait speed over 18 months. However, there was no association between baseline slow gait speed and cognition at follow-up. We also report a sig- nificant increase in falls risk over an 18 month period in those with baseline slow gait speed. Further studies should continue to explore the nature of the gait- cognition relationship specifically in those with Alzhei- mer Disease in helping to tease out which patients may be particularly vulnerable to further cognitive decline and adverse health outcomes. An important consideration in the present study is the nature of the included cohort. Notably, patients with other neurological disorders such as Parkinson’s Disease were excluded. Further, patients with a significant car- diac history, previous stroke etc. were all excluded. Thus, the cohort of included patients had fewer comorbidities which may impair cognition than in other naturalistic studies. This enhances the current study in assessing the relationship between gait and cognition specifically in mild-to-moderate AD. Abbreviations AD Al h i D AD: Alzheimer Disease; ADAS-Cog: Alzheimer’s Disease Assessment Scale, Cognitive Subsection; ATC: Anatomic Therapeutic Classification; BMI: Body Mass Index; CDR-Sb: Clinical Dementia Rating, Sum of Boxes; MCI: Mild Cognitive Impairment; NINCDS-ADRDA for AD: National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer’s Disease Criteria; NILVAD: A European Multicentre Double Blind-Placebo Controlled Phase III Trial of Nilvadipine in Mild-to-Moderate Alzheimer’s Disease Acknowledgements NILVAD Study Group. Other strengths of this study include its international nature as well as the high fidelity of follow up in in- cluded patients and the large amount of clinical and medical history data available. This enabled us to control for other causes of gait disorders such as arthritis and diabetes in our analysis. Further, it enabled us to include the effects of certain medications on incident slow gait speed and falls, such as benzodiazepines, which have been previously linked with falls in this vulnerable popu- lation [32]. Acknowledgements NILVAD Study Group. Poorer baseline cognition was associated Page 9 of 10 Page 9 of 10 Page 9 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Fatebenefratelli, Brescia, Italy; Anastasia Konsta, Aristotle University of Thessa- loniki (AUTH), First Psychiatric Department, Papageorgiou General Hospital, Greece; Orologas Anastasios, Ahepa University General Hospital of Thessalo- niki, Greece; Styliani Nenopoulou, Papanikolaou General Hospital of Thessalo- niki, Greece; Fani Tsolaki-Tagaraki, Papanikolaou General Hospital of Thessaloniki, Greece; Magdolna Pakaski, Department of Psychiatry, University of Szeged, Hungary; Olivier Dereeper, Centre Hospitalier de Calais, France; Vincent de la Sayette, Centre Hospitalier Universitaire de Caen, France; Olivier Se′ne’chal, Centre Hospitalier de Lens, France; Isabelle Lavenu, Centre Hospi- talier de Be′thune, France; Agnès Devendeville, Centre Hospitalier Universi- taire d’Amiens, France; Gauthier Calais, Groupement des Hoˆpitaux de l’Institut Catholique de Lille (GHICL), France; Fiona Crawford, Archer Pharma- ceuticals, Sarasota, Florida, and Roskamp Institute, Sarasota, Florida; Michael Mullan, Archer Pharmaceuticals, Sarasota, Florida, and Roskamp Institute, Sar- asota, Florida, Pauline Aalten, PhD, Department of Psychiatry and Neuro- psychology, School of Mental Health and Neurosciences, Alzheimer Center Limburg, Maastricht University, Maastricht, the Netherlands; Maria A. Ber- glund, RN, Sahlgrenska University Hospital, Gothenburg, Sweden; Jurgen A. Claassen MD, PhD, Department of Geriatric Medicine, Radboudumc Alzhei- mer Center, Donders Institute of Medical Neurosciences, Radboudumc, Nij- megen, the Netherlands; Rianne A. De Heus, MSc, Department of Geriatric Medicine, Radboudumc Alzheimer Center, Donders Institute of Medical Neu- rosciences, Radboudumc, Nijmegen, the Netherlands; Daan L. K. De Jong, MSc, Department of Geriatric Medicine, Radboudumc Alzheimer Center, Don- ders Institute of Medical Neurosciences, Radboudumc, Nijmegen, the Netherlands; Olivier Godefroy, MD, PhD, Centre Hospitalier Universitaire d’Amiens, France; Siobhan Hutchinson, MD, St. Funding F di f Funding from the NILVAD study was from the European Commission Framework 7 Programme Health Theme Collaborative Project (grant 279093; PI: Brian Lawlor). The funding agency had no role in the design, recruitment, conduct and data collection of the clinical trial and no input in the analysis, interpretation of data and presentation of primary/secondary trial results. 11. Dumurgier J, Artaud F, Touraine C, Rouaud O, Tavemier B, Dufouil C, et al. Gait speed and decline in gait speed as predictors of incident dementia. J Gerontol A Biol Sci Med Sci. 2017;72A:655–61. 12. Welmer A-K, Rizzuto D, Qiu C, Caracciolo B, Laukka EJ. Walking speed, processing speed, and dementia: A population-based longitudinal study. J Gerontol A Biol Sci Med Sci. 2014;69A:1503–10 12. Received: 9 July 2019 Accepted: 23 March 2020 Received: 9 July 2019 Accepted: 23 March 2020 Received: 9 July 2019 Accepted: 23 March 2020 Competing interests Th h d l h The authors declare that they have no competing interests. References ll 1. Puvill T, Lindenberg J, Gussekloo J, de AJM C, Slaets JP, RGJ W. 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Clin Rehabil. 2003;17:108–13. 8. Aditya BS, Sharma JC, Allen SC, Vassallo M. Predictors of a nursing home placement from a non-acute geriatric hospital. Clin Rehabil. 2003;17:108–13. AHD, BL, SPK: Conception and Design of Secondary Analysis, Statistical Analysis of Results and Writing/Revising the final manuscript. NILVAD Study Group (Principal Investigatory: BL): Design, analysis and conduct of the NILVAD clinical trial. The author(s) read and approved the final manuscript. 9. Wilson RS, Schneider JA, Beckett LA, Evans DA, Bennett DA. Progression of gait disorder and rigidity and risk of death in older persons. Neurology. 2002;58:1815–9. 10. Hackett RA, Davies-Kershaw H, Orrell CD, Steptoe A. Walking Speed, cognitive Function, and Dementia risk in the English Longitudinal Study of Ageing. 2018;66:1270–5. Acknowledgements NILVAD Study Group. James’s Hospital, Dublin, Ireland; Aikaterini Ioannou, MD, 1st Department of Neurology, Ahepa Univer- sity General Hospital, Aristotle University of Thessaloniki, Greece; Michael Jonsson, MD, PhD, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothen- burg, Sweden; Annette Kent, PhD, Trinity College Dublin (TCD), Dublin, Ireland; Ju¨rgen Kern MD, PhD, Department of Psychiatry and Neurochem- istry, Institute of Neuroscience and Physiology, Sahlgrenska Academy, Univer- sity of Gothenburg, Sweden; Petros Nemtsas MD, PhD, 1st Department of Neurology, Ahepa University General Hospital, Aristotle University of Thessa- loniki, Greece; Minoa-Kalliopi Panidou, BSc, MA, 1st Department of Neurology, Ahepa University General Hospital, Aristotle University of Thessaloniki, Greece; Laila Abdullah, PhD, Roskamp Institute, Sarasota, Florida; Daniel Paris, PhD, Roskamp Institute, Sarasota, Florida; Angelina M. Santoso, MD, MSc, Depart- ment of Geriatric Medicine, Radboudumc Alzheimer Center, Donders Institute of Medical Neurosciences, Radboudumc, Nijmegen, the Netherlands; Gerrita J. van Spijker, MSc, Department of Geriatric Medicine, Radboudumc Alzhei- mer Center, Donders Institute of Medical Neurosciences, Radboudumc, Nij- megen, the Netherlands; Martha Spiliotou MD, PhD, 1st Department of Neurology, Ahepa University General Hospital, Aristotle University of Thessa- loniki, Greece; Georgia Thomoglou, BSc, 1st Department of Neurology, Ahepa University General Hospital, Aristotle University of Thessaloniki, Greece; and Anders Wallin, MD, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothen- burg, Sweden. Acknowledgements NILVAD Study Group. Previous studies in the literature have demonstrated a relationship over a longer period of time and we cannot rule out that following this cohort for a longer period of time may demonstrate a significant rela- tionship between baseline gait speed and cognition at follow-up. boratory of Geriatric Neuropsychiatry, IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; D. William Molloy, University Col- lege Cork Centre for Gerontology and Rehabilitation, Cork, Ireland; Robert Coen, Mercer’s Institute for Research on Ageing, St. James’s Hospital, Dublin, Ireland; Matthias W. Riepe, Department of Geriatrics and Old Age Psychiatry, Psychiatry II, Ulm University at BKH Gu¨nzburg, Germany; Ja’nos Ka’lma’n, De- partment of Psychiatry, University of Szeged, Hungary; Rose Anne Kenny, De- partment of Medical Gerontology, Trinity College Dublin (TCD), Dublin, Ireland; Fiona Cregg, Department of Medical Gerontology, Trinity College Dublin (TCD), Dublin, Ireland; Sarah O’Dwyer, Mercer’s Institute for Research on Ageing, St. James’s Hospital, Dublin, Ireland; Cathal Walsh, Health Re- search Institute and MACSI, Department of Mathematics and Statistics, Uni- versity of Limerick, Ireland; Jessica Adams, Department of Old Age Psychiatry, King’s College London; Rita Banzi, Laboratory of Geriatric Neuropsychiatry, IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; Laetitia Breuilh, CHU Lille, Univ. Lille, DISTALZ Laboratory of Excellence, F-59000 Lille, France; Leslie Daly, CSTAR and School of Public Health, Physiotherapy and Sport Science, University College Dublin (UCD), Dublin, Ireland; Suzanne Hen- drix, Pentara Corporation, 2180 Claybourne Avenue, Salt Lake City, Utah; Paul Aisen, Department of Neurology, University of Southern California; Siobhan Gaynor, Molecular Medicine Ireland (MMI), Dublin, Ireland; Ali Sheikhi, Health Research Institute and MACSI, Department of Mathematics and Statistics, Uni- versity of Limerick, Ireland; Diana G. Taekema, Department of Geriatric Medi- cine, Rijnstate Hospital, Arnhem, the Netherlands; Frans R. Verhey, Department of Psychiatry and Neuropsychology, School of Mental Health and Neuroscience, Alzheimer Center Limburg, Maastricht University, Maas- tricht, the Netherlands; Raffaello Nemni, IRCCS Don Gnocchi Foundation- University of Milan, Italy; Flavio Nobili, Dept. of Neuroscience (DINOGMI), Uni- versity of Genoa, and IRCCS AOU Polyclinic, Hospital San Martino, Genoa, Italy; Massimo Franceschi, Neurology Department, Multimedica, Castellanza, Italy; Giovanni Frisoni, Centro San Giovanni di Dio—IRCCS Fatebenefratelli, Brescia, Italy; Orazio Zanetti, Centro San Giovanni di Dio—IRCCS In conclusion, we demonstrate a significant baseline association between cognition and slow gait speed in a large cohort of participants with mild to moderate Alz- heimer Disease. Ethics approval and consent to participate Ethical approval for the trial was granted from the appropriate National Competent Authorities, Independent Ethics Committees and Institutional Review Boards for all study sites. Ethical Approval for the study also included secondary analysis of NILVAD study data. Written consent was obtained from each participant/caregiver. Individual ethics committees that approved the original trial protocol are as follows: Comite de Protection des Personnes Nord Ouest III, Scientific Council of Papanikolaou Hospital Thessaloniki, Radbound Universitar Medisch Centrum, Concernstaf Kwaliteit en Veiligheid Comissie Mensgebonden Onderzoek Regio Arnhem-Nijmegen, Medical Re- search Council Ethics Committee for Clinical Pharmacology (KFEB), Comitato Etico Istituzioni Ospedaliere Cattoliche (CEIOC), Comitato Etico IRCCS Multi- Medica, Camitato Ethico dell’Aienda Ospedalier a Universitaria S Martino di Genova, Comitato Etico Regione Liguria, Comitato Etico Fondaione Don Carlo Gnocchi. Author details 1 f 1Department of Age-Related Healthcare, Tallaght University Hospital, Dublin, Ireland. 2Department of Medical Gerontology, School of Medicine, Trinity College Dublin, Dublin 2, Ireland. 3Mercer’s Institute for Research on Ageing, St James’s Hospital, Dublin, Ireland. Availability of data and materials 13. Taniguchi Y, Kitamura A, Seino S, Murayama H, Amano H, Nofuji Y, et al. Gait performance trajectories and incident disabling dementia among community-dwelling older Japanese. J Am Med Dir Assoc. 2017;18(2):192. e13–20. Due to the chance of individual participant identification in the current trial, data is not publicly available. Requests to obtain anonymised study data can be addressed to dyera@tcd.ie Page 10 of 10 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 Dyer et al. BMC Geriatrics (2020) 20:117 14. Doi T, Tsutsuminoto K, Nakakubo S, Kim MJ, Kurita S, Shimada H. Rethinking the relationship between spatiotemporal gait variables and dementia: a prospective study. J Am Med Dir Assoc. 2019;20(7):899–903. 15. Peel NM, Alapatt LJ, Jones LV, Hubbard RE. The association between gait speed and cognitive status in community-dwelling older people: a systematic review and meta-analysis. J Geront Ser A. 2019;74(6):943–8. 16. Kuate-Tegueu C, Avila-Funes JA, Simo N, LeGoff M, Amieva H, Dartigues JF, et al. Association of Gait Speed, psychomotor speed and dementia. J Alzheimers Dis 2017. 2017;60(2):585–92. 17. Taylor ME, Brodie MA, van Schooten KS, Delbaere K, JCT C, Payne N. (2019) Older People with Dementia Have Reduced Daily-Life Activity and Impaired Daily-Life Gait When Compared to Age-Sex Matched Controls. J Alzheimers Dis. 2019. https://doi.org/10.3233/JAD-181174. 18. Toots ATM, Taylor ME, Lord SR, Close JCT. Associations between gait speed and cognitive domains in older people with cognitive impairment. J Alzheimers Dis. 2019. https://doi.org/10.3233/JAD-18117. 19. Knapstad MK, Steihuag OM, Aaslund MK, Nakling A, Naterstad IF, Fladby T, et al. Reduced walking speed in subjective and mild cognitive impairment: a cross-sectional study. J Geriatr Phys Ther. 2018. https://doi.org/10.1519/JPT. 0000000000000157. 20. Hooghiemstra AM, Ramakers IHGB, Sistermans N, Pignenburg YAL, Aalten P, Hamel REG, et al. Gait speed and grip Srength reflect cognitive impairment and are modestly related to incident cognitive decline in memory clinic patients with subjective cognitive decline and mild cognitive impairment: findings from the 4C study. Jour Gerontol A. 2017;72(6):846–54. 21. Taylor ME, Lasschult DA, Lord SR, Delbaere K, Kurrie SE, Mikolaizak AS, et al. Slow gait speed is associated with executive function decline in older people with mild to moderate dementia: a one year longitudinal study. Arch Gerontol Geriatr. 2017;73:148–53. 22. Allali G, Verghese J. Management of Gait Changes and Fall Risk in MCI and dementia. Curr Treat Options Neurol. 2017;19:29. 23. Buchner DM, Larson EB. Availability of data and materials J Am Med Dir Assoc. 2019. https://doi.org/10.1016/j.jamda.2019.08.006. 32. Dyer AH, Murphy C, Lawlor B, Kennelly SP, NILVAD Study Group. Cognitive outcomes of long-term benzodiazepine and related drug (BDZR) use in people living with mild to moderate Alzheimer’s disease: results from NILVAD. J Am Med Dir Assoc. 2019. https://doi.org/10.1016/j.jamda.2019.08.006. Availability of data and materials Falls and fractures in patients with Alzheimer-type dementia. JAMA. 1987;257(11):1492. 24. vanDorn C, Gruber-Baldini AL, Zimmerman S, Hebel JR, Port CL, Baumgarten M, et al. Dementia is a risk factor for falls and fall injuries in nursing home residents. J Am Geriatr Soc. 2003;51(9):1213–8. 25. Taylor ME, Delbaere K, Lord SR, Mikolaizak AS, Brodaty H, Close JC, et al. Neuropsychological, physical and functional mobility measures associated with falls in cognitively impaired in cognitively impaired older adults. J Gerontol A Biol Sci Med Sci. 2014;69(8):987–95. 26. Taylor ME, Delbaere K, Mikolaizak AS, Lord SR, Close JC, et al. Gait parameter risk factors for falls under simple and dual-task conditions in cognitively impaired older people. Gait Posture. 2013;37(1):126–30. 27. Modarresi S, Divine A, Grahn JA, Overend TJ, Hunter SW, et al. Gait parameters and characteristics associated with increased risk of falls in people with dementia: a systematic review. Int Psychogeriatr. 2018:1–17. https://doi.org/10.1017/S1041610218001783. 28. Lawlor B, Kennelly S, O’Dwyer S, Cregg F, Walsh C, Cohen R, et al. NILVAD protocol: a European multicentre double-blind placebo-controlled trial of nilvadipine in mild-to-moderate Alzheimer’s disease. BMJ Open. 2014;4(10): e006364. 29. Meulenbroek O, O’Dwyer S, de Jong D, van Spijker G, Kennelly S, Cregg F, et al. 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https://openalex.org/W2969785912	https://sajbm.org/index.php/sajbm/article/download/1121/1062	English	null	Never-buyers of consumer non-durables	South African journal of business management	1,984	cc-by	5,593	Introduction In the field of consumer non-durables, an interesting segment of the market, known as 'never-buyers', has caused some dis- cussion in the literature. Ehrenberg (1972) claimed that, given a sufficiently long period of time, every consumer will eventually purchase a pro- duct at least once, and so the never-buyer does not exist. If a study shows a number of non-buyers in a period of time, Ehrenberg believes that, had the time of the study been longer, these non-buyers would not have been found. In fact, his Negative Binomial Distribution (NBD) theory which proposes that, provided the market is stationary frequently purchased consumer non-durables can be modelled using the NBD, (Equation Al) predicts this outcome. As time increases, the proportion of buyers of the product as predicted by the NBD tends to unity (Equation A2). (A brief description of the statistical distributions mentioned in the text is given in the Appendix.) Purely from a logical point of view one may seek to raise issue with Ehrenberg on the question of never-buyers. Non- smokers probably never buy cigarettes, non-drivers who do not own vehicles are unlikely to purchase petrol. Product classes can therefore be identified in which, by logical conside- rations alone, one would expect to find some never-buyers. Nevertheless, one should concede to Ehrenberg that such examples probably represent a small proportion of the con- sumer non-durables market. S. Afr. J. Bus. Mgmt. 1984, 15: 155- 161 In die artikel word die konsep van nooit-kopers van nle-duursame verbruikersgoedere bespreek. Ehrenbergse tradisionele Negatief· binomiaaldistribusie-benadering in verband met die vraag is voorgestel. Vorige ongepubliseerde werk uitgevoer by die Nagraadsebestuurskool, Universiteit van die Witwatersrand is bespreek, en hipoteses word voorgestel dat die waargenome groot nul-sel in die verkoops-frekwensiedistribusies miskien 'n gevolg mag wees van die bestaan van 'n groep nooit-kopers van die pro- duk, of as 'n gevolg van die opeenplasing van ten minste twee af- sonderlike koperpopulasies voorheen geidentifiseer as handels- naam-lojaal en handelsnaam-wlsselende gesinne. Die resultate van die navorsing wat gedoen is om die eerste hipotese te toets, word gegee. Twee sorgvuldig gekontroleerde stelsels data is gemodelleer met gebruik van die nulvermeerderde Negatief- binomiaal- en Sichel-distribusies. lndividuele handelsname in een datastesel (aankope van toiletseep) volg die voorspelling van die tra- disionele teorie - die proporsie van nie-verbruikers neem mettertyd af. C. Firer Graduate School of Business Administration, University of the Wit- watersrand, P.O. Box 31170, Braamfontein 2017, Republic of South Africa Never-buyers of consumer non-durables C. Firer Graduate School of Business Administration, University of the Witwatersrand, Johannesburg C. Firer Graduate School of Business Administration, University of the Witwatersrand, Johannesburg C e Graduate School of Business Administration, University of the Witwatersrand, Johannesburg In this article the concept of never-buyers of consumer non- durables is discussed. The traditional Negative Binomial Distribu- tion approach of Ehrenberg to the question is presented. Previous- ly unpublished work carried out at the Graduate School of Business Administration, University of the Witwatersrand, Is reviewed and hypotheses are put forward that the observed large zero cell in the purchase frequency distributions may be caused by the existence of a group of never-buyers of the product, or by the superimposition of at least two distinct buying populations, previously identified as brand-loyal and multibrand/brand-switchlng households. The results of the research aimed at testing the first hypothesis are presented here. Two carefully monitored data sets were modelled using zero-augmented Negative Binomial and Sichel distributions. The data were previously shown to exhibit the necessary mean households purchase/consumption stationarity. In- dividual brands in one data set (purchases of toilet soap) were shown to follow the predictions of the traditional theory - the proportion of non-buyers decreasing with time. In the second data set (consumption of packaged soup) the proportion of non- consumers of the brands fell towards zero as the length of the time period studied was increased, but at a rate faster than that predicted by the theory. The hypothesis of the existence of never- buyers/users of individual brands in these two product classes was therefore rejected. Introduction In die tweede datastelsel (verbruik van verpakte sop) het die proporsie van nie-verbruikers van die handelsname na nul gedaal met verlenging van die tydperk wat bestudeer is, maar teen 'n snelheid vinnlger as wat voorspel was deur die teorle. Die hipotese dat daar noolt-kopers/gebruikers van individuele handelsname in die twee produkklasse bestaan het, is dus verwerp. S.-Afr. Tydskr. Bedryfsl. 1984, 15: 155-161 Ehrenberg also claimed that his model is as applicable to an individual brand as it is to the product class as a whole. However, with respect to individual brands the situation may be quite different. Ehrenberg and other overseas writers have, with few published exceptions, only tested the NBD model in its univariate form. Where they found departures from the model, they ascribed them to effects which they labelled 'the shelving phenomenon' or 'the variance discrepency'. They believed this effect - an observed discontinuity in the frequen- cy of heavier purchases at or close to the number of units, equal to the number of weeks or period in the time period of the study- to be caused by a small sector of the market pur- chasing the product regularly. Sichel (1975) investigated this phenomenon using observa- tions on packaged-soup consumption. By their very nature, being consu.-nption-based, such data, according to Ehrenberg's theory, ought not to exhibit the effect described above. However, 'shelving' was observed in the data. C. Firer She also found that other statistical tests revealed signifi- cant departures from Ehrenberg's NBD model. As was subse- Accepted May 1984 S.-Afr. Tydskr. Bedryfsl. 1984, 15(3) 1S6 they were plotted in the fonn of frequency distributions, even- numbered purchase frequencies appeared high relative to the odd-numbered purchase frequencies, giving a zig-z.ag shaped distribution (Figure 1). This was attributed by Goodman to a consumer preference for purchasing the product in even- numbered lots. quently demonstrated by Zachos (l 977), although univariate distributions predicted by NBD theory may be accepted by the chi-square test, predictions in the bivariate fonn of the NBD often provide very unsatisfactory models. As a result, both of her own work and an earlier study by Tucker (1973), Sichel (1975) suggested that the true phen~ menon of repeat-consumption/buying may be represented by two distinctly different populations: 200 100 oL----~~~~~~:::::::c.-...:A::.. 0 2 4 6 8 10 12 14 No. OF OOTS PURCHASED Figure 1 Observed frequency distribution for Palmolive soap - eight months (N = 614) 200 100 oL----~~~~~~:::::::c.-...:A::.. 0 2 4 6 8 10 12 14 No. OF OOTS PURCHASED (a) A hard-core of stable, brand-loyal households which con- sume regularly in accordance with Ehrenberg's original model.' These therefore would be expected to follow the correlated NBD model (Equation A3). (a) A hard-core of stable, brand-loyal households which con- sume regularly in accordance with Ehrenberg's original model.' These therefore would be expected to follow the correlated NBD model (Equation A3). (b) 'A fair number of brand-switching, multibrand consum- ing households.' This group is statistically characterized by the uncorrelated bivariate NBD (Equation AS) in suc- cessive time periods. p She put forward the hypothesis that the observed bivariate distribution arose from the superimposition of these two populations. This hypothesis was supported by her work on the variation of market penetration, number of new and repeat consumers and correlation coefficients with increasing lengths of the observational period. She found that the observations usually lay between the predictions of the correlated and un- correlated models. Firer & Barnett (l 979) found similar results in the toilet soap market. Figure 1 Observed frequency distribution for Palmolive soap - eight months (N = 614) Joannides (l 976) fonnulated a binary model by combining the correlated and uncorrelated bivariate NBDs. He showed that his model could produce many of the observed shapes, patterns (including a shelO, and values of parameters which the original NBD theory could not. The two data sets analysed were: Soup consumption displayed a seasonal pattern. It dropped during spring and rose in autumn. Three separate stationary periods could thus be identified: Soup In the early 1970s the packaged-soup market in South Africa was dominated by the leading brand (here designated brand A) followed at some distance by the only other brand with a reasonable market share (designated brand B). Nine other brands made up the rest of the market. All brands were presented in unifonn packagings of four to six servings each. Joannides (1976) identified these populations as brand-loyal and multibrand, brand-switching households. However, the same distribution shape could arise from the superimposition of a buying population (which may, for a given time period contain non-buyers who will buy in a future period) and a group of never-buyers (whose distribution would consist of a certain frequency in the zero cell only). The data referred to as 'soup' in this paper were collected by Sichel (1975) as part of a South African National Dustbin Panel Survey. Housewives were provided with a bin into which they discarded empty packets of soup, the contents of which had been consumed by their families. Every twenty-eight days (i.e. at standard monthly intervals) the housewives were visited by interviewers who recorded the contents of their bins. The objective of the research reported in this article was to test these hypotheses by investigating the proportion of never- buyers in two frequently purchased South African consumer non-durables product classes. Accepted May 1984 For the purpose of testing a distribution fit to this data it was therefore necessary to group the cells. The z.ero cell was allowed to stand alone, the one-plus-two purchase frequency cells, the three-plus-four cells, etc., were grouped together. In practice, purchase/ consumption frequency distributions can be observed in which the size of the z.ero cell appears exces- sively large when compared to the distribution as a whole. This suggests that the distribution is not drawn from a single homogeneous population, but that at least two distinct popula- tions are involved. Data Infonnation relating to 512 households' consumption for sixteen twenty-eight day periods was collated. Table 1 Non-buyers of leading brands Table 1 Non-buyers of leading brands Table 1 Non-buyers of leading brands Proportions of non-buyers Brand over period studied Lux 0,450 Palmolive 0,492 Sunlight Mild 0,590 Breeze 0,651 Shield 0,694 Soup Brand A 0,203 Soup Brand B 0,309 According to Sichel (1980), individual brands need not neces- sarily follow the same distribution as that postulated for a total product field, and therefore Goodman's (1979) omission in not studying the zero-augmented NBD as a potential model for the individual brands is open to some criticism. This study therefore began by attempting to fit a zero- augmented NBD model to the individual brand data for Lux and Palmolive soaps. Earlier research (Firer & Barnett, 1979) had shown that for all time periods studied, the chi-square test rejected the hypothesis of an ordinary NBD model for the data grouped as suggested above. Parameter estimates for the model were made on ungrouped data. As can be seen from Table 2, the ungrouped data 'failed' the chi-square test. A detailed study of the tests revealed that the 'odd-even' purchase phenomenon was responsible for a substantial proportion of the total chi-square. The tests were thus repeated using grouped data. The zero-augmented Sichel distribution (-y = - 0,5) was then fitted to the same grouped data using the same tail-end grouping of cells of expected fre- quency less than 5,0. A visual inspection of the brand purchase frequency distribu- tions revealed certain interesting phenomena. First, in almost all cases a kink or a flattening out, and sometimes even a slight maximum, occurred at about the second or third cell. In the case of the toilet soap distributions, the problem of the con- sumers' favouring of even-numbered purchases (discussed above) may have been contributing towards this effect. Second- ly, in visually sweeping a smooth curve upwards through the data points, one would have expected such a curve to intercept the frequency axis at a much lower point than that actually recorded. An example from eacq data set is shown in Figures 1 and 2. From Table 2 it can be seen that, contrary to Goodman's expectations, the better fit was provided by the zero-augmented NBD model. An example of the fits obtained is shown in Figure 3. Systematic deviations of the two models were found. The Sichel distribution was first too low, then too high, then too low again, relative to the NBD. Soap The data referred to as 'soap' originate from the toilet soap product class. In South Africa this product class comprised more than thirty-five different brands, but was dominated by the two leading brands, Lux and Palmolive. Periods one through four (the first stationary winter period): - periods seven through ten (the stationary summer period); The data base used consisted of a sample of 614 White households' bimonthly purchases of toilet soap, over the period April to November 1978, details of which were collected by Market Research Africa using the consumer panel method. - periods thirteen through sixteen (the second stationary winter period). Similar mean consumption levels were found for the two winter periods. Goodman (1979) tested the data and concluded that pur- chasing rates over the eight-month period were stationary. The observations had one peculiarity worthy of mention. When This set of data differs fundamentally from the toilet soap observations in that it contains consumption and not purchase data. Thus idosyncrasies of individual households such as the 1so 100 so 6 8 10 12 14 No. Of OOTS Coo.HO Figure 2 Observed frequency distribution for combined brands A and B of soup - first four months (N = 614) S. Afr. J. Bus. Mgmt. 1984, 15(3) frequency of shopping and the number of units purchased on each shopping occasion are removed. Consumption should therefore produce a more uniformly distributed set of data. Table 1 Non-buyers of leading brands According to Sichel (1980) the nature of the deviations led one to the conclusion that decreas- ing the value of 'Y from - 0,5 to - 1,5 or - 2,5 would not significantly improve the fit. The hypothesis put forward is that these two effects are caus- ed by an 'excess' of zeros in the first cell of the distributions, and that this excess in fact comprises never-buyers/consumers of the brand. It was anticipated that the observations could best be model- led using a zero-augmented distribution (See (c) in Appendix). It was therefore concluded that, in the toilet soap field, in- dividual brand purchase frequencies are best modelled by the univariate zero-augmented NBD. Never-Buyers Never-buyers of the product field Never-buyers of the product field In the toilet soap product field, all of the 614 households in the sample had purchased some soap during the eight-month period of the survey. Thus no never-buyers of toilet soap ex- isted in the sample. However, in the packaged-soup market, after two winter periods, 20 of the 512 households had not yet consumed. Sichel (1975) concluded that, because there were so few non-consu- mers, these 20 households did not provide sufficient evidence of the existence of never-consumers. This conclusion is accepted by the author and the analysis below is thus devoted to indivi- dual brands. Never-buyers of individual brands: Never-buyers of individual brands: Figure 2 Observed frequency distribution for combined brands A and B of soup - first four months (N = 614) A study of the soap and soup data reveals that even after eight and sixteen months respectively, substantial proportions of the households in the samples had not purchased/ consumed the leading brands (Table 1). amined only the Sichel distribution when analysing the indivi- dual brands within the product field. Results Figure 4 is portions of n tions were de were made b observations In no case dicted by Eh the hypothes brands in th Analysis of In this analys period of the data did not Although t not correct, i portion of n seen from F Table 3 Soap: Estimates of proportion of never- buyers q derived from zero-augmented models the proportion of never-buyers in the sample, whereas the pro- portion of non-buyers as predicted by Ehrenberg's theory would tend to zero with lengthening time periods (Ehrenberg, 1972). Figure 4 is an example of the observed and expected pro- portions of non-buyers of Lux soap. The expected propor- tions were derived using Equation A4. The parameter estimates were made by the method of maximum likelihood using the observations of the first two-month period. In no case did the observed proportion lie above that pre- dicted by Ehrenberg's theory. As a result of these findings, the hypothesis of the existence of never-buyers for individual brands in the toilet soap market was rejected. Results Analysis of each of the data sets is discussed separately. One of the implications of the zero-augmented model is that estimates obtained for q, the proportion of never-buyers in the sample, should be independent of time. Table 3 shows the results obtained from the fitting of both the NBD and Sichel zero-augmented models. These estimates are certainly not constant. Analysis of the Soap Data Goodman (1979) found that the fit of the Sichel distribution (Sichel, 1971) with -y = -2,5 ((b) in Appendix) was generally superior to the NBD with respect to the toilet soap product class (all brands included). Results As a result of this finding, she ex- 1S8 Table 2 Soap: Results of chi-square tests for zero-augmented distributions NBD NBD Sichel ('y = - 0,5) Ungrouped Grouped Grouped Lax 1st 2 months P(x2 ~ 1,531 I 2)< =0,465 • a 1st 4 months P(x2 !l;: 16,379 I 5) < = 0,050 P(x2 ~ 2,023 I 2) = 0,364 P(x2~ 2,548 I 2)=0,275 6 months P(x2!l;:29,982 I 8)< =0,050 P(x2~5.747 I 3)=0,124 P(x2 ~ 7,791 I 3)=0,051 8 months P(x2 ~ 19,5(,6 I 11)=0.052 P(;x2~7,385 I 5)=0,194 P(x2 ~ 11,422 I 5)<0,050 Palmolive 1st 2 months P(x2 ~ 1,482 I 2)=0,477 a a 1st 4 months P(x2!l;:25,474 I 5)<0,050 P(x2!l;:2,536 I 1)=0,lll P(x2 ~ 2,916 I 1)=0,088 6 months P(x2!l;:28,420 I 7)<0,050 P(x2!l;:5,991 I 3)=0,112 P(x2 !l;: 8,398 I 3) < 0,050 8 months P(x2~21,031 I 9)<0.050 P(x2~9,181 I 4)=0,057 P(;x2!l;: 12,931 I 4)<0,050 'No degrees of flffllom left for chi-squatt tests Table 2 Soap: Results of chi-square tests for zero-augmented distributions l10 240 21) 11kl t; 150 z a ~120 a OOSERVED o ZEl{)-AUH:NTED NBD • ZERO-~NTED smn (y=-0.5) 'JO ZERO-AUMNTED NIil : 9=0.709 q=0.371 k=168 P(JCH 3855) =0.194 60 ZER>-AUMNTED S10£L: 8=0.814 q:O 406 p:1.87 (y=-0.5 a PRICH) Ptb11.422 l5l<O 05 J) 0 0 2 4 6 8 10 12 14 16 Nn CF LfflTS PLR:HASED and titted zero-augmented distributions for Lux soap - eight months (N = 614) l10 240 21) 11kl t; 150 z a ~120 a OOSERVED o ZEl{)-AUH:NTED NBD • ZERO-~NTED smn (y=-0.5) 'JO ZERO-AUMNTED NIil : 9=0.709 q=0.371 k=168 P(JCH 3855) =0.194 60 ZER>-AUMNTED S10£L: 8=0.814 q:O 406 p:1.87 (y=-0.5 a PRICH) Ptb11.422 l5l<O 05 J) 0 0 2 4 6 8 10 12 14 16 Nn CF LfflTS PLR:HASED Fl&are 3 Observed and titted zero-augmented distributions for Lux soap - eight months (N = 614) Fl&are 3 Observed and titted zero-augmented distributions for Lux soap - eight months (N = 614) Table 3 Soap: Estimates of proportion of never- buyers q derived from zero-augmented models Lux 1st 2 months 1st 4 months 6 months 8 months Palmolive 1st 2 months 1st 4 months 6 months 8 months Proportion of never-buyers NBD Siebel 0,634 0,635 0,498 0,515 0,410 0,440 0,37) 0,407 0,686 0,686 0,474 0,503 0,450 0,485 0,376 0,430 If never-buyers do exist, one would expect the observed pro- portion of non-buyers of a product to eventually level out at the proportio portion of n would tend t 1972). Analysis of the Soup Data In this analysis the non-consumer over the entire sixteen-month period of the survey were considered, despite the fact that the data did not exhibit stationarity over the period studied. Although the use of the sixteen-month period is theoretically not correct, it does at least give some guidance as to the pro- portion of non-consumers over a lengthy period. As will be seen from Figure S, the observed point plotted at sixteen If never-buyers do exist, one would expect the observed pro- portion of non-buyers of a product to eventually level out at S. Afr. J. Bus. Mgmt. 1984, 1S(3) 1,0 --o-- OBSERVED - EXPECTED 0.4 +-----,--"'T"""----,.-----r--- 0 2 4 6 8 LEt{iTH OF OBSERVATIONAL PERIOD I 11'.JNTHS I Figure 4 Proportion of non-buyers of Lux soap as a function of length of observational period (N = 614) 1,0 ---o--- OBSERVED ......,.__ ~XPECTED 159 0,0 +--,---.,..--.....----,--,--,---,---,--- 0 2 4 6 8 1l 12 14 16 LEt«iTH Of OBSER\ATIONAL PERIOO !l'llNTHSl Figure 5 Proportion of non-consumers of brand A soup as a function of length of observational period (N = S 12) S. Afr. J. Bus. Mgmt. 1984, 1S(3) 1,0 --o-- OBSERVED - EXPECTED 0.4 +-----,--"'T"""----,.-----r--- 0 2 4 6 8 LEt{iTH OF OBSERVATIONAL PERIOD I 11'.JNTHS I Figure 4 Proportion of non-buyers of Lux soap as a function of length of observational period (N = 614) S. Afr. J. Bus. Mgmt. 1984, 1S(3) S. Afr. J. Bus. Mgmt. 1984, 1S(3) 1,0 --o-- OBSERVED - EXPECTED 0.4 +-----,--"'T"""----,.-----r--- 0 2 4 6 8 LEt{iTH OF OBSERVATIONAL PERIOD I 11'.JNTHS I 1,0 ---o--- OBSERVED ......,.__ ~XPECTED 159 0,0 +--,---.,..--.....----,--,--,---,---,--- 0 2 4 6 8 1l 12 14 16 LEt«iTH Of OBSER\ATIONAL PERIOO !l'llNTHSl Figure 5 Proportion of non-consumers of brand A soup as a function of length of observational period (N = S 12) 159 1,0 ---o--- OBSERVED ......,.__ ~XPECTED 159 0,0 +--,---.,..--.....----,--,--,---,---,--- 0 2 4 6 8 1l 12 14 16 LEt«iTH Of OBSER\ATIONAL PERIOO !l'llNTHSl Figure 5 Proportion of non-consumers of brand A soup as a function of length of observational period (N = S 12) Figure 4 Proportion of non-buyers of Lux soap as a function of length of observational period (N = 614) theory. This theory does not provide for the existence of never- buyers. A similar calculation was made for the individual brands in the soup product field. months is well within the general sweep of the curve. Analysis of the Soup Data The consumption frequency distributions of the individual brands were not modelled except for the case of the combined brands A and B over the eight stationary winter months. The results of the chi-square tests on the two zero-augmented models were: Therefore, on the basis of the available evidence in two pro- duct fields, the never-buyer/consumer hypothesis was rejected. The Binary Model of Joannides (1976) should thus be con- sidered as the explanation for the observed distribution shapes. He showed that the marginal distributions obtained by sum- ming a correlated NBD, representing loyal repeat-buyers, and an uncorrelated NBD, representing brand-switching/multi- brand buyers, displayed high zero cell frequencies as well as points of inflection or secondary modes close to the zero cell. NBD Sichel P<x2 ~ 2S, 732 I 27) = 0,531 P(x2 ~33,304 I 26)=0,166 P<x2 ~ 2S, 732 I 27) = 0,531 P(x2 ~33,304 I 26)=0,166 The NBD model, which provided the better fit, gave an estimate of zero for the proportion of never-consumers. Because of this result, the fitting of models to the individual brands was omitted, and the graphs of observed and expected (via Ehrenberg's NBD theory) proportions of non-consumers of brand A, brand B and brands A and B combined were im- mediately studied. Thus the results obtained, together with a consideration of the Joannides model, lead one to the conclusion that perhaps brand-disloyal buyers/ consumers are better represented by a distribution of frequencies rather than the subset thereof (zero cell frequencies only) implied by the never-buyer hypothesis. These results have important implications for the marketers of consumer non-durables. Instead of devoting time and ef- fort in persuading never-buyers to use their products (in a generic sense), they would be strongly advised to undertake advertising campaigns aimed at wooing existing users of other brands of the product into trying their particular brand. The parameter estimates for brands A and B were based on averages of each of the first four months' estimates, calculated using the method of moments. For the combined brands the maximum likelihood parameter estimates for the first month were used. It was found that the observed pro- portions of non-consumers approached zero more rapidly than the expected proportions as predicted by the Ehrenberg theory. An example of the observed and expected proportions of non- consumers is shown in Figure 5. References Ehrenberg, A.S.C. 1972. Repeat buying: theory and applications. Amsterdam: North Holland Publishing Co. Analysis of the Soup Data Finally, this study highlighted the need to model the field of consumer non-durable purchase patterns from a multibrand perspective, rather than by looking at individual brands in isola- tion. Research in this area has been undertaken and will be reported on in the future. On the basis of this evidence, one must therefore also re- ject the hypothesis of the existence of never-consumers of in- dividual brands in the two data sets studied. (a) The NBD Repeat-buying Model (i) Univariate Case The model assumes that an individual household's purchases are characterized by a Poisson distribution. In order to study market behaviour as a whole, the individual household's distributions arc compounded using a 'Y distribution for the individual average purchase frequencies per unit time (Sichel, 1971). The result is the Negative Binomial Distribution (NBD): Ax I fJ = [kl(k + tm)lk rex + k) ltml(k + tm)]X (Al) r(k) r(x+ I) where xis the number of purchases in time t, 0< m < co ,0< k < oo , and x = 0,1,2, ... The proportion of buyers of the product in time t is given by b1 = 1 - (I +tmlkF" (A2) where a buyer is defined as one who buys at least one unit during time period t. (ii) The Bivariate Correlated NBD The bivariate model is obtained by mixing the product of two univariate Poisson distributions with the 'Y distribution, to form the bivariate correlated NBD model. In its symmetric form (where successive purchase periods are of equal length) it can be written Jtx I fJ = (l+Umlk)-1c r (x+y+k) [tml(k+Um)f+" (A3) ,Y r(k) r(x+ 1) re,,+ I) where k>O, m >0, k(fJ = k, m(t)= tm, x=0,1,2, ... , y=0,1,2, ... The market penetration (proportion of buyers of the product in time fJ is given by c"t = l -(1 + tmlk)-k (A4) (iii) The Uncorrelated Bivariate NBD Sichel (1980) derived the uncorrelated bivariate NBD in an attempt to model the behaviour of product-disloyal consumers (brand- switchers and multibrand buyers) whose consumptions are uncor- related between two time periods. The uncorrelated NBD is obtain- ed from the product of two univariate NBDs. It can be written in the form ,t, (r) represents a family of discrete distributions. Many of the bet- ter known discrete distributions such as the NBD, Poisson, geometric, etc., are special or limiting forms of 1/J(.r) (Sichel, 1974). Making gamma negative allows one to generate an entirely new set of discrete distributions. The distributions are mathematically dif- ficult to handle unless 'Y is a half-integer. -y could therefore be set, a priori, to - O,S and values of - l ,S or - 2,S would be used in order to try and improve the fit if 'Y = - O,S is not satisfactory. Two parameters, a and 0, have therefore to be estimated. Concluslons • r+} l r = 0,1,2, ... (A7) Sichel, H.S. 1980. Private Communication. _ (vf=e)l (a0/2)' K (a) 1/J(.r) - K (a(v' I - 0))r! • r+} l Sichel, N. 197S. Problem areas in repeat consumption models. Un- published M.B.A. dissertation, University of the Witwatersrand, Johannesburg. Tucker, A.E. 1973. The repeat purchase patterns of non-durable con- sumer goods in selected households. Unpublished M.B.A. disser- tation, University of the Witwatersrand, Johannesburg. r = 0,1,2, ... (A7) r = 0,1,2, ... (A7) (A7) It is derived by mixing the Poisson distribution with the flexible mixing distribution Zachos, G. 1977. Non-durable consumption patterns of individual households. Unpublished M.B.A. dissertation, University of the Witwatersrand, Johannesburg. ft>.) = j2v'{r=e)/ a0P • >. ~ - 1 • exp! _ ( _!_) >. _ a 2 e (AS) 2K (av'l -0) 0 4 } ' (AS) Appendix A brief description of the statistical distributions mentioned in the text is given here. In this distribution - oo < -y < oo , O < 0 < I and a >0 are the three parameters and K_r (.) is the modified Bessel function of the second kind or order (.). (a) The NBD Repeat-buying Model If the proportion of observed frequenci~ in the zero cell is large (general- ly above S00fo), the method of equating mean and zero cell propor- tions is fairly efficient (Sichel, 1973). If this is not the case, a maxi- mum likelihood method is available (Sichel, 1971). However, this method is very complicated, so when the i.ero cell is small, the method of moments is generally used (Sichel, 1974). (A2) (c) Concluslons Firer, C. 1980. The development of new mathematical models for multibrand buying. Unpublished M.B.A. dissertation, University of the Witwatersrand, Johannesburg. For the two data samples available, no evidence could be found to support the hypothesis that the apparent excessive size of the zero cell in the purchase/ consumption frequency distribu- tion was due to the superimposition of a buying/consuming population and a group of never-buyers/consumers. Firer, C. & Barnett, A.E. 1979. Testing the NBD repeat buying model for exposure periods of varying lengths. Unpublished Report. p Goodman, M.A. 1979. Multibrand purchasing behaviour - theory and applications. Unpublished M.B.A. dissertation, University of the Witwatersrand, Johannesburg. The q parameters of the z.ero-augmented NBD (which it was hoped would provide an estimate of the proportion of never- buyers of toilet soap) were not constant over the various time periods studied. In addition, with lengthening time periods, the observed proportion of non-buyers decreased at almost ex- actly the same rate as that predicted by the Ehrenberg NBD Joannides, J. 1976. A new distribution for repeat consumption. Un- published M.B.A. dissertation, University of the Witwatersrand, Johannesburg. g Morrison, D.G. 1969. Conditional trend analysis: a model that allows S.-Afr. Tydskr. Bedryfsl. 1984, 1S(3) 160 k>O, m>O, k(t)=tk, m(t)=tm, x=0,1,2, ... , y=0,1,2, ... for non-users. J. Market. Res., VI, 342-346. for non-users. J. Market. Res., VI, 342-346. Sichel, H.S. 1971. On a family of discrete distributions particularly Sichel, H.S. 1971. On a family of discrete distributions particularly suited to represent long-tailed frequency data. In Proceedings of the Third Symposium on Mathematical Statistics (N.F. Laubscher, In the case of this distribution, the proportion of buyers is ed.). S.A. C.S.l.R. (Pretoria), Sl-97. ed.). S.A. C.S.l.R. (Pretoria), Sl-97. ,/'r = I - (1 + ml k) - '" (A6) ,/'r = I - (1 + ml k) - '" (A6) Sichel, H.S. 1973. Statistical valuation of diamondiferous deposits . ,/'r = I - (1 + ml k) - '" The Sichel distribution (A6) (A6) Journal of the South African Institute of Mining and Metallergy, 23S-239. The Sichel distribution The Sichel distribution The Sichel distribution (b) Sichel, H.S. 1974. On a distribution representing sentence length in written prose. Journal of the Royal Statistical Society, 137(1), 2S-34. This distribution was also derived by Sichel in 1971. It can be writ- ten in the form _ (vf=e)l (a0/2)' K (a) 1/J(.r) - K (a(v' I - 0))r! Zero-augmented distributions A large number of observed purchase/consumption frequency distributions have an (what appears to the eye), excessively large mo cell. Irrespective of the true cause of this effect, one requires to be able to model such distributions. In order to do so, it is assumed that this cell is made up of two separate groups. The situation is illustrated graphically in Figure Al· An implication of this model is that, if a hard core of never-buyen does exist, the estimate of IJ should be independent of time. The values of IJ for different brands within a product field need not, of course, be similar. course, be similar. ~r ~q 1i / / / / / .... .... .... ( 1-q )do (0) -- 0 1 2 3 i. S r-.,_ OF I..HITS PIR:HASEO / CON9.Jt£0 fla11n Al A graphical representation of never-buyers ~r ~q 1i / / / / / .... .... .... ( 1-q )do (0) -- 0 1 2 3 i. S r-.,_ OF I..HITS PIR:HASEO / CON9.Jt£0 fla11n Al A graphical representation of never-buyers Sichel (1980) proposed a similar modd based on the Sichel distnbu- tion. Here, too, three parameters require estimation: a and 0 of the Sichel, and lj the proportion of never-buyers. The techniques of estimation of the parameters for both distribu- tions are discussed by Firer (1980). The techniques of estimation of the parameters for both distribu- tions are discussed by Firer (1980). Sichel (1980) proposed a similar modd based on the Sichel distnbu- tion. Here, too, three parameters require estimation: a and 0 of the Sichel, and lj the proportion of never-buyers. S. Afr. J. Bus. Mgmt. 1984, 15(3) 161 Morrison (1969) showed that the existence of never-buyers would introduce a systematic bias into the NBD model. He therefore pro- posed a model of the form given above. In this model three parameters require estimation: the k and m parameters of the NBD and lj, the proportion of never-buyers. Sichel (1980) proposed a similar modd based on the Sichel distnbu- tion. Here, too, three parameters require estimation: a and 0 of the Sichel, and lj the proportion of never-buyers. The techniques of estimation of the parameters for both distribu- tions are discussed by Firer (1980).
https://openalex.org/W4372361240	https://journal.formosapublisher.org/index.php/ajma/article/download/3934/3605	English	null	The Importance of Leadership in Enhancing Work Performance (Case: Education Bureau of Sidenreng Rappang Regency)	Asian Journal of Management Analytics	2,023	cc-by	5,962	Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 A B S T R A C T A R T I C L E I N F O Keyword: Leadership, Performance, Satisfaction, Motivation Leadership is one of the most important factors that could influence work performance. The leadership approach starts with the emotional aspect, which could effectively influence work performance in the short or long run. The purpose of this paper is to explore the importance of leadership in enhancing work performance. The paper consists of a quantitative study that looks at how leadership impacts work performance using numerical methodologies. The results of this paper show the positive and significant influence of leadership on work performance. This result reflected the real-life condition in which the quality of leadership affects work performance, since the main approach of leadership indirectly dives into the emotional aspect, which can spark motivation and satisfaction, which in turn lead to high work performance as the result of effort and hard work from the employee. Received : 18, February Revised : 20, March Accepted: 23, April ©2023 Saharuddin, Agit, Novianti: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. The Importance of Leadership in Enhancing Work Performance (Case: Education Bureau of Sidenreng Rappang Regency) Nugrawan Saharuddin1, Alamsyah Agit2, Dian Novianti3 Institut Agama Islam DDI Sidenreng Rappang Corresponding Author: Nugrawan Saharuddin nugrawantonrong0606@gmail.com Nugrawan Saharuddin1, Alamsyah Agit2, Dian Novianti3 Institut Agama Islam DDI Sidenreng Rappang Corresponding Author: Nugrawan Saharuddin nugrawantonrong0606@gmail.com INTRODUCTION Work performance is the key for one’s company to achieve their goals, these are due to the fact that work performance determines how effective employee perform, further, work performance is crucial for the success and sustainability of the company, since work performance have the function to monitor how well employee carry out their duty and responsibility, this information will help in improving company ability to form strategies to achieve organizational goals, high productivity, boosting employee morale, reduce costs, and develop competitive advantages. Therefore, by encouraging employee to work well, it will make one’s company to be more efficient, effective, engaged, and productive. Work performance often determine as a person effort to carried out a job using their skill, experience, and sincerity, for it to have best result (Niati et al., 2021). Performance also known or interpret as trade, which someone will be rewarded after they successfully completed their duty (Nur’aeni et al., 2022). Company is run by many people, as it has various different section for each process, this makes company run on interdependent on one another, therefore company needed employee with high performance which can be form by skills, experience, and knowledge (Sugiarti, 2021). There is a lot factors that could influence work performance which one of them being leadership, leadership can be determine as the ability to influence, inspire, and guide individuals or a group people into achieving a common goal, leader is someone with a clear vision, and should be able to communicate that vision for others to follow, leader should also be able to build trust and establish positive relationship with their workmate, empowering them to manage their job and work effectively in order to contribute for the company. Effective leadership require few aspects, which include, communication skills, precise decision making, strategic thinking, empathy, and adaptability. Good leader would reflect the ability to balance the needs of the employee and the organization goals, and also along the way, develop culture excellence, growth and innovation. ( DOI: https://doi.org/10.55927/ajma.v2i2.3934 ISSN-E: 2963-4547 https://journal.formosapublisher.org/index.php/ajma ( DOI: https://doi.org/10.55927/ajma.v2i2.3934 ISSN-E: 2963-4547 https://journal.formosapublisher.org/index.php/ajma 231 Saharuddin, Agit, Novianti INTRODUCTION Nowadays there is a lot of phenomenon that prove the importance of the right leadership style, which often found nowadays is the task-oriented leadership style, leadership style in which characterize by the behaviour of the leader that hold a strict supervise to their subordinates, making sure they carried out their duty, without coaching, and train the subordinates to develop their skill and ability to perform better in the future (Syahrani et al., 2022). The key to success for a company will be lacking when organizational leadership fails to demonstrate transactional leadership that employee would accept, which its main purpose should be easy to understand making employee easier to direct, control, and influenced to achieve company goals (Hajiali et al., 2022). Therefore, leadership is very importance due to its major impact toward the company and the employee, which these factors have to be understood by the executive that their leadership style is good and accepted by the employee. Leadership also requires someone with the knowledge to combine behaviour, and strategy, that apply philosophy, skills, traits, and attitudes to influence their subordinates to achieve company goals (Megawati et al., 2022). 232 Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 This mark the importance of leadership in order to influence employee performance, there are also several empirical studies that provide evidence of how leadership style affected work performance. There are a lot of leadership style, which has its own uniqueness, one of these leadership styles is Islamic leadership, not far different from others, this leadership apply Islamic materials, that help organization interact with stakeholders continuously, establish new partnerships, detecting new opportunities and develop capabilities to increase organizational performance (Kessi et al., 2022). Other form of leadership is the transformational leadership, this leadership style that are visionary for the future, which the leader will inspire the subordinates to harness their skills and develop their own leadership, this leadership are known to have the capabilities to increase work performance, and further prepare the employee to become future leader (Bakker et al., 2022). INTRODUCTION Meanwhile a commonly known leadership style, which is transactional leadership, which this leadership determine what employee must done in order to improve organizational performance and achieve its goal, which this organizational performance was build upon employee performance, this is the most common leadership style which most people are worked to get paid, but this leadership style is proven to have able to boost work performance (Nurlina, 2022). Despite of all its form, leadership known to have affected employee ability throughout its process, which every task given are meant to train the ability for the employee to finish, once its finish, it will give sufficient information to perform better in the next similar task, which by then the employee performance will improve in each task given (Ángeles López-Cabarcos et al., 2022). A similar statement comes from another research that stated, leadership affects work performance throughout its implementation, in order to perform more tasks which related to work performance, one must have extensive knowledge and skills about that work. This is obtained through those work, the more often one’s performing these tasks, the better they get, which is a good indication for higher work performance, and this cannot be separate from leader role (Qomariah et al., 2022). ) Reflecting from this empirical research and evidence, it proven that leadership is one of the keys in order for a company to achieve its goal, which company are established with the group of people as its employee, employee as we know have many specialty and various skills and knowledge for each individual. In order to utilize this, a good management will be needed, and leader is a part of good management, if a leader can blend well and manage to behave in a way that can influence employee, it will become a power that will translate into performance but does it really important. As we know work performance is influence by many factors, which makes leadership one of them, this requires an exploration specifically into leaderships aspect to prove whether leadership work universally in different location as a factor that influence work performance. 233 Saharuddin, Agit, Novianti LITERATURE REVIEW Leadership Style p y Leadership style is one of the many important factors that could influence work performance, according to Afrizal leadership is a behavioural norm used to influence the others, in for leadership to be effective, the individuals must know their surrounding this include, workplace, satisfaction and many other aspects that could motivates employee to work as hard as they can (Yusuf et al., 2022). What work most to create the best workplace atmosphere is the leadership style that demonstrated shared influence in determining though between supervisor and subordinates in order to conclude the best decision in the decision making (Khassawneh & Elrehail, 2022). There are several indicators that describe a good leadership style that one could embody, which is (1) motivating ability; (2) communicating ability; (3) decision making ability; (4) ability to control emotions; and (5) responsibility (Saputra & Mahaputra, 2022). Positive leadership style will manage influence someone through their mind, which will function as psychological empowerment, and brough positive self-evaluation traits throughout their work in order to improve themselves (Schermuly et al., 2022). It can’t be denied that leadership is important, as the leader supposed to use their power, knowledge, intelligence, and skills, to influence and motivated other for the betterment of themselves, and their organization, leadership have a very strong influence on employee attitudes, which also have direct effect toward their work performance (Jamali et al., 2022). Work Performance Work performance is a multidimensional concept, there are multiple components, as for its components consist of what specific to particular job and nonspecific performance component of work, other component can be specify as organizationally mandated performance, and innovative work behavior and more spontaneity, other than that its also complex due to its behavior, which consist of idea making, promoting ideas, and realizing ideas (Ramdhan et al., 2022). Work performance is influenced by interaction between ability and motivation which are formed by knowledge and expertise, while motivation form by attitudes and situations (Iis et al., 2022). Complete performance doesn’t only reflect one performance, but it has many groups of performance one must partake in order to make significant contribution, this performance including task performance, adaptive performance, and counterproductive work behavior (Krijgsheld et al., 2022). Performance itself achieved by employee that can be seen in their result of work whether by its quality or its quantity, that they manage to carry out in accordance with their responsibility, which also determine their success in their work (Andreas, 2022). There are several aspect that accessed in performance which is (1) technical ability, which describe the ability to utilize knowledge, methods, technique, and equipment in completing a task; (2) conceptual ability, this ability requires an individual to understand organizational problem as a whole, in which including their duties, functions, and responsibilities that they bear as an employee; and (3) interpersonal 234 Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 relationship skills, which a skill that requires an individual to implement team work and manage to work in group (Dwi & Ali, 2022). relationship skills, which a skill that requires an individual to implement team work and manage to work in group (Dwi & Ali, 2022). Conceptual Framework This paper focuses in exploring the importance of leadership in influencing work performance, which empirically have been research on, however, in order to prove whether or not the leadership aspect universally work despite the difference in location, as we know leadership influence employee to work as hard as they can, by managing resources as good as possible to complete the job and task given to them. There are several empirical research that showed result stating leadership influence work performance, which stated that leadership is the main factors in improving performance (Purwanto, 2022). Other supporting this finding by stating that leadership has a positive and significant effect toward work performance which indicates the importance of leadership in order to achieve high work performance (Tianingrum, 2022) (Sudiarso, 2022) (Herlambang et al., 2022). Another statement making clear that in order for leadership to be effective in influencing work performance, factors such motivation and compensation are needed to achieve high work performance (Hidayati & Zulher, 2022). Clarifying the purpose of this paper, conceptual framework is illustrated bellow: Picture 1. Conceptual Framework Leadership (X) Work Performance (Y) Work Performance (Y) METHODOLOGY The paper consists of quantitative study that looks at how leadership impacts work performance using numerical methodologies. The purpose of this research is to carry out an analysis to accomplish that objective. The research uses SPSS software and simple regression analysis to achieve this. There are 117 participants in the study, including civil servants and interns, and it focuses on the Sidenreng Rappang Regency's Education Bureau. g g g y Five distinct aspects for each variable were included in the questionnaire used to collect the data for this study. There were two statements for each component. Intelligence, behavior, the capacity to inspire others, communication skills, and the capacity for empowerment were the five pillars of leadership. Work quality, work quantity, time efficiency, teamwork, and discipline were the five components of effective work performance. The data will be summarized to make it easier to examine, and then the simple regression method will be used to identify the influences. Regression Analysis The purpose of the regression analysis is to evaluate and judge how strongly the variables are related. Leadership is the independent variable in this study, and work performance is the dependent variable. The results of this test can be used to determine how leadership affects work performance. The 235 Saharuddin, Agit, Novianti findings of this research will also be supported by the data, which will be evaluated and compared to previous empirical studies. Table 1. Simple Regression Result Coefficientsa Model B 1 (Constant) 10,138 X .306 Source: SPSS Output (2023) Table 1. Simple Regression Result Table 1. Simple Regression Result Coefficientsa According to table 1 the result of regression tests the possible equation to formulate are the following: Y1 = 10.138 + 0.306X1 + e Y1 = 10.138 + 0.306X1 + e The constant value in this study was determined to be 10.138, indicating that the value of work performance would be 10.138 if the leadership variable had the value of zero. Considering the regression coefficient was positive (0.135), an increase in leadership value would result in an increase in work performance value of 0.306 as well. Determinant Coefficient The determinant coefficient, also referred to as the coefficient of determination, determines the amount by which the independent variable partially influences the dependent variable. The correlation coefficient, on the other hand, measures the overall strength of the relationship between the independent and dependent variables. The regression test on the determinant coefficient in this study provided the following results. Table 2. Determinant Coefficient Result Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .601a .361 .294 5,808 a. Predictors: (Constant), X Source: SPSS Output (2023) The results of the correlation test revealed an R Square value of 0.361 based on the information in Table 2's data. This indicates that just 36.1% illustrate the ability of leadership toward work performance, which is are considered to be low given the existence of other factors that could significantly affect work performance. Other factors that were not examined in this study's calculations account for the remaining 63.9% of the total. The correlation coefficient value, however, was 0.601, showing that leadership has a 60.1% influence on work performance. 236 Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 Partial Test (t) When there are several independent factors, the partial test’s goal is to identify each independent variable’s unique impact on the dependent variable. However, there was only one independent variable and one dependent variable in this study, and the test was run to see how the use of leadership affected work performance. The test results will demonstrate the extent to which work performance is increased by the help of leadership, and these results will be evaluated in light of empirical research and the real-world circumstances in which this study was done. The partial test’s results are shown in the section below. Table 3. Partial Test Result Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 10.138 9.265 1.094 .281 X .306 .129 .467 2.378 .023 a. Dependent Variable: Y Source: SPSS Output (2023) The significant value shown in table 3, shows the value of 0.023, which is lower than 0.05, this marks the significant influence shown by leadership toward work performance. The value of regression coefficient also shown the positive value of 0.306, which shown positive correlation that illustrate leadership positive influence toward work performance, which interpreted as increasement in leadership will also lead to an increase on work performance. The real condition often illustrate this result, in which the condition where the leader have the capability of leader, they posses the requirement to influence people, this will certainly lead to well improved work performance, the right method and style of leadership, will increase the work performance through its human resources, leader will use their ability to influence both psychologically and physically, through this process of influence, it will lead to a slowly develop performance of the employee, either from their skills, their timing, and their knowledge, allowing them to work better and faster in the future. DISCUSSION The Influence of Leadership toward Work Performance p Leadership in its many form, is used to influence people in order to achieve common goals, in order for this factors to be effective, one must understand what can be done and what couldn’t, in term of a leader, they must first understand the character of their subordinates, these are done to gain the ability to utilize the human resources one company has, which as we know, not everyone have the same capability, same knowledge and same skills, therefore, leaders role are so important identify this differences. This paper provide result that stated leadership influence work performance significantly, this result illustrates that in a condition where leadership is effective and even increase its effectiveness, it 237 Saharuddin, Agit, Novianti will be followed by the increasing work performance. In term of performance, there is numerous factors that could influence it, which this performance also depends on technology in order to carried out the task done by human, but technology in one way or another also needed resourceful human, which this human resources should embody the knowledge and skills to manage and utilize technology, technology operating ability is certain must have ability for leader to embody (Abbas et al., 2022). Aside from this need of tools and skilful human resources. The result of this research is supported by the empirical evidence which stated that the leadership style is positively and significantly affect work performance, in which leadership style will influence someone into performing their best, this however needs certain strategy, the most real illustration toward this condition, is when the subordinates have given the chance to improvise in their task, allowing them to have room to growth (Abdelwahed et al., 2023). g g ( ) The influence of leadership has been illustrated in the past research, its explained that leadership, leadership style implication function to improve work performance, which the better the leadership style, it will also increase work performance (Alamanda et al., 2022). The Importance of Leadership The result of this research indicates the influence of leadership toward work performance positively and significantly, this marks the importance of leadership. In real life scenario, it can’t be denied that in order for something to be done, everyone need instructions, therefore this also reflect the importance of leadership. There is a certain situation for the employee to handle alone, but there is also some situation requires the role of leader, therefore its important for leader to interact with the employee, further tasked them with duty to manage crisis management, which is unpredictable, this approach will develop high quality human resources that have the capability to improvise many situation due to their mature judgement of the situation (Eichenauer et al., 2022). Following this finding, it’s important to remember that in the decision-making situation, there is a possibility where leader doesn’t quite remember all the important things to consider, therefore its very importance for a leader to always involve their employee in the decision-making situation, in order to view on wider aspect to consider in the company (Roberson & Perry, 2021). Since leader is the one to give out order, they must have dynamic personality to handle various personality and character of their employee, in term of performance, leader are capable to build a creative employee by gaining their trust and appreciate their work, therefore the importance of leadership in its process, are assume to manage many possibilities, one of them being are creativity that form through team work, knowledge sharing, and motivational learning (Khassawneh et al., 2022). g g The importance of leadership is crucial, when leader is misbehave or misdirecting the employee, it will lead to a damage that company have to suffer, therefore its important for leader to assure their self-quality before stepping up as a leader, in this situation, one can implement the self-leadership method to influence their selves before going onto real condition in a company, this method is meant for the leader to taking care their selves either physically and psychologically, and take sufficient recovery from work, this are very important, especially in order to prevent burnout from excessively work routine (Sjöblom et al., 2022). DISCUSSION Align to the result that this paper provide, its important to always improve good leadership, however there are few aspect that include in leadership style, which is the first place to look when one are going to improve leadership style, these include process of leader selection and requirement, training program and leadership development, providing reward and recognition for the leader that fulfil their responsibility well, evaluating potential future leader, provide coaching and counselling regularly to harness leader full potential, and lastly is to implement succession plan that aim to qualified future leader (Zulmariad et al., 2022). Another empirical research states that, as the leader is also a human, they also embody different personality and character, which its influence are determine by the leader, leader must be able to behave and act in order to motivate employee in improving their performance, this behaviour are values, norm, ethics, freedom, trustworthy, supervision, critical thinking, constructive suggestion, firm and respecting creativity, also innovation and motivation (Alie et al., 2022). Leadership is stated to be dependent on the style that leader adopt, there are several leadership styles, which one most commonly known as transactional leadership, that serve the purpose to compensate the employee for their work, which are fully under the command of the leader, which this leadership will lowly pay attention toward the growth of the employee. On the other hand, transformational leadership function as a style of leadership that will help employee in building their own leadership style, to prepare their selves for future leader possibility. Leadership work in way of psychological more than physical, which many aspects that lead to comfort and satisfaction, will influence the employee performance, there is also one more strong aspect that will motivate employee to work their best performance, which is appreciation, employee are like to be appreciate, therefore it will influence them to work hard (Budur & Demir, 2022). In order for leader to react effectiveness, leader also need the process of learning, they must recognize and adapt the best leadership style, that match their personality, character, skill, knowledge, and capability, not only that, 238 Asian Journal of Management Analytics (AJMA) Vol. 2, No. DISCUSSION 2, 2023: 231-244 leader must also take their environment and subordinates in consideration, in which leader must also recognize their environment, and their subordinates, in order to manage and utilize them to increase work performance and to achieve common organizational goals (Beauty & Aigbogun, 2022). The Importance of Leadership The importance of leadership can be interpreted as an investment toward the company, due to the fact that leadership firstly affect the employee well-being and environment, further, the influence of leadership is also known to affect mostly from the emotional side of the employee performance, like intention to quit, and the motivation to work harder (Lauritzen et al., 2021). Effective leadership plays a critical role in inspiring people to take a proactive approach toward contributing to their organization. Through providing their team members clear guidance, developing attainable goals, and providing the right tools, leaders who inspire their teams and create a pleasant work environment can motivate workers to go above and beyond their responsibility. Through this, staff members are given the support and empowerment they need and grow 239 Saharuddin, Agit, Novianti more committed in the expansion of their company (Amankwaa et al., 2022). Developing a workplace that supports diversity and accepts workers with a variety of social identities requires strong leadership. A sense of security and belonging among employees can be fostered by leaders, and this can have a number of advantageous effects. Leadership can also promote psychological safety, which allows workers to express themselves without worrying about criticism or retaliation. This will also encourage work group identification, which boosts team cohesion and enhances performance in general. Further leadership is recognized to be associated to higher employee job satisfaction, engagement, and dedication to the organization. Employees who feel psychologically empowered are also more inclined to take the initiative and become proactive problem solvers. Employees are more likely to experience a sense of loyalty to their company and stick with the firm in the long run when they believe that their distinctive ideas and contributions are valued (Shore & Chung, 2021). CONCLUSION AND RECOMMENDATION After performing this research, there are several findings that are align to the result of this paper, first the importance of leadership cannot be denied, especially in organization where between the supervisor whether the subordinates share common goals, also in its process, leadership isn’t an instant process, which one must have the capability to be an ideal leader, one must have the knowledge, skill and experience. Leadership also in one way or another will influence work performance, in this term leadership can influence work performance directly or indirectly. This paper obtain result that stated leadership influence work performance positively and significantly, which show that whenever the leadership aspect is increase, it will be follow by the work performance, in real life scenario, this can also happen, since leader have the capability to influence people to work hard, it then depends on his method, strategy and emotional approach. Several empirical findings also support the result of this paper which stated that leadership is important aspect in order to maximize work performance. The recommendation of this paper is focus on the leadership aspect, even though there are diversity of factors that could influence work performance, leadership consider to be very important, since leadership can be the origin source of satisfaction and motivation, therefore, its important to evaluate instrument to explore leadership deeper, and to develop strategies to create new methods of leadership in the future. LIMITATIONS This research only explores leadership aspect in general, while there are so many leadership styles one can implement, therefore, the limitation in this research locate in its depth, leadership style is unique and have diversity of style, its recommend for the next research to start from this, to explore various type of leadership and examinate which one is more effective than the other. 240 Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 Asian Journal of Management Analytics (AJMA) Vol. 2, No. 2, 2023: 231-244 REFERENCES Abbas, S. A., Alamsyah, Herman, B., Auliyah, I., & Saleh, W. (2022). Dampak Inovasi Teknologi Dalam Mendorong Kinerja Pegawai Pada Kantor Dinas Kependudukan Dan Pencatatan Sipil Kabupaten Soppeng. Jurnal Maneksi, 11(2), 332–341. Abdelwahed, N. A. A., Soomro, B. A., & Shah, N. (2023). Predicting Employee Performance Through Transactional Leadership And Entrepreneur’s Passion Among The Employees Of Pakistan. 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https://openalex.org/W2511368280	https://dash.harvard.edu/bitstream/1/34491842/1/5510262.pdf	English	null	Tympanic Plexus Neurectomy for Intractable Otalgia	Laryngoscope investigative otolaryngology	2,016	cc-by	4,342	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:34491842 Citation Citation Roberts, Daniel S., Alisa Yamasaki, Ahmad R. Sedaghat, Daniel J. Lee, and Edward Reardon. 2016. “Tympanic Plexus Neurectomy for Intractable Otalgia.” Laryngoscope Investigative Otolaryngology 1 (5): 135-139. doi:10.1002/lio2.31. http://dx.doi.org/10.1002/lio2.31. Roberts, Daniel S., Alisa Yamasaki, Ahmad R. Sedaghat, Daniel J. Lee, and Edward Reardon. 2016. “Tympanic Plexus Neurectomy for Intractable Otalgia.” Laryngoscope Investigative Otolaryngology 1 (5): 135-139. doi:10.1002/lio2.31. http://dx.doi.org/10.1002/lio2.31. Published Version doi:10.1002/lio2.31 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. From the University of Connecticut (D.S.R.), Division of Otolaryn- gology; the Department of Otolaryngology, Massachusetts Eye and Ear Infirmary (A.Y., A.R.S., D.J.L., E.R.); and the the Department of Otology and Laryngology, Harvard Medical School (A.Y., A.R.S., D.J.L., E.R.), Boston, Massachusetts, U.S.A. Editor’s Note: This Manuscript was accepted for publication 14 July 2016. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Laryngoscope Investigative Otolaryngology The authors have no funding, financial relationships, or conflicts of interest to disclose. INTRODUCTION complete resolution of their symptoms. An additional series supported the success of this technique.5 Tympanic neurectomy was first proposed by Lempert in 1946 for the treatment of tinnitus and was named tympanosympathectomy.1 Because this procedure failed to relieve tinnitus, it was not further considered. In 1962, Golding-Wood revived this technique, changing its name to the current tympanic neurectomy.2 The pro- cedure was used to relieve “paroxysms of agonizing and unbearable pain . . . felt deep in the ear.” This operation relieved pain in three of the four patients treated. The tympanic plexus is an arborization of nerves on the promontory of the middle ear. It is formed by the tympanic branch of the glossopharyngeal nerve, also known as Jacobson’s nerve, and the caroticotympanic branch of the internal carotid plexus. Jacobson’s nerve sends branches to the middle ear mucosa, Eustachian tube, and mastoid air cells—and sensory branches are distributed to the oval and round window area with branches to the tympanic membrane.5,6 Tympanic neu- rectomy is more widely described for the surgical man- agement of chronic parotitis, chronic parotid sialectasis, and Frey syndrome.7–9 In this study, we evaluate the long-term outcomes following tympanic plexus neurec- tomy for a cohort of patients with intractable otalgia. Tympanic plexus neuralgia is characterized by excru- ciating deep aural pain and was initially described by Reichert in 1933.3 Similar to trigeminal neuralgia, otalgia may be associated with a trigger area or with constant dull pain. In 1952, Rosen elicited the pain in two patients by stimulating Jacobson’s nerve with an electrode.4 Resec- tion of the tympanic plexus in both patients led to Tympanic Plexus Neurectomy for Intractable Otalgia Daniel S. Roberts, MD, PhD; Alisa Yamasaki, MD; Ahmad R. Sedaghat, MD, PhD; Daniel J. Lee; Edward Reardon, MD Objective: The goal of this study was to analyze whether tympanic plexus neurectomy is a successful surgical option in patients with intractable otalgia. Study Design: A retrospective single institution study from the experience of two surgeons was conducted. Methods: Records of adult patients with intractable unilateral otalgia of likely glossopharyngeal origin were reviewed, with institutional review board approval. Patients who responded to a tympanic plexus block were considered for tympanic neurectomy. Twelve patients (13 ears) underwent the procedure. Surgical outcomes and the presence of persistent otalgia were evaluated. Results: Persistent otalgia was present for 16.7 months 6 8.6 standard-error-of-the-mean months prior to an intervention. Narcotic medication was used in 41.7% of patients prior to surgery. Patients received tympanic plexus blocks (median: 1, range 1–3) prior to tympanic neurectomy to evaluate candidacy for surgery. Intractable otalgia resolved in six of 13 ears (46.2%) after one surgery, with an average follow-up of 25.5 months. A significant reduction in pain occurred in two of 13 ears (15.4%) after an initial surgery. One patient received no benefit from the initial procedure. Revision surgery occurred in four ears, resulting in pain relief in three of four cases. All together, nine of 13 ears received complete resolution of pain, and an additional two of 13 ears received partial benefit using our algorithm for treatment of intractable otalgia of tympanic plexus origin. Conclusion: Intractable otalgia treated with tympanic neurectomy is a viable treatment option in cases of failed medical management. These findings provide important information that will aid clinicians in counseling chronic otalgia patients. Key Words: Tympanic neurectomy, tympanic neuralgia, glossopharyngeal neuralgia, tympanic nerve, otalgia. Level of Evidence: NA. Send correspondence to Daniel Roberts, MD, PhD, 263 Farmington Avenue, Farmington, CT 06030. E-mail: droberts@uchc.edu Roberts et al.: Tympanic Neurectomy for Intractable Otalgia Laryngoscope Investigative Otolaryngology V C 2016 The Authors Laryngoscope Investigative Otolaryngology published by Wiley Periodicals, Inc. on behalf of The Triological Society Laryngoscope Investigative Otolaryngology 1: October 2016 RESULTS A total of 12 patients fulfilled our inclusion criteria after responding to tympanic plexus block and under- went tympanic plexus neurectomy in 13 ears. Patients received tympanic nerve blocks (median: 1, range 1–3) prior to being offered tympanic neurectomy. Table I shows the demographic characteristics of our patient cohort. The average age of the patients was 38.5 years. Female patients represented 58.3% (7 of 12) of the patients. Prior chronic pain conditions (chronic abdominal pain, interstitial cystitis, migraine) were pre- sent in three of 12 (25.0%) of the patients. The average duration of otalgia prior to surgical intervention was 16.7 months. Known prior narcotic use for otalgia occurred in five (41.6%) of the patients. TMJ—while the patient opened and closed the mandible— assessing for pain, crepitus, and inhibition of movement. A den- tal consultation was utilized in six of 12 patients, confirming the absence of TMJ disease. Imaging studies consisting of com- puted tomography (CT) of the temporal bone (10 of 12 patients) and magnetic resonance imaging (MRI) of the brain (7 of 112 patients) were performed as part of the evaluation in order to rule out other causes of otalgia. All patients were unsuccessful- ly treated with a medication targeting chronic neuropathic pain (i.e., gabapentin), and eight of 112 patients received an equivo- cal exam for determining the cause of pain from a neurologist. Patients were further deemed as surgical candidates based on their clinical response to a nerve block, which was created by injecting Xylocaine (AstraZeneca, Wilmington, DE) 1% with epi- nephrine 1:100,000 into the inferior aspect of the external ear canal, with vasoconstriction visualized medially along the tym- panic membrane. If intractable otalgia improved, tympanic plexus neurectomy was offered. Demographic and medical data, which included age, sex, history of otologic surgery, duration of otalgia, and history of narcotic use, were accessed. Patients were stratified into four categories based on clinical history: 1) No precipitating causes were cate- gorized as spontaneous otalgia and represented 50% of patients (6 patients, 7 ears); 2) three patients (25%) had undergone prior ear middle surgery with no other symp- toms other than persistent otalgia; 3) two patients devel- oped recalcitrant otalgia after deep neck space infections with surgical drainage; and 4) one patient had otalgia after a history of sudden sensorineural hearing loss. Surgical Procedure Under general anesthesia after injecting the ear canal with Xylocaine (AstraZeneca, Wilmington, DE) 1% with epi- nephrine 1:100,000, the operative microscope was used to raise a tympanomeatal flap, and Jacobson’s nerve was identified in all cases. A section of Jacobson’s nerve was removed using cold steel microinstruments beginning at the inferior tympanic cana- liculus, which extended superiorly to the area above the round window with adjacent nerves of the tympanic plexus; this proce- dure was termed simple neurectomy. The tympanomeatal flap was returned to the posterior canal, and gelfoam packing moist- ened with Ciprodex drops (Alcon Laboratories, Fort Worth, TX) was placed. However, over a 1-year period, otalgia recurred in four of six ears. All four patients had revision surgery that included a wide field approach, removing all visible neu- ral tissue with endoscopes, laser ablation of the tympan- ic plexus on the promontory, and closing the inferior and superior canaliculi with Otomimix (Olympus). Of the revision cases, three had complete control of persistent otalgia (follow-up 20 months to 7 years). One patient did not benefit from revision surgery. Overall, five of seven ears ultimately had complete control; one patient had a partial response; and one patient did not benefit from revision surgery. A more extensive surgical approach, termed wide field tympanic neurectomy, was developed for revision cases (four cases). An otologic endoscope was used in addition to microscop- ic visualization. Revision cases involved patients who had com- plete resolution of pain for several months, but the pain returned to preoperative levels. In these cases, there was visible regrowth of the nerve from the inferior tympanic canaliculus. All vestiges of the nerve and the plexus were removed from the hypotympanum and middle ear up to the point where Jacob- son’s nerve exited the mesotympanum. An argon laser was also used to ablate the plexus (Figure 1). In these cases, OtoMimix Bone Cement (Olympus, Germany) was used to occlude the MATERIALS AND METHODS This study was approved by the Human Studies Commit- tee of the Massachusetts Eye and Ear Infirmary (546787-2, PI: E. Reardon, MD). We performed a retrospective review of the medical and surgical records of patients over the period 2006 to 2015. Patients were included if they had 1) intractable otalgia of likely tympanic plexus origin, 2) failed medical management, and 3) responded to a tympanic plexus block with temporary improvement of otalgia. Patients were then offered tympanic plexus neurectomy. Otalgia was considered to be likely of glos- sopharyngeal origin (i.e., originating from the tympanic plexus) based on a detailed history of the pain quality and severity (deep lancinating ear pain, usually in the range of 7–10 out of 10) and was present for months to years. Pain originating from the temporomandibular joint (TMJ) was ruled out through physical examination in all patients by palpation of the DOI: 10.1002/lio2.31 Laryngoscope Investigative Otolaryngology 1: October 2016 135 inferior tympanic canaliculus and the superior opening of the nerve tract. TABLE I. Patient Demographics. N Average SE % Age 12 (13 ears) 38.5 3.7 – Female 7 – – 58.3 Other pain syndrome 3 – – 25.0 Duration of otalgia (months) – 16.7 8.2 – Known prior narcotic use 5 – – 41.7 Etiology Spontaneous 6 (7 ears) – – 50% Ear surgery 3 – – 25% Neck surgery 2 – – 16.7% Sudden hearing loss 1 – – 8.3% E 5 standard error. Spontaneous Otalgia p g Spontaneous otalgia with no other identifiable etiol- ogies occurred in six patients (7 ears) who underwent surgery. All patients had imaging studies (MRI and/or CT), with two cases showing sclerosis of the mastoid air space. Tympanic membranes and hearing was normal in these cases. All patients had neurological evaluations and failed medical management for persistent otalgia. For cases with a sclerotic mastoid (2 cases), a simple mastoidectomy was also performed at the time of tym- panic plexus neurectomy. Otalgia completely resolved in six of seven ears, and one ear had partial improvement. However, over a 1-year period, otalgia recurred in four of six ears. All four patients had revision surgery that included a wide field approach, removing all visible neu- ral tissue with endoscopes, laser ablation of the tympan- ic plexus on the promontory, and closing the inferior and superior canaliculi with Otomimix (Olympus). Of the revision cases, three had complete control of persistent otalgia (follow-up 20 months to 7 years). One patient did not benefit from revision surgery. Overall, five of seven ears ultimately had complete control; one patient had a partial response; and one patient did not benefit from revision surgery. Prior Ear Surgery Three ears had a history of ear surgery for chronic otitis media. Two patients had a history of tympanomas- toidectomy, and one patient previously had a tympano- plasty. Two patients had complete control of otalgia with simple neurectomies (follow-up 2 years for both Roberts et al.: Tympanic Neurectomy for Intractable Otalgia Laryngoscope Investigative Otolaryngology 1: October 2016 Roberts et al.: Tympanic Neurectomy for Intractable Otalgia Laryngoscope Investigative Otolaryngology 1: October 2016 136 postoperative follow-up time of 25.5 months. Pain was noticeably reduced in two of 13 patients (15.4) after one surgery at a mean follow-up time of 37.1 months, and these patients elected for no further interventions. Four patients elected for revision tympanic plexus neurectomy due to worsening otalgia after initial complete benefit, and three of these patients subsequently received full resolution of otalgia after a second surgery. One patient received no benefit from revision tympanic plexus neu- rectomy, and no further interventions for tympanic neu- ralgia were performed. One patient received no benefit from single tympanic plexus neurectomy. Overall, nine of 13 ears received complete resolution of pain; two of 13 ears received partial benefit; and two ears had no improvement, using our algorithm for treatment of intractable otalgia of tympanic plexus origin (Table III). TABLE II. Treatment and Success Rate for 12 Patients (13 Ears). N Average SE % Primary Surgery Complete responders 6 – – 46.2 Follow-up (m) – 25.5 10.7 – Partial responders 2 – – 15.4 Follow-up (m) 37.1 24.6 Initial total responder, otalgia recurred 4 – – 30.7 No improvement 1 – – 8.3 Revision Surgery Revision surgery rate 4 – – 30.7 Complete responders 3 – – 23.1 Follow-up (m) 15.7 4.6 – No improvement 1 – – 7.6 SE 5 standard error. There was no significant morbidity associated with the procedure. Notably, no patients described chronic dry mouth following surgery. No tympanic membrane perforations occurred following surgery. One patient reported self-limited bleeding following surgery. Audio- metric performance was unchanged postoperatively after tympanic plexus neurectomy for all patients. patients). One patient had a self-reported 50% improve- ment of persistent otalgia. An additional four patients were identified as hav- ing clinical symptoms of glossopharyngeal neuralgia but did not undergo surgery after undergoing tympanic plex- us block. One patient with a history of idiopathic otalgia did not respond to the tympanic plexus block. Prior Ear Surgery Further workup identified causes for otalgia in two patients (pituitary adenoma, ear canal cholesteatoma); therefore, tympanic plexus neurectomy was not offered. One patient with a history of multiple previous ear surgeries was treated biannually with tympanic plexus blocks to control persistent otalgia. DISCUSSION Tympanic plexus neurectomy is a surgical proce- dure that ablates the tympanic plexus with the goal of relieving persistent ear pain. In this study, we examined the outcomes of tympanic neurectomy, illustrating the largest series of patients undergoing the surgery for chronic intractable otalgia. To our knowledge, these data represent the first report of this surgical modality in 25 years. Otalgia Following Neck Surgery Two patients underwent tympanic plexus neurec- tomy with a history of neck surgery for the indication of a deep neck space infection. Persistent otalgia occurred in one patient after a level II deep neck space abscess that required surgical drainage. Simple tympanic plexus neurectomy resulted in complete control of the pain (fol- low-up 5.5 years). A second patient, with a history of mandibular split for mucoepidermoid cancer of the base of tongue and radiation 30 years prior, developed a deep neck infection resulting in intractable otalgia. Simple tympanic neurectomy resulted in the resolution of otal- gia (follow-up 6 years). Roberts et al.: Tympanic Neurectomy for Intractable Otalgia Otalgia After Sudden Hearing Loss A single patient developed persistent otalgia after sudden hearing loss. The otalgia had no other identifi- able causes. No improvement was noted after tympanic plexus neurectomy. Cumulative surgical outcomes are presented in Table II. A single surgical procedure resulted in resolu- tion of otalgia in six of 13 (46.2%) ears, with a mean Glossopharyngeal neuralgia is a rare condition and is estimated to be present in 0.2 to 0.7 per 100,000 peo- ple per year.10 Classic symptoms include severe chronic pain of the ear, base of tongue, tonsillar fossa, and angle of the jaw. Glossopharyngeal neuralgia of tympanic plex- us origin is a diagnosis of exclusion. Primary otologic disease must be ruled out with physical exam and imag- ing. Imaging should include CT temporal bone without contrast; CT neck with contrast, if there is a concern for malignancy; and MRI of the head with gadolinium, focusing on the root exit of cranial nerves IX and X, to rule out microvascular compression. Flexible endoscopy must rule out upper aerodigestive lesions before consid- eration of a tympanic plexus source. Neurologic and den- tal evaluations may be considered in cases of atypical pain syndromes or concern for TMJ syndrome. When TABLE III. Tympanic Plexus Neurectomy for the Indication of Otalgia. Resolution of Otalgia (Ears Treated) Complete Partial None Rosen, 1952.4 2 0 0 Golding-Wood, 1962.2 3 0 1 Friedman et al., 1974.5 1 0 0 Cook, 1990.17 2 0 0 Roberts et al., 2016* 9 2 2 Total 17 2 3 Current study includes data from revision surgery when indicated. Current study includes data from revision surgery when indicated. TABLE III. Tympanic Plexus Neurectomy for the Indication of Otalgia. TABLE III. Tympanic Plexus Neurectomy for the Indication of Otalgia. 137 Fig. 1. Tympanic neurectomy for intractable otalgia. (A–B) Examples of the tympanic plexus over the cochlear promontory of the middle ear. (C) Sharp dissection to ablate the neural tissue along the promontory. (D) Argon laser used to ablate the tympanic plexus. Fig. 1. Tympanic neurectomy for intractable otalgia. (A–B) Examples of the tympanic plexus over the cochlear promontory of the middle ear. (C) Sharp dissection to ablate the neural tissue along the promontory. (D) Argon laser used to ablate the tympanic plexus. reasonable surgical modality in appropriately selected patients with minimal morbidity. These findings are con- sistent with those of several smaller series (Table III). possible, an injection of local anesthetic into the inferior ear canal, with blanching of the skin to the tympanic membrane, may be utilized. The nerve block is nonspe- cific, and a positive response is consistent with glosso- pharyngeal neuralgia but is not pathognomonic. Symptoms must be differentiated clinically from other forms of otologic neuropathic pain originating from the Arnold’s nerve or sensory auricular branch of the facial nerve, which is characterized by external auditory canal (EAC) or conchal bowl symptomatology.11 In cases of EAC or conchal bowl pain, ablation of these nerves could also be considered. Partial responders or failures may represent patients experiencing otalgia due to other causes, for example, from Arnold’s nerve or from referred pain or a different etiology. Our diagnostic algorithm utilizes canal injection of Xylocaine (AstraZeneca, Wilmington, DE) into the inferior external ear canal, with vasoconstric- tion visualized at the tympanic membrane. Such an approach is capable of targeting sensory fibers of the tympanic plexus, which extend to the medial tympanic membrane and middle ear but also are capable of reach- ing sensory fibers of auriculotemporal nerve, sensory branches of the facial nerve, and Arnold’s nerve.6,11 The complex sensory innervation of the ear canal presents the possibility of false positives, possibly accounting for our rate of partial responders and failures. Further, evaluation included both CT of the temporal bone and MRI of the brain in the majority of patients; however, both imaging modalities were not used in all cases. It is possible that an alternative disease process was missed in partial responder or failure cases. Roberts et al.: Tympanic Neurectomy for Intractable Otalgia Roberts et al.: Tympanic Neurectomy for Intractable Otalgia Laryngoscope Investigative Otolaryngology 1: October 2016 TABLE III. Tympanic Plexus Neurectomy for the Indication of Otalgia. Both imaging Neurosurgical management is indicated in refracto- ry cases with rhizotomy and microvascular decompres- sion at the level of the root exit of cranial nerves IX and X. For these procedures, pain control rates range from 85% to 96%, with permanent cranial neuropathy rates of 6% to 18%. Here, we report the outcomes of patients with isolated ear pain, a subset of patients representing 11.3% of patients with glossopharyngeal neuralgia.10 Our findings show complete resolution of otalgia in 69.2% of cases, with a 15.4% rate of partial benefit sug- gesting that tympanic plexus neurectomy may be a Roberts et al.: Tympanic Neurectomy for Intractable Otalgia Laryngoscope Investigative Otolaryngology 1: October 2016 138 modalities are advocated because CT will show lesions with bone involvement, whereas MRI is best suited for the identification of vascular loops at the root exit of cra- nial nerves IX and X. outcomes. Rather, the subjective interpretations of two clinicians were utilized. Three patients received tympan- ic plexus neurectomy at the time of surgery for chronic otitis media, introducing the possibility for selection bias. A prospective study at our institution with a larger number of patients will incorporate such instruments. Finally, these data represent two surgeons’ experience with tympanic plexus neurectomy. It is also possible that the tympanic plexus was not adequately ablated in our partial responders or failure cases due to the known complexity of tympanic plexus arborization to include variable hypotympanic branch- ing.12 For our failure cases, simple neurectomy was uti- lized as an initial modality. Subsequent complete resolution of symptoms occurred after wide field tympan- ic neurectomy in three of four cases, possibly illustrating the benefit of the wide field approach. Anatomical stud- ies also indicate that Jacobson’s nerve may be entirely encased in a bony canal in the middle ear identified in two of 16 cases, possibly leading to surgical failure.13 Regrowth of the tympanic plexus has not been studied, but our case series illustrates possible regrowth requir- ing revision surgery. Indeed, presumed regrowth was visualized with the aid of an endoscope. In our revision cases, (Olympus) was placed in the proximal and distal bony canal openings for Jacobson’s nerve, and the Argon laser was used to ablate all visible fibers. Acknowledgment We would like to acknowledge Dr. Saumil Merchant and Dr. Robert Lofgren, who provided guidance for our first tympanic neurectomy cases. We would like to acknowledge Dr. Saumil Merchant and Dr. Robert Lofgren, who provided guidance for our first tympanic neurectomy cases. CONCLUSION Tympanic plexus neurectomy appears to be a safe and efficacious procedure in properly selected patients with chronic otalgia. These observations provide useful guidance for clinicians counseling patients with chronic otalgia who are considering tympanic neurectomy. BIBLIOGRAPHY 1. Lempert J. Tympanosympathectomy; a surgical technic for the relief of tin- nitus aurium. Arch Otolaryngol 1946;43:199–212. 1. Lempert J. Tympanosympathectomy; a surgical technic for the relief of tin- nitus aurium. Arch Otolaryngol 1946;43:199–212. 2. Golding-Wood PH. Tympanic neurectomy. J Laryngol Otol 1962;76:683– 693. 2. Golding-Wood PH. Tympanic neurectomy. J Laryngol Otol 1962;76:683– 693. eichert FL. Tympanic plexus neuralgia. JAMA 1933;100:1744–1746 4. Rosen S. Surgery and neurology of Meniere’s disease. I. Role of the chorda tympani nerve in tinnitus, vertigo, and deafness. Arch Otolaryngol 1952;56:152–160. 5. Friedman WH, Swerdlow RS, Pomarico JM. Tympanic neurectomy: a review and an additional indication for this procedure. Laryngoscope 1974;84:568–577. 6. Arslan M. The innervation of the middle ear. Proc R Soc Med 1960;53: 1068–1074. 7. Motamed M, Laugharne D, Bradley PJ. Management of chronic parotitis: a review. J Laryngol Otol 2003;117:521–526. Review. An alternative modality to test for otalgia of tym- panic plexus nerve origin is middle ear injection of a local anesthetic described by W. H. Slattery and D. E. Brackmann (oral communication, September, 2015).15 This modality has an added level of specificity by cir- cumventing anesthesia of the Arnold’s nerve, which is also targeted by a canal injection. However, the added specificity of intratympanic anesthesia must be balanced with a possible risk of temporary facial nerve paralysis in the case of a dehiscent facial nerve; vertigo, which may occur in some cases; or temporary impairment of auditory function, previously illustrated by alterations in transiently evoked otoacoustic emissions.16 y g 8. Daud AS, Pahor AL. Tympanic neurectomy in the management of parotid sialectasis. J Laryngol Otol 1995;109:1155–1158. Review. 9. Hays LL. The Frey syndrome: a review and double blind evaluation of the topical use of a new anticholinergic agent. Laryngoscope 1978;88:1796– 1824. 10. Rey-Dios R, Cohen-Gadol AA. Current neurosurgical management of glos- sopharyngeal neuralgia and technical nuances for microvascular decom- pression surgery. Neurosurg Focus 2013;34:E8. p g y g 11. Eshraghi AA, Buchman CA, Telischi FF. Sensory auricular branch of the facial nerve. Otol Neurotol 2002;23:393–396. 12. Donaldson. Surgical anatomy of the tympanic nerve. J Laryngol Otol 1980;94:163–168. 13. Tekdemir I, Aslan A, Tuccar E, et al. An anatomical study of the tympanic branch of the glossopharyngeal nerve (nerve of Jacobson). Ann Anat 1998;180:349–352. ; 14. Lima JA, Wilpizeski CR. Effectiveness of the CO2 laser in experimental tympanic neurectomy. Laryngoscope 1980;90:414–422. tympanic neurectomy. Laryngoscope 1980;90:414–422 15. Slattery WH, Brackmann, D. TABLE III. Tympanic Plexus Neurectomy for the Indication of Otalgia. Such use of the laser is supported by experimental evidence, show- ing that CO2 laser may be utilized to perform tympanic plexus neurectomy by means of obliterating Jacobson’s nerve in the promontory region without sensorineural hearing loss.14 Laser-assisted tympanic plexus neurec- tomy with an argon laser appears to be safe in our small cohort. All future tympanic plexus neurectomy proce- dures will use wide field tympanic neurectomy approach due to our success with revision cases and no added morbidity. Laryngoscope Investigative Otolaryngology 1: October 2016 Roberts et al.: Tympanic Neurectomy for Intractable Otalgia 139 BIBLIOGRAPHY Use of intratympanic lidocaine to identify glossopharyngeal neuralgia. Personal Communication to DSR. 2016. Several limitations of our study are acknowledged. Because this is a retrospective study, there is the risk of bias from this type of evaluation. We also did not use pain or quality-of-life surveys in the assessment of 16. Laurikainen EA, Johansson RK, Kileny PR. Effects of intratympanically delivered lidocaine on the auditory system in humans. Ear Hear 1996; 17:49–54. 6. Laurikainen EA, Johansson RK, Kileny PR. Effects of intraty 17. Cook JA, Irving RM. Role of tympanic neurectomy in otalgia. J Laryngol Otol 1990;104:114–117. Laryngoscope Investigative Otolaryngology 1: October 2016 139
https://openalex.org/W4386617156	https://acp.copernicus.org/articles/23/10117/2023/acp-23-10117-2023.pdf	English	null	Morphological features and water solubility of iron in aged fine aerosol particles over the Indian Ocean	Atmospheric chemistry and physics	2,023	cc-by	16,997	Morphological features and water solubility of iron in aged ﬁne aerosol particles over the Indian Ocean Sayako Ueda1, Yoko Iwamoto2, Fumikazu Taketani3, Mingxu Liu1, and Hitoshi Matsui1 1Graduate School of Environmental Studies, Nagoya University, Nagoya, 464-8601, Japan 2Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima, 739-8521, Japan 3Japan Agency for Marine-Earth Science and Technology, Yokohama, 236-0001, Japan Received: 3 March 2023 – Discussion started: 22 March 2023 Revised: 27 July 2023 – Accepted: 27 July 2023 – Published: 11 September 2023 Abstract. Atmospheric transport of iron (Fe) in ﬁne anthropogenic aerosol particles is an important route of soluble Fe supply from continental areas to remote oceans. To elucidate Fe properties of aerosol particles over remote oceans, we collected atmospheric aerosol particles over the Indian Ocean during the RV Hakuho Maru KH-18-6 cruise. After aerosol particles were collected using a cascade impactor, particles of 0.3–0.9 µm aero- dynamic diameter on the sample stage were analyzed using transmission electron microscopy (TEM) with an energy-dispersive X-ray spectrometry analyzer. The particle shape and composition indicated that most particles collected north of the Equator were composed mainly of ammonium sulfate. Regarding the particle number frac- tion, 0.6 %–3.0 % of particles contained Fe, which mostly co-existed with sulfate. Of those particles, 26 % of Fe occurred as metal spheres, often co-existing with Al or Si, regarded as ﬂy ash; 14 % as mineral dust; and 7 % as iron oxide aggregates. Water dialysis analyses of TEM samples indicated Fe in spherical ﬂy ash as being almost entirely insoluble and Fe in other morphological-type particles as being partly soluble (65 % Fe mass on aver- age). Global model simulations mostly reproduced observed Fe mass concentrations in particulate matter with a diameter of less than 2.5 µm (PM2.5) collected using a high-volume air sampler, including their north–south contrast during the cruise. In contrast, a marked difference was found between the simulated mass fractions of Fe mineral sources and the observed Fe types. For instance, the model underestimated anthropogenic aluminosili- cate (illite and kaolinite) Fe contained in matter such as ﬂy ash from coal combustion. Our observations revealed multiple shapes and compositions of Fe minerals in particles over remote ocean areas and further suggested that their solubilities after aging processes differ depending on their morphological and mineral types. Proper consideration of such Fe types at their sources is necessary for accurately estimating atmospheric Fe effects on marine biological activity. Research article Atmos. Chem. Phys., 23, 10117–10135, 2023 https://doi.org/10.5194/acp-23-10117-2023 © Author(s) 2023. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Even today south Asian regions have severe air pollution, at- tributable to anthropogenic and natural sources (Guttikunda et al., 2014; Chen et al., 2020; Dhaka et al., 2020; Kanawade et al., 2020; Ojha et al., 2020; Takigawa et al., 2020). Sea areas around the KH-18-6 cruise route are not areas with Fe limitations on primary production by marine mi- croorganisms (Mahowald et al., 2018) but are suitable for catching aerosols in long-range-transported polluted air from south Asia. Transmission electron microscopy (TEM) anal- ysis with EDS and water dialysis were used for examina- tion of samples of ﬁne particles that can contain combustion aerosols. Speciﬁcally, we investigated Fe-containing parti- cles’ morphological features related to the particle origin and atmospheric aging processes. As basic data for aerosols, bulk aerosol sampling of particulate matter with a diameter of less than 2.5 µm (PM2.5) was conducted to measure ions and met- als. After describing the methods used for this study (Sect. 2), the mass concentration and number fraction of Fe and their relation with other major aerosol components co-existing in- ternally and externally with Fe are presented, based on anal- yses of bulk samples and TEM observations (Sect. 3.1 and 3.2). Then, typical morphological features of Fe-containing Actually, Fe is emitted as aerosol particles having various morphologies, with their mineralogy and size distributions according to their sources (e.g., Jeong et al., 2014; Ingal et al., 2018; Umo et al., 2019; Ohata et al., 2018; Rathod et al., 2020). In addition, changes in Fe solubility with particle aging processes depend on many factors such as Fe miner- alogy and size, atmospheric and meteorological conditions, and particle acidity (Wiederhold et al., 2006; Journet et al., 2008; Cwiertny et al., 2008; Shi et al., 2009, 2015; Ito and Feng, 2010; Li et al., 2017; Sakata et al., 2022). This ear- lier knowledge about relations between solubility, Fe min- eral species, and aging processes has usually been based on bulk sample measurements, laboratory experiment ﬁndings, and simulation results. To evaluate model results, Fe mass concentrations and solubilities measured during numerous observation studies and chemical analyses of bulk samples have been used (Mahowald et al., 2009; Wang et al., 2015; Myriokefalitakis et al., 2018; Rathod et al., 2020). Neverthe- less, data of bulk samples alone do not provide adequate in- formation about the source, mineralogy, atmospheric aging, or solubility of individual Fe-containing particles. 1 Introduction Nevertheless, some observations made in leeward areas of polluted regions have revealed trace metals in individual particles (Hidemori et al., 2014; Li et al., 2017). For instance, Li et al. (2017) investigated individual Fe-containing parti- cles aged for 1–2 d using single-particle analysis of samples collected under polluted air over the East China Sea. Us- ing scanning transmission electron microscopy (STEM) and nanoscale secondary ion mass spectrometry, they found the presence of iron sulfate in a sulfate coating around iron ox- ide (FeOx) as evidence of Fe aging. Sample collection lee- ward of polluted regions and recent microscopic techniques have made it possible to identify small amounts of Fe in aged particles. As a microscopic analysis technique, water dialy- sis is a powerful tool for investigating ratios of water-soluble and insoluble materials, even in individual particles (Okada, 1983; Miki et al., 2014; Ueda et al., 2018). This method includes a comparison of morphological observations made before and after water dialysis of aerosols. Combined with energy-dispersive X-ray spectrometry (EDS), the method can quantify water-soluble elements in individual particles (Ueda et al., 2022). Recent sophisticated global aerosol modeling studies have evaluated global climatic effects of anthropogenic Fe (e.g., Scanza et al., 2018; Matsui et al., 2018b; Rathod et al., 2020). Matsui et al. (2018b) demonstrated that the atmospheric bur- den of anthropogenic combustion Fe is 8 times greater than earlier estimates had suggested. Simulations conducted us- ing a soluble Fe mechanism designed for Earth system mod- els by Scanza et al. (2018) have incorporated consideration of changes in Fe solubility that occur with atmospheric pro- cesses that affect Fe in dust and combustion aerosols. Those simulations indicated that, in many remote ocean regions, sources of Fe from combustion and dust aerosols are equally important. Moreover, Rathod et al. (2020) released a revised emission inventory of anthropogenic combustion Fe that was produced using a technology-based methodology. However, the accuracy of current model-based estimates remains un- clear because of the lack of information related to the min- eral composition, morphological structure, and solubility of actual Fe-containing particles in the atmosphere, especially in remote ocean areas. ) For this study, we conducted sampling to investigate aged Fe-containing particles over the Indian Ocean during the RV Hakuho Maru KH-18-6 cruise in November 2018. 1 Introduction al., 2009). Transport and deposition of atmospheric aerosol particles constitute an important route supplying Fe to re- mote ocean areas (e.g., Uematsu et al., 1983; Jickells and Moore, 2015; Ito et al., 2019). Chief sources of soluble Fe in the atmosphere are Fe-containing mineral dust and Fe emitted from anthropogenic combustion and biomass burn- ing (combustion Fe) (e.g., Guieu et al., 2005; Mahowald et al., 2009). Mineral dust is borne aloft mainly as coarse par- ticles by dust storms occurring in arid and semi-arid con- tinental areas (Zhang et al., 2003; Zhao et al., 2010; Ma- Iron (Fe) is recognized as an essential micronutrient for ocean primary productivity. The addition of water-soluble Fe into remote oceans, designated as high-nutrient and low- chlorophyll regions, stimulates phytoplankton blooms. Iron can thereby change the marine environment, alter biological diversity, and inﬂuence the global carbon cycle (Martin and Fitzwater, 1988; de Baar et al., 1995; Harrison et al., 1999; Jickells et al., 2005; Tsuda et al., 2003, 2007; Iwamoto et ublished by Copernicus Publications on behalf of the European Geosciences Union. 10118 S. Ueda et al.: Morphology and solubility of aged ﬁne iron howald et al., 2014). In contrast, combustion Fe is emitted as both ﬁne and coarse particles through evaporation of metals at higher temperatures in thermal sources and condensation processes that occur with diffusion and cooling (Markowski and Filby, 1985; Liu et al., 2018; Ohata et al., 2018). Al- though many earlier studies have implicated mineral dust as an important source of soluble Fe transferred to oceans (e.g., Uematsu et al., 1983; Mahowald et al., 2005; Iwamoto et al., 2011), anthropogenic Fe has also attracted increasing atten- tion recently as a source supplying water-soluble Fe, steadily and efﬁciently, by long-range transport (Chuang et al., 2005; Sedwick et al., 2007; Luo et al., 2008; Takahashi et al., 2013; Ito, 2015; Matsui et al., 2018b). on marine environments necessitates the elucidation of de- tails of atmospheric Fe properties in remote areas far from the Fe sources. Compared with major aerosol components such as sea salt and sulfates, Fe is a trace element that accounts for only a small fraction among components, especially in remote, weakly affected areas that are from the source (Seinfeld and Pandis, 2006). This relative scarcity of Fe makes it difﬁcult to ﬁnd and investigate Fe-containing particles in aerosol sam- ples. 2.3 Individual particle analyses using an electron microscope We collected PM2.5 samples on Teﬂon ﬁlters (WP500-50; Sumitomo Electric Fine Polymer, Inc.) and prebaked (900 ◦C for 3 h) quartz ﬁber ﬁlters (QR-100; Advantec Toyo Kaisha Ltd.) using two high-volume samplers (HV-700F; Shibata Science Co. Ltd.) with a custom-made particle-size separa- tor at about 12 or 24 h intervals at a ﬂow rate of 500 L min−1 on the compass deck located at about 14 m altitude from sea level. Collection of particles from the ship exhaust (the ship’s funnel was located at the rear of the sampling position) was avoided carefully: the pumping of aerosol samplers was controlled automatically using a wind sector to operate only when the relative wind direction was −80 to 80◦of the bow and when the relative wind speed was higher than 3 m s−1. After collection, the Teﬂon and quartz ﬁber ﬁlter samples (for ionic species and trace metals) were stored before chemical analyses, respectively, at 4 and −18 ◦C. Mass concentrations of water-soluble ionic species (Cl−, NO− 3 , SO2− 4 , Na+, NH+ 4 , 2.2 Chemical composition of PM2.5 2.2 Chemical composition of PM2.5 1 Introduction Enhancing our understanding of the roles and effects of atmospheric Fe Atmos. Chem. Phys., 23, 10117–10135, 2023 https://doi.org/10.5194/acp-23-10117-2023 S. Ueda et al.: Morphology and solubility of aged ﬁne iron Figure 1. Ship tracks of the KH-18-6 cruise of the RV Hakuho Maru as well as the rain site (a) and 5 d horizontal air-mass backward trajectory (b). The black dots in (a) represent 00:00 of each day at universal time. The red squares in (a) represent sites where rain was observed. Air-mass backward-trajectory calcula- tions started from 500 m a.s.l. above the site. The thin gray lines show trajectories of every 6 h. The orange circles in (b) represent TEM sampling sites. The red and blue lines show the trajectories for TEM sampling. particles are explained in terms of their relation to the Fe source (Sect. 3.3). Additionally, this paper describes global model simulations for each Fe source and comparison with results obtained from observations (Sect. 3.4). Finally, dif- ferences in measured solubility for morphologically catego- rized Fe are presented with a discussion of their relation to atmospheric aging, along with implications posed by these Fe simulation results (Sect. 3.5). 2.1 Atmospheric observations on board and air-mass backward trajectories 2.1 Atmospheric observations on board and air-mass backward trajectories Figure 1. Ship tracks of the KH-18-6 cruise of the RV Hakuho Maru as well as the rain site (a) and 5 d horizontal air-mass backward trajectory (b). The black dots in (a) represent 00:00 of each day at universal time. The red squares in (a) represent sites where rain was observed. Air-mass backward-trajectory calcula- tions started from 500 m a.s.l. above the site. The thin gray lines show trajectories of every 6 h. The orange circles in (b) represent TEM sampling sites. The red and blue lines show the trajectories for TEM sampling. Atmospheric observations were conducted over the Indian Ocean during the RV Hakuho Maru KH-18-6 cruise of 6– 28 November 2018. Figure 1 portrays ship tracks of the RV Hakuho Maru cruise, rain site, 5 d air-mass backward trajec- tories, and sampling locations of TEM samples. The back- ward trajectories were computed using the Hybrid Single- Particle Lagrangian Integrated Trajectory (HYSPLIT) model developed by the National Oceanic and Atmospheric Admin- istration (NOAA) Air Resources Laboratory (ARL) (Stein et al., 2015; Rolph et al., 2017). The settings of the tra- jectory duration, starting height, vertical-mode calculation method, and dataset were chosen, respectively, as 5 d, 500 m above sea level, model vertical velocity, and Global Data Assimilation System meteorological data. Air masses of the northern Indian Ocean (6–16 November 2018) origi- nated from India. Those around the Equator arrived from the east (17–19 November 2018); those of the southern Indian Ocean were from the sea around Western Australia, mov- ing counterclockwise to the observation sites (20–28 Novem- ber 2018). During observation periods, precipitation of less than 6 mm h−1 was observed on 11 and 12 November 2018. K+, Mg2+, and Ca2+) on quartz ﬁber ﬁlter samples (12 sam- ples collected during 8–28 November 2018) were analyzed using ion chromatography. Non-sea-salt (nss) concentrations of SO2− 4 , K+, and Ca2+ were estimated from Na+ concentra- tions in the samples using the bulk seawater ratios described by Wilson (1975). Metals in PM2.5 (Na, Al, K, Ca, Ti, V, Mn, Fe, Ni, and Zn) were analyzed using inductively coupled plasma mass spectrometry (ICP-MS, 7700X, G3281A; Agi- lent Technologies, Inc.) with microwave-assisted extraction in a mixture of nitric acid, hydroﬂuoric acid, and hydrogen peroxide using the Teﬂon ﬁlter sample (14 samples collected during 8–30 November 2018). S. Ueda et al.: Morphology and solubility of aged ﬁne iron at Nagoya University. For this study, seven samples (nos. 01– 07) of stage 3 were used for analyses. Sample collection sites and 5 d backward trajectories are presented in Fig. 1b. Sam- ple details are presented in Table 1. To obtain the elemental compositions of individual particles, the MFX of each element was obtained manually for selected areas according to the particle shape and size. Background noise effects were eliminated using X/Pd. If the difference between the X/Pd value of a particle area and that of a near background area was greater than 3 standard deviations of multiple background spectra in the same sample, then the value was treated as a signiﬁcant spectrum of the particle. p p Particles collected on the collodion ﬁlm were pho- tographed using TEM (200 keV, JEM-2100 Plus; JEOL Ltd.) at 1200 × and 6000 × magniﬁcations. To measure the heights of individual particles on the collection surface, par- ticles were coated with a Pt/Pd alloy at a shadowing angle of 26.6◦(arctan 0.5) before being micrographed. The Pt/Pd coating thickness was about 7 Å. The EDS analyses were conducted using TEM operated in STEM mode at 200 keV. The EDS hyperspectral imaging (HSI) data were sampled at greater than 20 000 magniﬁcation for 10–30 frames (20 s per frame) and were kept for each dot of 256 × 256 pixels us- ing software (NSS3; Thermo Fisher Scientiﬁc Inc., Hamp- ton, NH, USA). The dot size was 26×26 nm in the observed ﬁeld at 20 000 magniﬁcation. Although estimating the mass of each element from EDS analysis is difﬁcult, software can estimate the mass fraction (MFX) of an element X of all ana- lyzed elements from measurement results of X-ray counts as values relative to detected elements. Elemental analyses were conducted fundamentally for C, N, O, Na, Mg, Al, Si, P, S, Cl, K, Ca, Ti, V, Mn, Fe, Cu, Zn, Pd, and Pt. Although the software can identify other elements automatically if EDS spectra have speciﬁc X-ray peaks, other elements were de- tected only rarely. The X-ray count sensitivity when detected using an EDS analyzer depends on the analytical conditions for analyses, such as the frame number and adjustment of the electron beam and detector. Because some conditions can change, evaluating the X-ray count values as absolute val- ues is difﬁcult for samples analyzed on different days or un- der different settings. 2.3.2 Water dialysis and estimation of the water-soluble Fe fraction (fWSFe) For samples collected from the Indian Ocean (sample nos. 01–05) north of the Equator, a water dialysis tech- nique (Mossop, 1963; Okada, 1983; Okada and Hitzenberger, 2001; Ueda et al., 2011a, b) was applied to observe and estimate the volume fraction of water-soluble and water- insoluble materials in particles. The TEM grid with parti- cle samples was ﬂoated on an ultrapure water drop (approxi- mately 0.3 mL) on a Petri dish at about 25 ◦C for 3 h with the collection side upward. After water dialysis, some areas were photographed again. Unfortunately, a large part of the collo- dion ﬁlm tore during water dialysis. For sample no. 03 only, some EDS analysis data for Fe-containing particles were ob- tained from the same particle area after water dialysis. For this study, we expanded the water dialysis technique and EDS analysis to quantify changes in the fraction of water-soluble Fe to total Fe in individual Fe-containing par- ticles. Because Pd is a water-insoluble material coated onto the top surface of samples for this study, the mass of Pd for a selected area after water dialysis (mPd_after) can be regarded as equivalent to that for the same area before water dialysis (mPd_before). mPd_before = mPd_after (3) (3) The fraction of the water-insoluble Fe (WIFe) mass to the total Fe mass in a particle, fWIFe, can be represented as MFX = mX mtotal (1) fWIFe = mWIFe mFe × 100 [%] = mFe_after mFe_before × 100[%], (4) MFX = mX mtotal MFX = mX mtotal MFX = mX mtotal (1) (4) Therein, mX and mtotal, respectively, represent the mass of an element X in a selected area and the mass of all analyzed (selected) elements in the area. For this study, TEM sam- ples were coated uniformly by Pd/Pd using the shadowing method before water dialysis. Usually, Pd is not included in general aerosols. Sample nos. 01–07 were vapor deposited by Pt/Pd at one time. Therefore, the MFX value standard- ized by MFPd, X/Pd, can be regarded as a value that is inde- pendent from mtotal, which changes according to the aerosol composition, collodion ﬁlm thickness, and distance from the Cu grid. where mFe_before and mFe_after, respectively, denote the Fe mass before and after water dialysis for an Fe-containing par- ticle. Under Eq. (3), Eq. S. Ueda et al.: Morphology and solubility of aged ﬁne iron Although estimating the mass of each element is not possible, analyzing EDS spectra using soft- ware can estimate the mass fraction (MF) of each element from measurement results of X-ray counts as values relative to the selected elements. The mass fraction of element X is described as shown below. 2.3.1 Observation and elemental analysis After diffusion drying, aerosols were collected on carbon- coated nitrocellulose (collodion) ﬁlms using cascade im- pactors. Then the morphologies and compositions of parti- cles were analyzed using TEM. The aluminum cascade im- pactor designed by the authors has three impaction stages, each with a single jet nozzle. The respective nozzle diame- ters for stages 1, 2, and 3 were 1.2, 0.8, and 0.4 mm. The 50 % cutoff diameters at a ﬂow rate of 1.0 L min−1 were, respec- tively, 1.6, 0.9, and 0.3 µm, as calculated based on Eq. (5.28) presented by Hinds and Zhu (2022). Aerosol samples were collected on the compass deck at the upwind side of the ship for 10–30 min. About one to two TEM samples were taken per day. They were stored under dry conditions at room tem- perature (about 25 ◦C) until TEM analyses were conducted https://doi.org/10.5194/acp-23-10117-2023 Atmos. Chem. Phys., 23, 10117–10135, 2023 10120 2.4 Global model simulation of Fe ern Africa was used to model global-scale atmospheric iron concentrations. The size distribution of anthropogenic Fe was referred from observation results reported by Moteki et al. (2017) from aircraft measurements using a single-particle soot photometer for magnetite over eastern Asia. Iron emis- sions from combustion from open biomass burning were cal- culated based on a report by Luo et al. (2008). Dust Fe was not treated in our model, but we assumed a constant iron con- tent of 3.5 % in natural dust (Duce and Tindale, 1991; Jickells et al., 2005; Shi et al., 2012). We conducted global model simulations using the Commu- nity Atmosphere Model version 5 with the Aerosol Two- dimensional bin module for foRmation and Aging Simu- lations (CAM–ATRAS), with modiﬁcations for particulate iron (Matsui et al., 2014, 2018a; Matsui and Mahowald, 2017; Matsui, 2017; Liu and Matsui, 2021a, b; Liu et al., 2022). The model setting for this study was described by Liu et al. (2022). Brieﬂy, the model incorporates emissions, gas-phase chemistry, condensation or evaporation of inor- ganic and organic species, coagulation, nucleation, activa- tion of aerosols and evaporation from clouds, aerosol forma- tion in clouds, dry and wet deposition, aerosol optical prop- erties, aerosol–radiation interactions, and aerosol–cloud in- teractions. Aerosol particles were resolved with 12 size bins from a 0.001 to 10 µm dry diameter. The model was run with horizontal resolution of 1.9◦× 2.5◦and 30 vertical layers from the surface to approximately 40 km. The near-surface layer of model results was used for this study. S. Ueda et al.: Morphology and solubility of aged ﬁne iron 1. TEM samples used for this study, analyzed particles, and Fe-containing particle numbers. Atmospheric EDS- Sample Sampling time Location conditions analyzed Fe-containing ID Start time Period Lat Long Temp. RH particles particles hh:mm min N deg. E deg. ◦C % Number Number % No. 01 10 Nov 2018 10 14.99 87.99 28.8 66 535 3 0.6 02:43 No. 02 12 Nov 2018 10 10.02 87.98 28.9 81 305 2 0.7 01:51 No. 03 13 Nov 2018 20 9.24 88.00 28.8 80 703 17 2.4 02:01 No. 04 14 Nov 2018 20 5.01 87.98 28.8 74 507 10 2.0 02:36 No. 05 16 Nov 2018 20 1.01 89.72 29.5 69 336 10 3.0 02:31 No. 06 18 Nov 2018 25 −0.01 89.06 32.3 60 136 2 1.5 08:05 No. 07 21 Nov 2018 31 −6.92 88.00 29.3 71 106 1 0.9 07:56 2.4 Global model simulation of Fe 2.3.2 Water dialysis and estimation of the water-soluble Fe fraction (fWSFe) (4) can be described using Fe/Pd before and after water dialysis of the same area for an Fe- containing particle (respectively, Fe/Pdbefore and Fe/Pdafter), which is based only on measurable values. fWIFe = Fe/Pdafter Fe/Pdbefore × 100[%] (5) (5) Finally, the fraction of water-soluble Fe to total Fe in a parti- cle, fWSFe, can be estimated as presented below. X/Pd = MFX MFPd = mX mPd (2) fWSFe = 100 −fWIFe [%] (6) (2) (6) Atmos. Chem. Phys., 23, 10117–10135, 2023 Atmos. Chem. Phys., 23, 10117–10135, 2023 https://doi.org/10.5194/acp-23-10117-2023 https://doi.org/10.5194/acp-23-10117-2023 10121 3.2 Individual particle features and co-existing states with Fe of sulfate and soot The val- ues of nss K+ also tended to be higher north of the Equa- tor. South of the Equator, the nss SO2− 4 and NH+ 4 concentra- tions were lower. Consequently, the fractions of sea salt com- ponents (i.e., Na+ and Cl−) were higher. The PM2.5 ionic amounts in equivalent concentrations of total cations with- out H+ were comparable to or greater than 75 % of that of total anions (Fig. S1a). For non-sea-salt components, the re- lations between the doubled nss SO2− 4 molar concentration and the NH+ 4 plus nss K+ molar concentration were usu- ally between 1 : 1 and 2 : 1 (Fig. S1b), suggesting that nss SO2− 4 originated from ammonium sulfate, ammonium bisul- fate, and potassium sulfate rather than from sulfuric acid. mass concentration. North of the Equator, the nss SO2− 4 and NH+ 4 concentrations were especially high. These mass frac- tions were, respectively, 70 %–76 % and 18 %–22 %, except for data of 11 November 2018, when nss SO2− 4 and NH+ 4 were less around rain event occurrences (Fig. 1a). The val- ues of nss K+ also tended to be higher north of the Equa- tor. South of the Equator, the nss SO2− 4 and NH+ 4 concentra- tions were lower. Consequently, the fractions of sea salt com- ponents (i.e., Na+ and Cl−) were higher. The PM2.5 ionic amounts in equivalent concentrations of total cations with- out H+ were comparable to or greater than 75 % of that of total anions (Fig. S1a). For non-sea-salt components, the re- lations between the doubled nss SO2− 4 molar concentration and the NH+ 4 plus nss K+ molar concentration were usu- ally between 1 : 1 and 2 : 1 (Fig. S1b), suggesting that nss SO2− 4 originated from ammonium sulfate, ammonium bisul- fate, and potassium sulfate rather than from sulfuric acid. Figure 4a portrays a STEM image and EDS mapping of C, O, Al, Si, S, K, Ca, and Fe of an area of sample no. 03 and examples of the X-ray count spectrum for each particle. Ball-like and clustered particles were composed mainly of S, C, and O, indicating sulfate with organic components. K signals in the particles were often found at the same posi- tion as the detected S signal. Number fractions of particles detecting each element are depicted in Fig. 5. 3.1 Horizontal variation in PM2.5 components Figure 2 shows horizontal distributions of the mass concen- trations of nss SO2− 4 , NH+ 4 , and Fe in PM2.5. The values are shown as mass mixing ratios (the measured mass concentra- tion divided by the atmospheric density (1.18–1.21 kg m−3) calculated using the daily average of temperature and atmo- spheric pressure measured on board), with the same unit of model output, as discussed later (Sect. 3.4). The 25th per- centile and the 75th percentile mass concentrations of ions and metals north and south of the Equator at 87–90◦E are presented, respectively, in Tables 2 and 3. Scatterplots show- ing concentrations of Fe and the other elements are presented in Figs. S1 and S2 in the Supplement. Among the measured ions in PM2.5, the sum of the mass fractions of nss SO2− 4 , NH+ 4 , Na+, and Cl−was larger than 84 % of the total ion The model explicitly treats Fe from biomass burn- ing and anthropogenic combustion. Five Fe sources and minerals (biomass burning and four anthropogenic Fe sources (magnetite (Fe3O4), hematite (Fe2O3), kaolin- ite (Al2Si2O5(OH)4), and illite ((K, H3O)(Al,Mg,Fe)2(Si, Al)4O10)) are considered in addition to eight other aerosol species: sulfate, nitrate, ammonium, dust, sea salt, primary and secondary organic aerosol, black carbon, and water. The anthropogenic Fe emission inventory developed by Rathod et al. (2020) with the update by Liu et al. (2022) for south- https://doi.org/10.5194/acp-23-10117-2023 Atmos. Chem. Phys., 23, 10117–10135, 2023 10122 10122 S. Ueda et al.: Morphology and solubility of aged ﬁne iron Table 2. Average values of PM2.5 ion mass concentrations at 87– 90◦E. 0–16◦N 0–20◦S ng kg−1 ng kg−1 Cl− 44 [16–31]∗ 175 [24–248]∗ NO− 3 53 [28–75]∗ 49 [35–65]∗ SO2− 4 11 059 [7052–16 192]∗ 1266 [394–1403]∗ Na+ 191 [157–227]∗ 177 [67–296]∗ NH+ 4 3165 [1892–4124]∗ 257 [24–294]∗ K+ 445 [163–656]∗ 38 [7–55]∗ Mg2+ 33 [26–38]∗ 22 [13–32]∗ Ca2+ 59 [38–75]∗ 36 [26–48]∗ nss SO2− 4 10 010 [6994–16 135]∗ 1222 [329–1374]∗ nss K+ 438 [158–648]∗ 32 [0–51]∗ nss Ca2+ 52 [32–67]∗ 29 [15–46]∗ ∗Values in square brackets are 25th and 75th percentile value ranges. Table 2. Average values of PM2.5 ion mass concentrations at 87– 90◦E. 5–1023 ng kg−1, respectively. The Fe concentrations were high (10–137 ng kg−1) north of the Equator but low (< 19 ng kg−1) south of the Equator. 3.1 Horizontal variation in PM2.5 components The Fe mass concen- trations were positively well correlated with nss K+ (R2 = 0.95), nss SO2− 4 (R2 = 0.93), and Ca (R2 = 0.90) mass con- centrations (Fig. S2). The concentrations of V and Ni, which originate from heavy oil combustion by ships, were closely correlated (R2 = 0.96). However, their correlations with Fe concentration (R2 values of 0.69 and 0.83, respectively) were weaker than the correlation between V and Ni or the corre- lations of Fe found with nss K+, nss SO2− 4 , and Ca. These results suggest that a large fraction of the observed Fe had been transported from around the continental atmosphere with dust, nss K+, and nss SO2− 4 . However, good correla- tion with continental elements implies that Fe was trans- ported together with the continental air mass. It does not imply that emission sources for each element are the same. Therefore, details of the composition and morphological fea- tures of individual Fe-containing particles were investigated as described hereinafter in the text. ∗Values in square brackets are 25th and 75th percentile value ranges. Table 3. Average values of PM2.5 metal mass concentrations at 87– 90◦E. 0–16◦N 0–20◦S ng kg−1 ng kg−1 Na 306.9 [231.8–377.5]∗ 221.6 [72.7–348.7]∗ Al 119.3 [48.2–139.2]∗ 13.1 [0.0–26.5]∗ K 445.6 [163.7–466.6]∗ 38.8 [7.1–58.5]∗ Ca 37.3 [17.4–42.3]∗ 7.9 [4.6–12.4]∗ Ti 5.0 [1.9–6.2]∗ 0.3 [0.0–0.5]∗ V 1.6 [0.7–1.9]∗ 0.4 [0.0–0.6]∗ Fe 66.7 [26.6–85.7]∗ 4.3 [0.1–6.7]∗ Ni 0.7 [0.3–0.8]∗ 0.1 [0.0–0.2]∗ Zn 66.8 [27.6–71.5]∗ 3.2 [0.0–3.6]∗ ∗Values in square brackets are 25th and 75th percentile value ranges. Table 3. Average values of PM2.5 metal mass concentrations at 87– 90◦E. 3.2 Individual particle features and co-existing states with Fe of sulfate and soot Photographs of all samples (nos. 01–07) are presented in Fig. S3. The morphological features of sample nos. 01–06 were similar. Electron microphotographs of samples no. 01 and no. 07 and the number fractions of the morphological types for all samples are presented in Fig. 3. The morpho- logical types were classiﬁed based on a report by Ueda et al. (2016) and features of particles on the samples. For sam- ple nos. 01–06, many particles in all samples had a rounded shape (ball) or were clustered into ball shapes (clustered), such as the blue- and white-arrowed particles of sample no. 01 in Fig. 3. Dome-shaped particles have less height to area (dome). Chain aggregation of small globules (ca. 30 nm) is regarded as a particle composed mainly of soot (soot), al- though they accounted for only a small fraction. For sample no. 07, some particles constructed of cubic parts having a high contrast to an electron beam were also found. They can usually be regarded as sea salt (SS)-shaped. Photographs of all samples (nos. 01–07) are presented in Fig. S3. The morphological features of sample nos. 01–06 were similar. Electron microphotographs of samples no. 01 and no. 07 and the number fractions of the morphological types for all samples are presented in Fig. 3. The morpho- logical types were classiﬁed based on a report by Ueda et al. (2016) and features of particles on the samples. For sam- ple nos. 01–06, many particles in all samples had a rounded shape (ball) or were clustered into ball shapes (clustered), such as the blue- and white-arrowed particles of sample no. 01 in Fig. 3. Dome-shaped particles have less height to area (dome). Chain aggregation of small globules (ca. 30 nm) is regarded as a particle composed mainly of soot (soot), al- though they accounted for only a small fraction. For sample no. 07, some particles constructed of cubic parts having a high contrast to an electron beam were also found. They can usually be regarded as sea salt (SS)-shaped. ∗Values in square brackets are 25th and 75th percentile value ranges. mass concentration. North of the Equator, the nss SO2− 4 and NH+ 4 concentrations were especially high. These mass frac- tions were, respectively, 70 %–76 % and 18 %–22 %, except for data of 11 November 2018, when nss SO2− 4 and NH+ 4 were less around rain event occurrences (Fig. 1a). 3.2 Individual particle features and co-existing states with Fe of sulfate and soot Moreover, S was detected in > 92 % of all particles for samples collected north of the Equator. K was detected in 16 %–44 % of par- ticles. For other elements, particles containing sea salt (Na, Mg, and Cl) were often detected (in 3 %–48 % of all parti- cles). In addition, Al and Si were detected in about 5 % of Among metals measured using ICP-MS, the Na and K mass concentrations were high: 57–1077 ng kg−1 and https://doi.org/10.5194/acp-23-10117-2023 Atmos. Chem. Phys., 23, 10117–10135, 2023 S. Ueda et al.: Morphology and solubility of aged ﬁne iron 10123 Figure 2. Horizontal variations in mass concentrations of (a) nss SO2− 4 , (b) NH+ 4 , and (c) Fe in PM2.5. Each sample was collected contin- uously for 12 or 24 h under control by the wind sector. Data are shown at averaged locations for latitude and longitude during the sampling period. S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron 10123 Figure 2. Horizontal variations in mass concentrations of (a) nss SO2− 4 , (b) NH+ 4 , and (c) Fe in PM2.5. Each sample was collected contin- uously for 12 or 24 h under control by the wind sector. Data are shown at averaged locations for latitude and longitude during the sampling period. Figure 3. Electron micrographs for sample nos. 01 (a) and 07 (b) and pie charts showing the number fractions of morphological types (c). The blue-arrowed particles are ball-like-shaped particles. The white-arrowed particles are clusters of balls. The red arrows indicate soot as well as dome-shaped and sea-salt (SS)-shaped particles. particles collected north of the Equator. The ﬁndings show that Fe was detected in 1 %–3 % of all particles. As shown by particles A and B in Fig. 4a, Fe signals were often found from a partial area with attached or coated sulfate. For this study, metal-congested areas containing Fe are designated as Fe-containing parts to distinguish a term Figure 4. STEM image and an elemental map of sample no. 03 for the same region (a) before and (b) after water dialysis and the ex- tracted X-ray count spectrum for arrowed particles A and B and for the same region after water dialysis (A’ and B’). 3.2 Individual particle features and co-existing states with Fe of sulfate and soot Phys., 23, 10117–10135, 2023 S. Ueda et al.: Morphology and solubility of aged ﬁne iron 10124 Figure 5. Number fractions of element-containing particles. Sin- gle type denotes particles other than cluster-shaped particles. The bracketed numbers in the legend are analyzed particles. (Ueda et al., 2011b;Ueda, 2021). Most particles of samples examined for this study had a ball-like shape or a cluster. This result indicates that sulfates in the particles were more closely related to neutralized sulfate such as ammonium sul- fate than to sulfuric acid, which also corresponds with the result found for PM2.5, as shown in Sect. 3.1. Particles com- posed mainly of ammonium sulfate can be present as solid or liquid under a metastable humidity condition between ef- ﬂorescence and deliquescence humidities, according to their atmospheric humidity experiences. Furthermore, solid am- monium sulfate particles tend to transform to rectangular regular shapes when experiencing metastable humidity con- ditions (Ueda, 2021). However, rectangular particles were rarely observed in this study, although the atmospheric rela- tive humidity (60 % RH–81 % RH) at the sampling time (Ta- ble 1) is usually a metastable condition of ammonium sulfate (35 % RH–80 % RH). Therefore, sulfate particles in our sam- ples are regarded as having existed as droplet particles in the atmosphere. Based on single soot mass measurement analyses reported by Corbin et al. (2018), much soot originating from combus- tion of heavy fuel oil in ship engines can include Fe along with V, Na, and Pb. Some soot particles that are less coated by sulfate were also found in our samples. However, metals, including Fe, were not detected from less-coated soot par- ticles in this study, as shown in Fig. S4. For our PM2.5 re- sult, as explained in Sect. 3.1, Fe was somewhat correlated with V, although the correlation was weaker than that be- tween V and Ni or that between Fe and nss SO2− 4 , nss K+, or Ca. In addition to Fe from transport from continental ar- eas, some Fe might have originated in the ship’s exhaust, but no strong evidence of ship exhaust contamination was found from our individual particle observations, including analyses of Fe-containing particles presented hereinafter. Figure 5. Number fractions of element-containing particles. Sin- gle type denotes particles other than cluster-shaped particles. The bracketed numbers in the legend are analyzed particles. 3.2 Individual particle features and co-existing states with Fe of sulfate and soot Asterisked (∗) el- ements represent elements included in the background area without particles. Figure 4. STEM image and an elemental map of sample no. 03 for the same region (a) before and (b) after water dialysis and the ex- tracted X-ray count spectrum for arrowed particles A and B and for the same region after water dialysis (A’ and B’). Asterisked (∗) el- ements represent elements included in the background area without particles. Figure 3. Electron micrographs for sample nos. 01 (a) and 07 (b) and pie charts showing the number fractions of morphological types (c). The blue-arrowed particles are ball-like-shaped particles. The white-arrowed particles are clusters of balls. The red arrows indicate soot as well as dome-shaped and sea-salt (SS)-shaped particles. Figure 3. Electron micrographs for sample nos. 01 (a) and 07 (b) and pie charts showing the number fractions of morphological types (c). The blue-arrowed particles are ball-like-shaped particles. The white-arrowed particles are clusters of balls. The red arrows indicate soot as well as dome-shaped and sea-salt (SS)-shaped particles. Figure 4. STEM image and an elemental map of sample no. 03 for the same region (a) before and (b) after water dialysis and the ex- tracted X-ray count spectrum for arrowed particles A and B and for the same region after water dialysis (A’ and B’). Asterisked (∗) el- ements represent elements included in the background area without particles. particles collected north of the Equator. The ﬁndings show that Fe was detected in 1 %–3 % of all particles. As shown by particles A and B in Fig. 4a, Fe signals were often found from a partial area with attached or coated sulfate. For this study, metal-congested areas containing Fe are designated as Fe-containing parts to distinguish a term from whole particles (Fe-containing particles) such as parti- cles with a co-existing Fe-containing part and sulfate. From some Fe-containing parts, Si, Al, and/or Ca were also de- tected. Detailed compositions of Fe-containing particles and types of Fe-containing parts are presented hereinafter in the text. Figure 4b shows a STEM image, EDS mapping, and X-ray count spectra after water dialysis for the same area as that portrayed in Fig. 4a. A large part composed of S and K disappeared with dissolution by water dialysis, suggesting that they are water-soluble materials. However, many parts composed of Si, Al, Fe, or C remained on ﬁlm. https://doi.org/10.5194/acp-23-10117-2023 Atmos. Chem. 3.3.1 Fe in metal sphere (spherical ﬂy ash) 3.3.1 Fe in metal sphere (spherical ﬂy ash) As shown for particle A in Fig. 4 and for the particle in Fig. 7a, Fe-containing spheres with Al and/or Si were found in nine particles (21 % of Fe-containing particles) from sam- ple nos. 03 and 04, affected by air masses from the east- ern coast of India (Table S1 and Fig. 1b). In addition, Fe spheres without Al or Si were found in two particles (5 % of Fe-containing particles) from sample no. 05, affected by air masses from the Maldives. All particles containing metal spheres of both types co-existed with sulfate. In the STEM image, such metal spheres have a high contrast compared with sulfate. Such spheres remained as a residue after wa- ter dialysis. Spherical shapes of metals reﬂect their forma- tion through evaporation at high temperatures and subse- quent rapid condensation. Moreover, the shape coincides with often-encountered features of ﬂy ash particles origi- nating from coal combustion at electrical generation facili- ties (e.g., Fisher et al., 1978; Yao et al., 2015; Umo et al., 2019). For this study, metal spheres composed mainly of Fe, Si, or Al in our atmospheric aerosol samples are desig- nated as spherical ﬂy ash to distinguish them from ﬂy ash related to emission sources that are not classiﬁed based on morphological characteristics. In coal, Fe generally exists as pyrite or aluminosilicate (Tomeczek and Palugniok, 2002). Aluminosilicate Fe, such as kaolinite and illite, generally un- dergoes high-temperature melting and fragmentation, lead- ing to formation to ﬂy ash (Rathod et al., 2020). Although low-temperature combustion processes such as residential uses might not engender Fe volatilization (Flagan and Se- infeld, 2012), power plants can emit large amounts of ﬂy ash (Rathod et al., 2020). Although the mass concentration peak of ﬂy ash emitted from power plants is super-micrometric (1–10 µm), submicrometer ﬂy ash particles are also emitted together. They have been observed and described in the liter- ature (Markowski and Filby, 1985; Liu et al., 2018; Umo et al., 2019). Figure 6. Mixing states and morphological features of Fe- containing particles of samples collected north of the Equator. (a) Number fraction of each element-containing particle in Fe- containing particles. (b) Pie chart of mixing type with sulfate. (c) Pie chart of Fe-containing part types based on constituent ele- ments and morphology. Section 3.3 presents methods of classifying Fe-containing part types. S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron 10125 10125 Figure 6. Mixing states and morphological features of Fe- containing particles of samples collected north of the Equator. (a) Number fraction of each element-containing particle in Fe- containing particles. (b) Pie chart of mixing type with sulfate. (c) Pie chart of Fe-containing part types based on constituent ele- ments and morphology. Section 3.3 presents methods of classifying Fe-containing part types. Single type in (a) denotes particles other than cluster-shaped particles. elemental map, and X-ray spectra for typical Fe-containing particles are portrayed in Fig. 7. Details of the respective morphological types are explained later in Sect. 3.3.1–3.3.4. 3.3.1 Fe in metal sphere (spherical ﬂy ash) Single type in (a) denotes particles other than cluster-shaped particles. can originate from biomass burning in addition to sea salt and mineral dust. Adachi et al. (2022) reported ash-bearing par- ticles found in biomass burning smoke based on TEM anal- ysis of ﬁne (< 2.5 µm) particles. They deﬁned particles con- taining both Ca and Mg (> 5 weight %) as ash-bearing par- ticles. According to their report, ash-bearing particles from biomass burning smoke commonly exhibit aggregated shapes with complicated compositions, predominantly calcium with other elements (e.g., C, O, Mg, Al, Si, P, S, and Fe). However, in our study, an Fe-containing particle in which both Ca and Mg were detected was only one (2 % of Fe-containing par- ticles). In addition, morphological features of Fe-containing particles were not like those of ash-bearing particles reported by Adachi et al. (2022). As shown in Fig. 4a and b, Fe-containing parts had a few different morphological types, such as spherical or ir- regular shapes and homogeneous or heterogeneous mixing with other elements. Such morphological types can be re- lated to their Fe emission source. Based on the contrast of STEM images and EDS mapping, we divided Fe-containing parts into three morphological types associated with an emis- sion source (spherical ﬂy ash, mineral dust, FeOx aggre- gate) and the other two types (differently shaped Fe and small Fe thickly coated by sulfate). The average ± stan- dard deviation values of Fe/Pd for each type above were, re- spectively, 1.1±2.2, 0.18±0.14, 0.73±0.81, 1.7±0.6, and 0.07±0.05. Number fractions for the morphological types for Fe-containing particles are presented in Fig. 6c. For sample no. 03, the morphological features found after water dialysis were classiﬁed. In addition, examples of STEM images, the 3.3 Fe-containing particles: composition and morphological features implying their source Co-existing Fe-containing parts and sulfate were found from TEM samples. Such sulfates on Fe-containing parts can be formed by condensation from sulfuric gaseous materials or coagulation of sulfate particles. Good correlation between Fe and nss SO2− 4 , as explained in Sect. 3.1, also suggests that a large mass of Fe was transported to the ocean with sulfate particles and their precursor gases. Relations of sulfate par- ticle shapes on samples to their acidity have been reported from some earlier microscopic studies, as explained here- inafter. Acidic sulfate particles such as sulfuric acid (H2SO4) and bisulfate (NH4HSO4) are usually found as droplets hav- ing a satellite structure because of their property of retain- ing water, even if they were collected after diffusion drying (Waller et al., 1963; Frank and Lodge, 1967; Gras and Ay- ers, 1979; Bigg, 1980; Ferek et al., 1983). In contrast, par- ticles composed mainly of ammonium sulfate collected af- ter passing the diffusion dryer have been found in the form of ball-like shapes, rectangular regular shapes, and clusters Figure 6a and b portray number fractions of particles con- taining the respective elements and mixing types with sul- fate for Fe-containing particles (42 particles) observed in ﬁve samples (nos. 01–05) affected by air masses from south Asia. Most Fe-containing particles were mixed with sulfate (Fig. 6b). Actually, S was detected in 90 % of Fe-containing particles (Fig. 6a). In addition, K was found in half of the Fe- containing particles (Fig. 6a), suggesting that biomass burn- ing affected the Fe-containing particle compositions. How- ever, many K signals were detected from the sulfate coat- ing. Therefore, such a K signal implies secondary formation on Fe-containing parts rather than a source of Fe-containing parts. Both Al and Si were detected in 20 % and 30 % of Fe- containing particles. Very little V originating from heavy oil combustion of ships was detected in Fe-containing particles. Moreover, Na, Mg, Ca, Mn, and Zn were detected in less than 7 %–10 % of Fe-containing particles. Both Ca and Mg Atmos. Chem. Phys., 23, 10117–10135, 2023 https://doi.org/10.5194/acp-23-10117-2023 S. Ueda et al.: Morphology and solubility of aged ﬁne iron p gy y g Figure 7. Examples of STEM images, elemental maps, and X-ray count spectra of typical Fe-containing particles. (a) Example of a particle having an Fe-containing part in the metal sphere (spherical ﬂy ash). The metal sphere part of this particle is coated by sulfate. (b) Example of an Fe-containing particle co-existing with Si, Al, or Ca heterogeneously (mineral dust). The Fe, Si, Al, and Ca parts of this particle are coated by sulfate. (c) Example of an FeOx aggregate particle. (d) Example of a particle having a small Fe-containing part thickly coated by sulfate. Asterisked (∗) elements in the X-ray count spectrum represent elements included in the background area without particles. Figure 7. Examples of STEM images, elemental maps, and X-ray count spectra of typical Fe-containing particles. (a) Example of a particle having an Fe-containing part in the metal sphere (spherical ﬂy ash). The metal sphere part of this particle is coated by sulfate. (b) Example of an Fe-containing particle co-existing with Si, Al, or Ca heterogeneously (mineral dust). The Fe, Si, Al, and Ca parts of this particle are coated by sulfate. (c) Example of an FeOx aggregate particle. (d) Example of a particle having a small Fe-containing part thickly coated by sulfate. Asterisked (∗) elements in the X-ray count spectrum represent elements included in the background area without particles. Aggregated FeOx co-existing with soot was also found at urban sites (Ohata et al., 2018; Ueda et al., 2022). From the present study, aggregated FeOx particles (designated as FeOx aggregates) were found without a C-rich part in three particles (7 % of Fe-containing particles) in sample nos. 02, 03, and 04, affected by air masses from India (Table S1 and Fig. 1). Two FeOx aggregates co-existing with sulfate were found. As explained in Sect. 3.2, soot-containing metals were also minor. Actually, FeOx can be emitted from blast fur- naces at iron-smelting facilities (Machemer, 2004) and as ex- haust from motor vehicles (Kukutschová et al., 2011; Liati et al., 2015). However, considering FeOx without soot, FeOx aggregates examined for this study might have originated mainly from the former source. fore, heterogeneous structures including Fe, Al, Si, Ca, and others imply that they are mineral dusts with no combustion- related origin. Such Fe-containing parts regarded as mineral dust were found in six particles (14 % of Fe-containing par- ticles) in sample nos. S. Ueda et al.: Morphology and solubility of aged ﬁne iron 01, 03, 04, and 05 (Table S1). Most particles of this type (ﬁve particles) co-existed with sulfate. Based on water dialysis of sample no. 03, Ca in particle B in Fig. 4a dissolved with water dialysis, as portrayed in Fig. 4b. Although calcite in dust is poorly soluble, chemical transfor- mation from calcite into calcium sulfate or calcium nitrate with atmospheric aging can alter its solubility (Okada et al., 1990, 2005; Zhang and Iwasaka, 1999; Matsuki et al., 2005). Aggregated FeOx co-existing with soot was also found at urban sites (Ohata et al., 2018; Ueda et al., 2022). From the present study, aggregated FeOx particles (designated as FeOx aggregates) were found without a C-rich part in three particles (7 % of Fe-containing particles) in sample nos. 02, 03, and 04, affected by air masses from India (Table S1 and Fig. 1). Two FeOx aggregates co-existing with sulfate were found. As explained in Sect. 3.2, soot-containing metals were also minor. Actually, FeOx can be emitted from blast fur- naces at iron-smelting facilities (Machemer, 2004) and as ex- haust from motor vehicles (Kukutschová et al., 2011; Liati et al., 2015). However, considering FeOx without soot, FeOx aggregates examined for this study might have originated mainly from the former source. 3.3.3 FeOx aggregate Some aggregate components comprising Fe and O without other metals were found as shown for a particle in Fig. 7c. They are regarded as being FeOx, such as magnetite and illite. Similar aggregated FeOx nanoparticles have been re- ported by several studies applying electron microscopy to the observation of urban atmospheres (Hu et al., 2015; Adachi et al., 2016; Ohata et al., 2018), roadside environments (Sander- son et al., 2016), and polluted remote seas (Li et al., 2017). 3.3.2 Fe co-existing with Al, Si, or Ca heterogeneously (mineral dust) As shown for particle B in Fig. 4 and for the particle in Fig. 7b, some non-spherical parts containing Fe, Si, and Al were also found. Unlike the Fe-containing sphere parts explained earlier, Ca was usually detected from such non- spherical Fe-containing parts. Additionally, they have some domains of different concentrations of Fe, Al, Si, and Ca. The Fe, Al, Si, and Ca are the main elements of silicate minerals. Mineral dust is usually composed of a few different mineral species (Conny, 2013; Jeong and Nousiainen, 2014; Jeong et al., 2014, 2016; Conny et al., 2019; Ueda et al., 2020). There- https://doi.org/10.5194/acp-23-10117-2023 Atmos. Chem. Phys., 23, 10117–10135, 2023 10126 S. Ueda et al.: Morphology and solubility of aged ﬁne iron 3.3.4 Differently shaped Fe and small Fe thickly coated by sulfate 3.3.4 Differently shaped Fe and small Fe thickly coated by sulfate For Fe-containing parts of three particles (7 % of Fe- containing particles), different shapes were found in terms of the features described above: spherical ﬂy ash, mineral dust, and FeOx aggregates. They were designated as dif- ferently shaped Fe. Of those, two particles were without Atmos. Chem. Phys., 23, 10117–10135, 2023 https://doi.org/10.5194/acp-23-10117-2023 S. Ueda et al.: Morphology and solubility of aged ﬁne iron 10127 Figure 8. Spatial distributions of PM2.5 Fe mass concentrations simulated using the CAM–ATRAS model for (a) total, (b) anthropogenic (AN), (c) mineral dust (Dust), and (d) biomass burning (BB) Fe. Values are monthly averaged surface mass concentrations for Novem- ber 2018. S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron 10127 Figure 8. Spatial distributions of PM2.5 Fe mass concentrations simulated using the CAM–ATRAS model for (a) total, (b) anthropogenic (AN), (c) mineral dust (Dust), and (d) biomass burning (BB) Fe. Values are monthly averaged surface mass concentrations for Novem- ber 2018. Figure 9. PM2.5 Fe mass concentrations simulated by CAM– ATRAS. (a) Averaged PM2.5 Fe simulated value for 87.5◦E and observed PM2.5 Fe of 87–90◦E during the KH-18-6 cruise. The lower and upper error bars, respectively, stand for the 25th and 75th percentiles. (b) Pie chart of averaged mass fraction simulated PM2.5 Fe for each Fe species of 0–16◦N at 87.5◦E. Simulated values are monthly averaged mass concentrations for November 2018. with the observed values. The averages of observed values and model results were, respectively, 66.7 and 45.7 ng kg−1 at 0–16◦N and 4.3 and 1.0 ng kg−1 at 0–20◦S. The model simulations reproduce the contrast of the Fe mass that was found north (high concentration) and south (low concentra- tion) of the Equator during the cruise well. Although the sim- ulated Fe mass tends to be lower than the observed Fe mass (Fig. 9a), our simulations using the recent anthropogenic Fe inventory data reported by Rathod et al. (2020) show higher Fe mass concentrations and better agreement with observa- tions than earlier estimates producing older emission inven- tories (Liu et al., 2022). Large uncertainties exist in Fe emis- sion inventories from all sources, including anthropogenic sources, dust, and biomass burning. Moreover, the different spatial and temporal scales found between observation and models might inﬂuence the comparisons presented herein. Figure 9. 3.3.4 Differently shaped Fe and small Fe thickly coated by sulfate PM2.5 Fe mass concentrations simulated by CAM– ATRAS. (a) Averaged PM2.5 Fe simulated value for 87.5◦E and observed PM2.5 Fe of 87–90◦E during the KH-18-6 cruise. The lower and upper error bars, respectively, stand for the 25th and 75th percentiles. (b) Pie chart of averaged mass fraction simulated PM2.5 Fe for each Fe species of 0–16◦N at 87.5◦E. Simulated values are monthly averaged mass concentrations for November 2018. The CAM–ATRAS results show that anthropogenic Fe was dominant in the PM2.5 Fe concentration within the area presented in Fig. 8. The averaged mass fractions of Fe source/mineral types in the model estimated for 0–15◦N at 87.5◦E are depicted in Fig. 9b. The anthropogenic Fe (mag- netite, hematite, illite, and kaolinite), dust Fe, and biomass burning Fe were, respectively, 80 %, 19 %, and 1 % of the mass of PM2.5 Fe. In particular, anthropogenic Fe around In- dia has a high concentration in Fig. 8, suggesting that the anthropogenic activities taking place in that area affected Fe concentrations over the Indian Ocean. sulfate. For the other 19 Fe-containing particles, discrimi- nating the shapes of Fe-containing parts was difﬁcult be- cause such parts were much smaller than the sulfate coat- ing, as illustrated in Fig. 7d. Such Fe-containing particles were classiﬁed as small Fe thickly coated by sulfate sepa- rately from differently shaped Fe. The number fraction was 45 % of the Fe-containing particles. However, the Fe/Pd val- ues (0.07 ± 0.05) from such small Fe were usually less than those of the other types. Anthropogenic Fe was mostly (74 % in mass of PM2.5 Fe) estimated as FeOx (magnetite and hematite). The mass frac- tion of anthropogenic aluminosilicate Fe (illite and kaolin- ite), which mainly derives from coal combustion, was only 6 % in the model. These simulation results can be expected to underestimate the fraction of aluminosilicate Fe and to overestimate the fraction of FeOx compared with the TEM results. In the TEM samples, spherical ﬂy ash having sufﬁ- cient Fe/Pd values was often found (26 % of Fe-containing particles), as depicted in Fig. 6. In addition, most of the ﬂy ash particles (8 in 11 spherical ﬂy ash particles) contained https://doi.org/10.5194/acp-23-10117-2023 3.5 Water solubility of Fe Figure 10 portrays the relation between Fe/Pd before and af- ter water dialysis (14 particles) for sample no. 03. The sym- bols were made according to the composition and shape of residues after water dialysis. For particles containing metal sphere residues regarded as ﬂy ash, the values of Fe/Pd after water dialysis presented in Fig. 10a and b correspond well to the values found before water dialysis at a 1 : 1 ratio. Be- cause the Pd mass after water dialysis can be regarded as equal to that before water dialysis, such a 1 : 1 relation indi- cates the Fe in spherical ﬂy ash as almost insoluble in water. For particles aside from metal spheres containing residues, the detected values of Fe/Pd decreased, suggesting that some Fe contents are soluble in water. Figure 11 portrays fWSFe for Fe-containing particles on sample no. 03. The averages of fWSFe were 48 %, 6 %, 58 %, and 65 %, respectively, for all Fe-containing particles, spherical ﬂy ash, mineral dust, and Fe-containing particles except spherical ﬂy ash. Bermuda, and the eastern Mediterranean (Crete). Their frac- tions of water-soluble Fe were low (< 5 %) under samples in- ﬂuenced by Saharan dust, but the fractions for samples from Noida (3 %–20 %) and samples inﬂuenced from Europe were high (17 %–35 %), indicating an anthropogenic contribution of soluble Fe. From measurements taken of the remote ocean, Baker et al. (2013) reported an Fe solubility of 2.4 %–9.1 % for aerosol collected in the Atlantic Ocean during research cruises. Compared with the Fe solubility found from the stud- ies described above, the water-soluble Fe fraction for indi- vidual Fe-containing particles in this study tended to be esti- mated as higher. This ﬁnding could be attributed to the fact that our analyzed samples comprised smaller particles (0.3– 0.9 µm sample stage diameter) with a greater surface relative to the particle mass and aged particles collected over the re- mote ocean, as explained below. Some measurement-based studies examining bulk aerosol samples have reported fractions of water-soluble Fe in all Fe (Kumar and Sarin, 2010; Baker et al., 2013; Ingall et al., 2018). After Kumar and Sarin (2010) also measured Fe in PM2.5 at a high-altitude site in a semi-arid region of western India, they reported fractions of water-soluble Fe as 0.06 %– 16.1 %. Ingall et al. S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron 10128 Figure 10. Scatterplot of the Fe/Pd before and after water dialysis for Fe-containing particles on sample no. 03. Panel (b) shows an enlarged graph of the light gray square area of panel (a). Pd is a coating material in alloy for giving shadowing to samples. Al and/or Si. The mineralogy of Fe from coal combustion in the emission inventory by Rathod et al. (2020), which is used in our model, was estimated from a reference to mineralog- ical measurements of bulk samples using Mössbauer spec- trometry or from the X-ray absorption near-edge structure. Although fuels and combustion temperatures can affect the mineralogical transformation from fuels to by-products, the Fe contents estimated from mineralogical measurements of coal ﬂy ash at electrical generation facilities tend to include both oxides (such as magnetite and hematite) and clay (such as kaolinite and illite) (Hinckley et al., 1980; Szumiata et al., 2015; Waanders et al., 2003; Oakes et al., 2012; Rathod et al., 2020). However, even if Fe in spherical ﬂy ash in our TEM samples were assumed to be composed of aluminosilicates and equivalent FeOx, the mass fraction of aluminosilicate Fe inferred from TEM results would be larger than 13 %, which is higher than our model-simulated ﬁndings. Multiple factors such as the fossil fuel composition and combustor can affect the chemical composition and size–mass distribution of coal ﬂy ash particles at the source (Markowski and Filby, 1985; Liu et al., 2018). The lack of clarity in assessing those factors can also affect the uncertainty in the simulation for the emis- sions of Fe originating from coal combustion and transport according to the particle size. Such underestimation of alu- minosilicates is expected to be one factor causing the under- estimation of the total Fe mass concentration in our model. Figure 10. Scatterplot of the Fe/Pd before and after water dialysis for Fe-containing particles on sample no. 03. Panel (b) shows an enlarged graph of the light gray square area of panel (a). Pd is a coating material in alloy for giving shadowing to samples. Figure 11. Boxplot of the water-soluble Fe fraction (fWSFe) of Fe- containing particles for sample no. 03. The lower boundary of the box shows the 25th percentile. The line within the box represents the median. The upper boundary of the box stands for the 75th per- centile. S. Ueda et al.: Morphology and solubility of aged ﬁne iron The whiskers above and below the box, respectively, show the 90th and 10th percentiles. The white circles show values for the respective particles. Figure 11. Boxplot of the water-soluble Fe fraction (fWSFe) of Fe- containing particles for sample no. 03. The lower boundary of the box shows the 25th percentile. The line within the box represents the median. The upper boundary of the box stands for the 75th per- centile. The whiskers above and below the box, respectively, show the 90th and 10th percentiles. The white circles show values for the respective particles. 3.4 Fe simulation for each source by the global model To elucidate the origins of Fe and the model performance, Fe simulation results obtained using the CAM–ATRAS model were compared with observation results. Figure 8 shows the monthly mean simulated mass concentrations in PM2.5 for the total Fe and Fe from each source at the surface. The color scale of 0–150 ng kg−1 is the same as that presented for Fig. 2c. The averages of the total Fe mass concentra- tions for areas north and south of the Equator along the ship tracks (0–16◦N and 0–20◦S at 87.5◦E) are shown in Fig. 9a Atmos. Chem. Phys., 23, 10117–10135, 2023 https://doi.org/10.5194/acp-23-10117-2023 3.5 Water solubility of Fe (2018) measured the water solubility of total Fe in bulk aerosol samples taken from multiple lo- cations in the Southern and Atlantic oceans, Noida (India), The values of fWSFe found from this study were 20 %– 100 % in Fe-containing particles, except for spherical ﬂy ash. Among Fe-containing minerals, the fractions of water- https://doi.org/10.5194/acp-23-10117-2023 Atmos. Chem. Phys., 23, 10117–10135, 2023 10129 10129 S. Ueda et al.: Morphology and solubility of aged ﬁne iron soluble Fe in FeOx (magnetite, goethite, and hematite) and clays (e.g., kaolinite and illite) are very low (respectively, < 0.01 % and 1.5 %–4 %), but those of ferrous and ferric sul- fate are higher (50 %–90 %) (Desboeufs et al., 2005; Jour- net et al., 2008; Schroth et al., 2009; Rathod et al., 2020). The Fe solubilities for many Fe-containing particles in this study were comparable with those of ferrous and ferric sul- fates. Our earlier study found non-/less-sulfate-coated FeOx in aerosol samples collected in urban Tokyo, for which we applied the same analysis (Ueda et al., 2022). However, few decreases in Fe with water dialysis were found. The differ- ence in Fe solubility found from this urban study can be at- tributed to atmospheric aging processes because of the coat- ing of secondary aerosol materials and Fe oxidation during transport. Some results of experimentation and simulation studies have shown that Fe in minerals composed of Fe ox- ide and clays can be oxidized and enhanced in an acidic liq- uid phase, such as in aerosol droplets and clouds (Shi et al., 2009, 2015; Chen et al., 2012). For the present study, most of the observed Fe-containing particles co-existing with sulfate were regarded as droplets in the atmosphere. Therefore, Fe in particles was regarded as being oxidized in liquid droplet particles in the atmosphere, after which they were collected as water-soluble Fe, such as Fe sulfate. For Fe-containing particles in this study, the number fraction of FeOx aggre- gates was smaller, whereas that of categorized particles to small Fe thickly coated by sulfate was higher. The loss of the water-insoluble Fe shape occurring because of the change to water-soluble Fe can be expected to affect this result. strated that Fe in coal ﬂy ash can dissolve in H2SO4 acidic aqueous solutions of pH 1 and 2. 3.5 Water solubility of Fe Our observation results obtained from these TEM sam- ples suggest ﬂy ash as an important component of ﬁne Fe- containing particles. In addition, water dialysis results sug- gest that Fe in aged particles over the remote ocean tends to exist as partly or mostly water-soluble Fe, whereas Fe in spherical ﬂy ash can maintain water insolubility. Model sim- ulations have led to estimates that FeOx is a major compo- nent in PM2.5 Fe of this area and that Fe from anthropogenic aluminosilicates from sources such as coal combustion is mi- nor. Underestimation of the mass fraction of Fe which re- mained water insoluble during atmospheric transport can be a factor affecting the estimation error in water-soluble Fe de- position. In addition, although Fe mineralogy is often used for the simulation of solubility of anthropogenic Fe in per- centage terms in some models, the spherical shape of ﬂy ash particles and the results obtained from water dialysis imply that Fe solubility and the change in the atmosphere depend not only on mineralogical features but also on morphological and structural features of ﬂy ash originating from emission processes. The presence of ﬂy ash in ﬁne aerosols should be noted for model simulations of water-soluble Fe and estima- tions based on size-segregated samples of Fe concentrations. All water-insoluble residues in Fe-containing particles of fWSFe < 15 % were spherical ﬂy ash. Although all of them co-existed with sulfate, water dialysis results indicated that they retained the water insolubility of Fe. Our observed in- soluble sphere residues imply structural and morphological features as reasons for the insolubility of the Fe contained in aged ﬂy ash particles. Spherical particles have minimal surface area. In addition, their Fe is distributed in particles with other insoluble materials composed of Si and Al in the sphere. This distribution would physically block the oxida- tion and dissolution of Fe. However, results of several stud- ies have suggested that Fe in similar spherical ﬂy ash parti- cles can dissolve in acidic particles (Chen et al., 2012; Li et al., 2017). Li et al. (2017) observed similar submicrometer Fe-rich spheres coated by sulfate in samples collected over the Yellow Sea affected by the eastern Asian continental out- ﬂow. They used nanoscale secondary ion mass spectrometry and elemental mapping with STEM to analyze sulfate-rich particles containing Fe-rich parts and reported the presence of dispersed FeS−(Fe sulfate) around the FeO−(Fe oxide)- rich part. 3.5 Water solubility of Fe These results of earlier studies suggest that some Fe in ﬂy ash can exist as water- soluble Fe in sulfuric acid particles. However, the results of our experiments have indicated that soluble Fe in spherical ﬂy ash was considerably less present or nonexistent com- pared with insoluble Fe. From the present study, as explained in Sect. 3.2, sulfate particles were found as neutralized par- ticles such as ammonium. Such atmospheric conditions ob- served in the present study might not have decreased pH suf- ﬁciently to enhance the Fe solubility of ﬂy ash. soluble Fe in FeOx (magnetite, goethite, and hematite) and clays (e.g., kaolinite and illite) are very low (respectively, < 0.01 % and 1.5 %–4 %), but those of ferrous and ferric sul- fate are higher (50 %–90 %) (Desboeufs et al., 2005; Jour- net et al., 2008; Schroth et al., 2009; Rathod et al., 2020). The Fe solubilities for many Fe-containing particles in this study were comparable with those of ferrous and ferric sul- fates. Our earlier study found non-/less-sulfate-coated FeOx in aerosol samples collected in urban Tokyo, for which we applied the same analysis (Ueda et al., 2022). However, few decreases in Fe with water dialysis were found. The differ- ence in Fe solubility found from this urban study can be at- tributed to atmospheric aging processes because of the coat- ing of secondary aerosol materials and Fe oxidation during transport. Some results of experimentation and simulation studies have shown that Fe in minerals composed of Fe ox- ide and clays can be oxidized and enhanced in an acidic liq- uid phase, such as in aerosol droplets and clouds (Shi et al., 2009, 2015; Chen et al., 2012). For the present study, most of the observed Fe-containing particles co-existing with sulfate were regarded as droplets in the atmosphere. Therefore, Fe in particles was regarded as being oxidized in liquid droplet particles in the atmosphere, after which they were collected as water-soluble Fe, such as Fe sulfate. For Fe-containing particles in this study, the number fraction of FeOx aggre- gates was smaller, whereas that of categorized particles to small Fe thickly coated by sulfate was higher. The loss of the water-insoluble Fe shape occurring because of the change to water-soluble Fe can be expected to affect this result. 3.5 Water solubility of Fe They concluded that such an Fe sulfate was formed from Fe dissolution of ﬂy ash in an acidic aqueous phase be- cause no other atmospheric source of Fe sulfate or process engenders its formation. Using bulk samples of coal ﬂy ash composed mainly of spherical particles, Chen et al. (2012) also investigated Fe dissolution. Their experiments demon- Atmos. Chem. Phys., 23, 10117–10135, 2023 Competing interests. The contact author has declared that none of the authors has any competing interests. Competing interests. The contact author has declared that none of the authors has any competing interests. Global model simulations using a recent emissions inven- tory mostly reproduced the observed PM2.5 Fe concentra- tions in the north and south during the cruise. The model simulations suggested that PM2.5 Fe over the observation site was inﬂuenced strongly by anthropogenic Fe emissions around India. In contrast, comparison with the morphological features of observed Fe suggests that the simulations overes- timate the fractions of anthropogenic FeOx and underesti- mate the fraction of Fe in aluminosilicates originating from coal combustion. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. We are indebted to staff members of the Hakuho Maru for assisting with our work on board and to Kazuo Osada of Nagoya University for support and technical advice. We also extend our gratitude for technical support from the High- Voltage Electron Microscope Laboratory of Nagoya University. We gratefully acknowledge the NOAA Air Resources Laboratory (ARL) for providing the HYSPLIT transport model (http://www. arl.noaa.gov/ready.html, last access: 8 March 2018). Mingxu Liu acknowledges the support of JSPS Postdoctoral Fellowships for Re- search in Japan (standard). Water dialysis analysis of a TEM sample indicates that about half of the Fe in Fe-containing particles was soluble in water. However, Fe in spherical ﬂy ash particles was al- most insoluble in water, even when co-existing with sulfate. Dissolution and oxidation of Fe in spherical ﬂy ash were re- garded as having been blocked by the small surface of the sphere and the structure of Fe dispersion in other insoluble aluminosilicates. Our results obtained from shipboard observations and in- dividual ﬁne aerosol analysis indicate that Fe of various types, such as ﬂy ash, FeOx, and mineral dust, co-exists with sulfate over the remote Indian Ocean and that the solubility of Fe differs among the materials. Although the model sim- ulations show good agreement with the observed Fe mass concentrations, the results indicate marked differences in the mass fractions of mineral sources of model simulations com- pared with the observed Fe types. Results of earlier studies have demonstrated that anthropogenic ﬁne Fe tends to be composed mainly of FeOx, with increased solubility occur- ring along with aging. However, our results suggest that Fe in spherical ﬂy ash can stand out in ﬁne aerosols over the remote ocean and can retain water insolubility. Competing interests. The contact author has declared that none of the authors has any competing interests. For an accu- rate estimation of the effects of atmospheric Fe on marine biogeochemical activity, greater attention must be devoted to morphological and mineral types of Fe depending on its source and especially to insoluble Fe in ﬂy ash. Financial support. This study was supported by the Min- istry of Education, Culture, Sports, Science and Technol- ogy and the Japan Society for the Promotion of Science (MEXT/JSPS; KAKENHI grant nos. 18H03369, 18J40204, 19K20438, 22K18023, JP19H04253, JP19H05699, JP19KK0265, JP20H00196, JP20H00638, JP21K12230, JP22H03722, JP22F22092, JP23H00515, JP23H00523, and JP23K18519); the MEXT Arctic Challenge for Sustainability II (ArCS II; grant no. JPMXD1420318865) projects; and the Environment Research and Technology Development Fund 2–2003 (grant no. JPMEERF20202003) and 2–2301 (grant no. JPMEERF20232001) of the Environmental Restoration and Conservation Agency. Review statement. This paper was edited by Luis A. Ladino and reviewed by Weijun Li and two anonymous referees. References 4 Summary and conclusions To elucidate the mixing states and the water solubility of Fe- containing particles in remote marine areas, we conducted shipborne aerosol observations over the Indian Ocean during the RV Hakuho Maru cruise. After TEM samples for individ- ual particle analysis were obtained, they were analyzed using EDS and water dialysis. Most of the particles were composed mainly of sulfate neutralized by ammonium or potassium: the particle number fraction, 0.6 %–3.0 %, of particles on a sam- ple stage of 0.3–0.9 µm diameter contained Fe. They mostly included co-existing sulfate. Air-mass backward-trajectory analyses suggest that air masses south of the Equator were transported from southern India. Both the correlations of the respective elements mea- sured using chemical analysis for bulk PM2.5 samples and the absence of V and soot for individual Fe-containing par- ticles imply that the Fe in particles was transported mainly from around the continent rather than from ship exhaust. The Fe in particles was found to include the following: 26 % of https://doi.org/10.5194/acp-23-10117-2023 Atmos. Chem. Phys., 23, 10117–10135, 2023 10130 S. Ueda et al.: Morphology and solubility of aged ﬁne iron S. Ueda et al.: Morphology and solubility of aged ﬁne iron metal spheres, often co-existing with Al or Si, regarded as ﬂy ash; 14 % as irregularly shaped heterogeneously co-existing with Si, Al, or Ca, regarded as mineral dust; and 7 % as FeOx aggregates. ical analyses of PM2.5 samples. HM and ML conducted numerical model simulations using CAM–ATRAS. S. 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https://openalex.org/W4285235660	https://www.shs-conferences.org/articles/shsconf/pdf/2022/11/shsconf_mtde2022_03003.pdf	English	null	The use of distance learning technologies at the university in the new reality	SHS web of conferences	2,022	cc-by	5,571	The use of distance learning technologies at the university in the new reality Anastasiya Pesha, Marina Shavrovskaya, and Tatyana Kamarova Department of Labor Economics and Human Resources Management, Ural State University of Eco- nomics, Yekaterinburg, 620144, Russian Federation Anastasiya Pesha, Marina Shavrovskaya, and Tatyana Kamarova Abstract. The purpose of the paper is, on the basis of empirical research, to identify problems and growth areas in the field of effective use of distance learning technologies in universities in the context of COVID-19. The authors used theoretical and empirical research methods: literary analysis, generalization, methods of descriptive statistics. The main method for collecting empirical data is to survey students' opinions. The study involved 1578 students from more than 70 Russian universities. The authors substantiated the problems and opportunities for learning with the use of distance learning technologies. The paper shows not only the current difficulties of learning in an online format, but also identifies their main advantages. It presents the change in the attitude of students to the online format of education for a period of 1 year, their vision of the effectiveness of distance learning technologies in higher educational institutions. The authors substantiated the objectivity and reliability of the results obtained through their correlation with the results of similar studies conducted by scientists in various countries. The materials of the article can be useful for university teachers implementing the curriculum of disciplines using distance learning technologies, as well as for all those interested in higher education issues. * Corresponding author: myrabota2011@gmail.com SHS Web of Conferences 141, 03003 (2022) MTDE 2022 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction p g g [ ] Despite the different social, economic and technological realities, scientists from different countries discuss the problems and opportunities of distance education and education using distance technologies at universities. For example, P. Gregori, V. Martínez, J. J. Moyano- Fernández analyze possible actions that will help reduce the percentage of "dropout" students in distance learning in Spain [10], A. T. Ragusa, A. Crampton explore the feeling of isolation of Australian students [11], critical issues of distance learning in Russia are pointed out in a study by E.N. Zaborova, I.G. Glazkova and T.L. Markova [7]. Studying the problems and opportunities of distance education during the Pandemic on an international scale is important not only from the point of view of national factors, but also for using successful experiences and taking into account mistakes. Currently, the authors are analyzing the consequences of a rapid transition to distance learning, as well as measures that need to be taken to prevent possible problems associated with this transition. For example, M. Rizun and A. Strzelecki demonstrate the results of an empirical study, where they describe the effectiveness of the transition of Polish students to distance learning [12]. Scientists from Indonesia R. Hamid, I. Sentry & S. Hasan, based on the analysis of students' opinions, studied the factors of the effectiveness of online learning during the Pandemic [13]. The study by Y.Nenko, N.Кybalna, Y. Snisarenko reveals the most popular tools used in distance learning for students of Ukrainian universities, as well as the factors of distance learning implementation [14]. M.W.Marek, C.S.Chew, W.C.V.Wu showed that teachers coped with the challenge due to the ability to adapt and plan their activities, while paying special attention to the needs of students [15]. A.V. Pesha and T.A. Kamarova highlighted a number of advantages associated with the transition of universities to online education during the Pandemic - the convenience of studying the material with the student's individual choice of time and place to study, the erasure of geographical boundaries of education, flexibility in terms of material support of the student, age, health status [16]. The authors conducted research focusing on the tools – emotional, technical, social [14,17], described the problems of transition to distance learning [13,18,19], the competence of teachers and the technical readiness of universities to switch to online learning [2, 10, 19]. 1 Introduction The development and spread of digital technologies opens up the horizons of strategic restructuring of the higher education system, in terms of improving the educational environment of universities, modernizing business processes and revising organizational design, as well as revealing the competence potential of the teaching staff and university students. This is noted in a number of studies aimed at revealing various aspects of the digital transformation of didactics. For example, this fact is revealed in an article by R. Brigstock devoted to the idea of forming a new educational model of university education focused on the development of students' abilities that they need in the present and will need in the future. [1], in the works of C. Brooks et al., S. A. Popenici et al. where the impact of the use of digital technologies on the transfer of knowledge in higher education is studied [2,3], C. Buabeng-Andoh, where the author focuses on the analysis of factors that encourage teachers to use digital technologies in their lessons [4]. The works of L. A. Danchenok [5] and O.I. SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 Popova are devoted to the transformation of models for the implementation of educational programs in the digital economy [6]. Scientists note the influence of teachers' abilities to integrate ICT (information and communication technologies) on student satisfaction and keeping them at the university [7]. There is a wide range of terms related to distance learning and education, including: e- learning (e-learning), online learning, blended learning (blended learning). In our work, we will adhere to the S.Guri-Rosenelt approach, which shows that distance learning is all learning, when teaching and learning activities are distributed in time and space, which does not require a teacher and a student to be in the same place at the same time [8, p. 5]. Online learning is one of the distance learning forms implemented using digital educational technologies. Another term characterized by I.E. Allen, S. Jeff, G. Richard is mixed learning which is one of the most important in our study [9]. According to the researcher’s, blended learning is a form of organization of the learning environment, in which 30-80% of training is implemented using information and communication technologies via internet [9]. 1 Introduction In the study, the results of which are showed in this article, we focus on changing students' perception of the transition to learning using distance learning technologies over time and depending on their form of study at the university: full-time, part-time, part-time. The nature of distance education during the emergence and course of COVID-19 accelerated the processes of digital modernization of higher education and revealed a number of problems that hinder the effective translation of knowledge, the development of skills and abilities of students. Scientists all over the world are implementing a large number of studies in which they present the results of studying the problems that have arisen in the new reality of COVID-19, the speed and results of the adaptation of universities in general and teaching 2 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 staff to learning using distance learning technologies [13, 14, 15]. At the same time, the issue of students’ adaptation of various education forms to new conditions of educational activity with a certain time interval is insufficiently presented. staff to learning using distance learning technologies [13, 14, 15]. At the same time, the issue of students’ adaptation of various education forms to new conditions of educational activity with a certain time interval is insufficiently presented. y The purpose of the article is based on empirical research to identify problems and growth areas in the field of effective use of distance learning technologies at universities in the new reality of COVID-19. 2 Materials and methods The methodological basis of the study was personality-oriented and environmental approaches in education, theoretical and empirical research methods. The data were collected among undergraduate and graduate students of various forms of education. The sample population of the first stage was 249 students from 28 universities in Russia. There were 1,350 responses in the second stage of the study. After checking the results of the second stage of the study, 21 responses were rejected (20 students of secondary specialized educational institutions and one of a Chinese university). Total 1,578 students took part in the study (according to the stages of the study: n1=249, n2 = 1,329). p y ( g g y , , ) 484 questionnaires of the second stage were included in the data analysis and corresponded to the number of responses required for efficiency in relation to the total number of Russian university students 4049333 people. The confidence level of the exact sample was 95% (p <0.05), and based on the total number of students 4049333 and the sample size of 384 people, the margin of error was determined as 5%. In order to include a larger number of respondents in the end, the final sample was formed from the calculation nfinal = nnessessarycoefficient 1.25, which amounted to 484 people. To ensure the reliability of the sample, the number of respondents in federal districts was determined based on the percentage ratio of the number of students in the district to the total number of students in the Russian Federation. We did not set out to compare the opinions of students from different regions, our task is to gather the general opinion of students depend on the forms of education. The distribution of participants by form of study was representative of the total number of students at the time of data collection. Based on the appropriate responses, a representative profile of the participants in the second stage of the study is presented in Table 1. 484 questionnaires of the second stage were included in the data analysis and corresponded to the number of responses required for efficiency in relation to the total number of Russian university students 4049333 people. The confidence level of the exact sample was 95% (p <0.05), and based on the total number of students 4049333 and the sample size of 384 people, the margin of error was determined as 5%. 3 Results There are the data in comparison of respondents' opinions on the 5 questions asked in the second part of the questionnaire, depending on the stage of the survey in Fig. 1. Fig. 1. Respondents' answers to the question "Has the quality of university education changed in your opinion during the pandemic?" (in % of the total number of respondents, according to two stages of the survey). Fig. 1. Respondents' answers to the question "Has the quality of university education changed in your opinion during the pandemic?" (in % of the total number of respondents, according to two stages of the survey). Fig. 1. Respondents' answers to the question "Has the quality of university education changed in your opinion during the pandemic?" (in % of the total number of respondents, according to two stages of Fig. 1. Respondents' answers to the question "Has the quality of university education changed in your opinion during the pandemic?" (in % of the total number of respondents, according to two stages of the survey). Fig. 1. Respondents' answers to the question "Has the quality of university education changed in your opinion during the pandemic?" (in % of the total number of respondents, according to two stages of the survey). Fig. 1. Respondents' answers to the question "Has the quality of university education changed in your opinion during the pandemic?" (in % of the total number of respondents, according to two stages of the survey). We can see that respondents' opinions on the impact of the pandemic on the quality of education at the university were divided. At the very beginning of the Pandemic and the transition to a distance learning format, students of all forms of education tended to have a negative impact of changes on the quality of education. The negative impact was noted by more than half of the full-time survey participants (54.3%) and a third of part-time respondents (30.7%), as well as 66.7% of evening part-time students. A high percentage of negative opinions may be associated with the first reaction to changes, as well as with a sense of the unfair cost of this training format. Students noted the convenience of offline learning. 2 Materials and methods In order to include a larger number of respondents in the end, the final sample was formed from the calculation nfinal = nnessessarycoefficient 1.25, which amounted to 484 people. To ensure the reliability of the sample, the number of respondents in federal districts was determined based on the percentage ratio of the number of students in the district to the total number of students in the Russian Federation. We did not set out to compare the opinions of students from different regions, our task is to gather the general opinion of students depend on the forms of education. The distribution of participants by form of study was representative of the total number of students at the time of data collection. Based on the appropriate responses, a representative profile of the participants in the second stage of the study is presented in Table 1. Table 1. Profile of research participants at the second stage (people) Federal District Required number of respondents Number of participant s (total) Sample population (coefficient 1.25) Number of respondents by forms of training full- time part - time evening part - time South 38 48 48 Required sample size Central 122 198 152 230 134 20 Far Eastern 17 41 22 Uralsky 28 726 35 Number of participants (total) Northwest 40 53 50 1068 207 54 Siberian 45 138 57 Sample population (coefficient 1.25) North Caucasian 20 29 25 Privolzhsky 76 96 95 290 169 25 Total 384 1329 484 Table 1. Profile of research participants at the second stage (people) 3 3 https://doi.org/10.1051/shsconf/202214103003 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 3 Results 42.6% of part-time students say that there is no impact of the transition to online learning on its quality, commenting that not much has changed for part-time students, most of the technologies of the digital educational environment have been used by them before to prepare assignments and receive feedback from teachers. At the same time, the absence of changes was noted by 23.7% of full-time respondents, for whom the majority of the technologies and tools were new. About 20% of part-time students spoke about the positive impact of the transition on the quality of education, while evening part-time students unanimously did not choose these answers. A year later, fewer students talked about the negative impact. In particular, 40.5% of full- time students noted a negative impact, but, in comparison with the 1st stage of research, the vast majority of negative responses are associated with a slight negative impact on the quality of education by switching to an online format. The percentage of part-time students who gave negative feedback of the impact of the transition to online learning has also decreased (25.8%). As for the evening part-time respondents, the number of responses with a minus sign also decreased significantly and amounted to 40.8% of the number of survey participants. The percentage of responses "no, it has not changed" increased from 26.1% to 4 4 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 31.8%. A significant decrease in negative responses and an increase in the number of opinions regarding the positive impact of changes on the quality of education during the Pandemic may indicate the adaptation of students in the learning process to digital technologies. 31.8%. A significant decrease in negative responses and an increase in the number of opinions regarding the positive impact of changes on the quality of education during the Pandemic may indicate the adaptation of students in the learning process to digital technologies. The second question was asked to clarify the students’ opinions regarding the problems they had to face during the transition to learning using distance technologies. In this question, respondents could choose several answers. The main problems identified by students at the very beginning and a year later are presented in Fig. 2. Evening part-time students (n=2, 2020) noted the lack of necessary and sufficient technical support, the number of tasks to disciplines and the lack of feedback from the teacher. 3 Results The difference in students' perception of the advantages of online learning is well visualized in the figure. The main advantage highlighted by the majority of respondents is the opportunity to study from home (from 71.4% to 78.1% of respondents by forms of education). Also, the students chose one of the main advantages of saving time, which is associated with travel to the place of study and with the speed of switching to various tasks, including non-academic ones. "Having a flexible schedule", as one of the main advantages for full-time and part-time students, is perceived less significantly for evening part-time students, which basically implies flexibility. Also, fewer evening part- time students are interested in the development of digital skills, as a plus of switching to an online learning format. New digital tools attracted 33.2% of part-time students in online education, which is significantly more than the percentage of students of other forms of education who chose this plus. p Fig 3 Answers of respondents to the question "What are the advantages of online learning?" (in% of Fig. 3. Answers of respondents to the question "What are the advantages of online learning?" (in% of the total number of respondents). Fig. 3. Answers of respondents to the question "What are the advantages of online learning?" (in% of the total number of respondents). Fig. 3. Answers of respondents to the question "What are the advantages of online learning?" (in% of the total number of respondents). Fig. 3. Answers of respondents to the question "What are the advantages of online learning?" (in% of the total number of respondents). The views of students of education various forms on the prospects of learning in the digital educational environment demonstrate a significant increase in the popularity of this format of interaction at the university in 1 year (Fig. 4). The majority of full-time students agree with the statement about the great prospects of online learning in combination with the traditional format of work in the classroom (57.6%), and almost half of the part-time students agree with them (42.9%). An equal approach to all students is important to a greater number of part-time and evening part-time students, in comparison with full-time students, which shows their choice in answering this question. 3 Results The key problem noted by students of all education forms at both stages of the study is related to the lack of communication with each other and with the teacher (32% at the first stage and 26.8-49% at the second one). At the second stage of the study, an increase in participants indicating a lack of communication is visible, especially among full-time students (from 32% to 49%). Evening part-time students are less likely than others to highlight the problem of communication lack. The second problem, which is noted many students of all forms of education, concerns problems with Internet communication. Students stress the presence of such problems, both on the part of teachers and problems with home Internet. Fig. 2. Answers of respondents to the question "What problems have you faced in working in a digital educational environment?" (in% of the total number of respondents, according to two stages of the survey). The abrupt transition of all active life to an online environment has put Internet service providers in a difficult position, which has also affected the learning process using distance learning technologies, especially with synchronous interaction. A large number of full-time students, both at the first and second stages of the survey, express their opinion about the poor technical training of teachers, which has become one of the key problems of the qualitative organization of the educational process in the circumstances. Evening part-time students also mention this problem, but not so often. At the second stage, part-time students more often than other groups of respondents chose the option "there were no problems" 5 5 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 (23%). The positive dynamics of the number of students' opinions about the absence of problems in answering this question, among other things, may indicate a change in attitude to the circumstances, their acceptance and conscious attitude to student learning. (23%). The positive dynamics of the number of students' opinions about the absence of problems in answering this question, among other things, may indicate a change in attitude to the circumstances, their acceptance and conscious attitude to student learning. , p g Next, we asked the students about the advantages of learning using distance learning technologies. The question also suggested the possibility of choosing several answers. The results are presented in Fig. 3. Fig. 4. Answers of respondents to the question "What, in your opinion, are the prospects for learning in a digital educational environment?" (in% of the total number of respondents, according to two stages of the survey). Resuming the analysis of the survey’s results, we can make a conclusion that there are significant differences in the opinions of students on all the questions asked, received in March 2020 and a year later. In particular, the number of negatively colored comments of students has decreased (22% and 14% of negative comments of full-time and part-time students in 2020, against 3% and 5% of representatives of these aggregates in 2021). Positive dynamics is also observed in the responses of respondents regarding the impact of online learning on the quality of education among all groups of respondents. However, there is a specificity of students' opinions on this issue, according to the form of study. So, at the first stage, 42.6% of correspondence students wrote about the lack of impact of the transition to online learning on its quality, at the same time, only 23.7% of full-time respondents noted the absence of changes, for which the vast majority of the used technologies and tools were new. A year later, the percentage of respondents in all forms of education who noted the absence of changes leveled off and amounted to about a third of students. As for the positive impact of the transition to learning with the use of distance learning technologies, here the opinions of students also have significant differences in the forms of education. 22.1% and 26% of full-time students expressed these points of view; the percentage of evening part-time students who reacted positively to the transition to the online format was about 25% at both stages. At the same time, the share of students who believe that the quality of education has become higher among correspondence students has grown over the year from 19.1% to 3 Results When choosing the "other" options, respondents wrote a year ago that the prospects are great, provided that universities are interested in developing this format of work with students, and also provided that the curriculum is clearly planned. A year later, the number of free-form responses increased, and their degree became more negative. For example, answers were received among part-time students about the lack of prospects for the online format, despite the fact that it is not being canceled, as well as about the low quality of education. Full-time students used the following judgments in their responses: "it's a pity", "a decrease in the level of education", "I really 6 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 hope that they will be canceled soon". Nevertheless, despite the number of negatively colored answers in free form (8, or 0.6% of respondents), the number of choices of the option "no, they will soon be canceled" regarding the prospects of online education in a year decreased more than twice (31.3% in 2020, 15% in 2021). At the first stage of the study, both respondents of the full-time and part-time form chose the lack of prospects and the imminent cancellation of online education. hope that they will be canceled soon". Nevertheless, despite the number of negatively colored answers in free form (8, or 0.6% of respondents), the number of choices of the option "no, they will soon be canceled" regarding the prospects of online education in a year decreased more than twice (31.3% in 2020, 15% in 2021). At the first stage of the study, both respondents of the full-time and part-time form chose the lack of prospects and the imminent cancellation of online education. 3 Discussion The conducted review of the literature aimed at studying the problems of emergency transition to education using distance learning technologies allowed us to correlate the results obtained with the data of existing studies. Thanks to the analysis, we have formed possible development zones in terms of the organization of the educational process at the university in the online format. Similar results to our study regarding students' satisfaction with learning during this period in the online format are also noted by other researchers. For example, in the work of Ukraine colleagues Y.Nenko, N.Kybalna, Y. Snisarenko, 59.9% indicated full satisfaction with online education during the Pandemic and self-isolation [14]. About 85% of the surveyed Russian students in the study by I. R. Gafurov noted satisfaction with the transition to online learning [20]. The greatest dissatisfaction with the quality of education during the emergency transition to the online format of interaction was expressed by full-time students in comparison with students of other forms. There is a possibility that this difference in quality and satisfaction ratings is due to the difference in study schedules and the distribution of workload hours for students of various forms of study, as well as higher demands of full- time students for personal interaction with teachers and their dissatisfaction with the high cost of educational services in a distance format. Among the main problems noted by students, the main ones are a sense of isolation and lack of communication with colleagues and teachers, which is consistent with the results of other works of this period, for example in the work of M. W.Marek, C. S.Chew, W. C. V. Wu [15]. Technical problems, as the key ones, were also highlighted by students who took part in other studies, which is reflected in the works of S. El Firdoussi and her colleagues [19], for students of Ukraine, according to N. Y.enko, N.Kybalna, Y.Snisarenko, technical problems have become the main obstacle to effective learning during quarantine – 44.5% of respondents chose this fact [14], in a large-scale study by I. A. Aleshkovsky and his colleagues, 24.4% of Russian university students also noted these problems [18]. In our study, the choice among the main problems associated with the use of ICT among full-time students significantly exceeds the number of choices by part-time students and evening part- time ones. 7 7 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 39.5%. That is, part-time students, more than others, express satisfaction and readiness to study online. 39.5%. That is, part-time students, more than others, express satisfaction and readiness to study online. y The analysis of the key problems and opportunities of online learning identified by students in 2020 and 2021 showed that the main one for all respondents at both stages was the lack of communication with each other and with the teacher (32% at the first stage and 26.8-49% at the second one). At the second stage of the study, an increase in participants indicating a lack of communication is visible, especially among full-time students (from 32% to 49%). Evening part-time students are less likely than others to highlight the problem of lack of communication. The second problem concerns the difficulties with Internet communication. A large number of full-time students, both at the first and second stages of the survey, express their opinion about the poor technical training of teachers. At the second stage, students of the part-time department to a much greater extent than the other groups of respondents chose the option "there were no problems" (23%). The positive dynamics of the number of students' opinions about the absence of problems in answering this question, among other things, may indicate a change in attitude to the circumstances, their acceptance and conscious attitude to student learning. SHS Web of Conferences 141, 03003 (2022) MTDE 2022 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 Analyzing the respondents' answers about the prospects of online education, we received a positive answer. At the same time, full-time students in this regard show much more confidence compared to part-time (42.9%) and evening part-time students (32.1%). The relevance of this result is found in the article by I. A. Aleshkovsky and his colleagues, where 16.2% of students note high prospects and readiness to fully switch to online learning [18]. These results correlate with the results of surveys among high school teachers conducted by researchers from the USA, Malaysia, Taiwan (Marek M. W., Chew C. S., Wu W. C. V. [15]), which indicate that teachers highly appreciate the potential of using distance learning technologies in traditional education. The results of our study show a high level of students’ readiness to study online, which is confirmed in the works of other authors, for example, M. L. Hung, C. Chou, C. H. Chen, & Z. Y. Own [21], I. A. Aleshkovsky and his colleagues [18]. The respondents' answers obtained in our study coincide with the prevailing point of view of Moroccan students revealed in the work of S. El Firdoussi and her colleagues, that blended learning in the ratio of 50/50% of full-time and online learning can be the best alternative to existing educational formats [19], which is confirmed by the research of I. A. Aleshkovsky and his colleagues [18], as well as in the work of B. Means, Y. Toyama, R. Murphy, M. Baki [22]. [ ] y p y [ ] We share the point of view of M. Babinčáková, P. Bernard that despite all the shortcomings, the challenge to higher education given by the pandemic served to gain new experience and prospects, and also became a stimulus for its development [23]. Our results show that students have adapted to the online format, learned new tools and identified its advantages for themselves. In addition, over the past year, the efficiency of the system that ensures the quality of education in universities has increased, algorithms have been worked out and work has been done on the errors and shortcomings of the emergency transition to online learning. Acknowledgments: The reported study was funded by RFBR, project number 19-29-07435 3 Discussion We can assume that they are more independent, responsible and ready to ensure the effectiveness of their work from a technical point of view, which is due to the socio- economic specifics of most of this group. 8 https://doi.org/10.1051/shsconf/202214103003 References 1. R. Bridgstock, In Grad. Empl. in cont., 339 (2017). https://doi.org/10.1057/978-1-137- 57168-7_16 2. C.Brooks, S. Gibson, Can. J. of Learn. and Tech. 38(2), (2012). https://doi.org/10.4324/9781315853529 3. S. A. Popenici, S. Kerr, Res. and Prac. in Tech. Enh. Learn. 12(1), (2017). https://doi.org/10.1186/s41039-017-0062-8 4. C. Buabeng-Andoh, Inter. J. of Ed. and Dev. using ICT 8(1), 136 (2012). https://www.academia.edu/11106469/Factors_influencing_teachers_adoption_and_inte gration_of_information_and_communication_technology_into_teaching_A_review_of _the_literature L. A.Danchenok, A. S. Zaitseva, N. V. Komleva, Open Ed. 23(1), 34 (2019). https://doi.org/10.21686/1818-4243-2019-1-34-45 p g 6. O. I. Popova, Manag. is. 5(54), 158 (2018). https://www.elibrary.ru/item.asp?id=36714636 7. E. N. Zaborova, I. G. Glazkova, T. L. Markova, Sociol. Stud. 2(2), 131 (2017). https://elibrary.ru/item.asp?id=28790355 8. S. Guri-Rosenelt, NY: Nova Scien. Pub. Inc., (2009). https://www.academia.edu/20619431/digital_technologies_in_higher_education_sweep ing_expectations_and_actual_effects 9 SHS Web of Conferences 141, 03003 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214103003 9. I. E.Allen, S.Jeff, G. Richard, Builds on a series of annual reports on the state of online ed. in US Higher Ed., 1 (2007). https://files.eric.ed.gov/fulltext/ED529930.pdf 9. I. E.Allen, S.Jeff, G. Richard, Builds on a series of annual reports on the state of online ed. in US Higher Ed., 1 (2007). https://files.eric.ed.gov/fulltext/ED529930.pdf 10. P.P.Gregori, V.Martínez, J. J. Moyano-Fernández, Eval. and progr. Plan. 66, 48 (2018). https://doi.org/10.1016 / j.evalprogplan.2017.10.004 10. P.P.Gregori, V.Martínez, J. J. Moyano-Fernández, Eval. and progr. Plan. 66, 48 (2018). https://doi.org/10.1016 / j.evalprogplan.2017.10.004 11. A.T.Ragusa, A. Crampton, Rural Soc. 27(2), 125 (2018). https://doi.org/10.1080/10371656.2018.1472914 12. M.Rizun, A.Strzelecki, Inter. J. of Environ. Res. and Pub. Health 17(18), 6468, (2020). https://doi.org/10.3390/ijerph17186468 13. R.Hamid, I.Sentryo, S.Hasan, Jur. Prima Eduk. 8(1), 85 (2020). https://doi.org/10.21831/jpe.v8i1.32165 14. Y.Nenko, N.Кybalna, Y. Snisarenko, Rev. Brasil. de Educ. do Campo 5, (2020). https://doi.org/10.20873/uft.rbec.e8925 15. M. W.Marek, C. S.Chew, W. C. V. Wu, Inter. J. of Dist. Ed. Tech. 19 (1), 40 (2021). https://doi.org/10.4018/IJDET.20210101.oa3 p g 16. A.V.Pesha, T.A. Kamarova, Kadrovik 7, 73 (2020). https://www.elibrary.ru/item.asp?id=44127347 17. V.Abou-Khalil, S.Helou, E.Khalifé, M. A.Chen, R.Majumdar, H.Ogata, Ed. Sciences, 11(1), 24 (2021). https://doi.org/10.3390/educationsci11010024 18. A. Aleshkovsky, A.T. Gasparishvili, O.V. Krukhmaleva, Higher ed. in Russia 29(10), 86 (2020). https://doi.org/10.31992/0869-3617-2020-29-10-86-100 19. S.El Firdoussi, M.Lachgar, H.Kabaili, A.Rochdi, D.Goujdami, L. El Firdoussi Ed. Research Inter. 13, (2020). DOI: https://doi.org/10.1155/2020/8890633 20. I. R.Gafurov, G. I. Ibragimov, A. M.Kalimullin, T. B. Alishev, Higher ed. in Russia 29 (10), 101 (2020). DOI: https://doi.org/10.31992/0869-3617-2020-29-10-101-112 21. M. L. Hung, C.Chou, C. H. Chen, Z. Y. Own, Comp. & Ed. 55 (3), 1080 (2010). https://doi.org/10.1016/j.compedu.2010.05.004 22. B. Means, Y. Toyama, R. Murphy, M. Baki, Teach. college record 115(3), 1 (2013). https://www.learntechlib.org/p/156867/ ̌ 23. M. Babinčáková, P.P. Bernard, J. of chem. ed. References 97(9), 3295 (2020). DOI: https://doi.org/10.1021/acs.jchemed.0c00748 10
W4380287997.txt	https://zenodo.org/records/8027463/files/ADE12.pdf	fr		Problèmes de Compréhension de Lecture Rencontrés Par les Étudiants à l'Université de Samuel Adegboyega	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	3,023	GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. Problèmes de Compréhension de Lecture Rencontrés Par les Étudiants à l’Université de Samuel Adegboyega Adebayo Akinsanya Atchrimi Department of Languages, College of Humanities, Samuel Adegboyega University, Ogwa, Edo State, Nigeria. 08114068601 aaatchrimi1@gmail.com Akinnola Faith Idowu Department of French, School of Languages, Osun State College of Education, Ilesha, Osun State, Nigeria. 08036187175 Faithidowuakinnola65@gmail.com And Omotayo Julius Languages and Literature Lead City University, Toll-gate, Ibadan sessyjunior@gmail.com 08023831364 D.O.I: 10.5281/zenodo.8027463 Date of Publication: 04-12-2021 Journal Website: https://gjollaas.com/index.php/pub Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 109 GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. RÉSUMÉ Cet article vise à étudier les problèmes de compréhension de lecture rencontrés par les étudiants de l’Université de Samuel Adegboyega. Cette étude vise également à mieux comprendre les facteurs affectant la compréhension en lecture des élèves. À cette fin, le chercheur a utilisé un questionnaire qui comprend des énoncés généraux sur les principaux problèmes auxquels les élèves sont confrontés dans la compréhension de la lecture. Les participants à cette étude sont 20 (vingt) étudiants (10 (dix) hommes et 10 (dix) femmes) du Département des langues de l’Université de Samuel Adegboyega. Les résultats de l'étude indiquent que les élèves ont une estimation élevée de certains problèmes qu'ils rencontrent dans la compréhension de la lecture en raison de la complexité des textes, de l'anxiété et de la reconnaissance des mots (décodage). Les résultats de l'étude ont également révélé qu'il y avait des différences statistiquement significatives entre les élèves de sexe masculin et féminin en ce qui concerne leurs problèmes de compréhension de lecture. Mots clés: Compréhension, lecture, étudiants, université, problèmes Introduction La lecture joue un rôle important dans nos vies. C'est une partie importante de la vie quotidienne que l'on ne peut pas imaginer la vie sans elle. Quatre compétences linguistiques sont utilisées pour atteindre la maîtrise d’une langue: écouter, parler, lire et écrire Chawwang (2008). La lecture est une compétence importante que les apprenants devraient connaître. Les apprenants lisent des textes pour différents objectifs allant de l'obtention d'informations au plaisir. Il aide les apprenants à se familiariser avec les matières de leurs majors et à améliorer leurs connaissances linguistiques Gilakjani et Sabouri (2016). La lecture est un processus interactif dans lequel l'écrivain et le lecteur dialoguent à travers un texte. Barnett (1989) et Omaggio, 1993) définissent la lecture comme communication, comme processus mental, comme participation active du lecteur à la création de sens et comme manipulation de stratégies. De plus, Day et Bamford (2000) postulent que la lecture est la construction du sens à partir d'un message imprimé ou écrit. La lecture est importante car c'est l'une des compétences linguistiques les plus fréquemment utilisées dans la vie quotidienne, comme en témoigne l'utilisation d'Internet. Les étudiants recherchent des informations sur le Web et la plupart sont publiées en Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 110 GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. français. «Étant donné qu'une compétence de lecture est clairement l'une des plus importantes, en fait, dans de nombreux cas à travers le monde, nous pouvons affirmer que la lecture est la compétence étrangère la plus importante, en particulier dans les cas où les étudiants doivent lire du matériel français pour leur propre matière spécialisée ne jamais avoir à parler la langue; de tels cas sont souvent qualifiés français comme langue de bibliothèque » McDonough et Shaw (1993, p. 89) Pressley (2002), les caractéristiques d'un «bon lecteur» peuvent être résumées comme suit: a) sont actifs pendant la lecture et ont des objectifs explicites liés au texte. b) Regardez à travers le texte avant de lire et tout en lisant, ils font toujours des suppositions sur ce qui va se passer lors de la prochaine session. c) Regardez à travers les passages et ils essaient de deviner le sens et la structure des mots à partir du contexte. d) Utiliser leurs connaissances de base et vérifier leur compréhension. e) Restructurez le sens, répétez-le et remettez-le en question. f) Pensez aux personnages et aux événements lors de la lecture de textes de fiction. Ils ont tendance à résumer des textes informatifs. g) Supposons que la lecture soit un processus productif. Les compétences en compréhension de la lecture sont importantes pour que les élèves deviennent des lecteurs efficaces. Ces compétences nous permettent de lire avec compétence et d'apprendre efficacement Grabe et Stoller (2002). La plupart des apprenants du français peuvent rencontrer des problèmes de compréhension lors de la lecture d'un texte, mais les lecteurs expérimentés y feraient face en appliquant des stratégies de lecture efficaces pour résoudre les problèmes de compréhension (Tobing, 2013). Définition des termes Compréhension écrite: La compréhension en lecture fait référence à la capacité de lecture ou à la compétence en lecture, et les termes sont souvent utilisés de manière interchangeable. Il est défini comme le processus d'extraction et d'intégration d'informations à partir d'un texte écrit en anglais tout en appliquant les connaissances de base des lecteurs et la connaissance de la langue anglaise Koda 2005) Birsch (2011) a défini la compréhension en lecture comme la capacité à obtenir un sens à partir de ce qui est lu. Selon Gilakjani & Sabouri (2016), la compréhension de la lecture nécessite différentes compétences en lecture telles que la reconnaissance de mots, la maîtrise, la connaissance lexicale et les connaissances préexistantes à entreprendre rapidement afin que le lecteur acquière des connaissances à partir du texte. Tompkins (2011) a défini la compréhension en lecture comme le niveau de compréhension d'un texte. Il soutient que la compréhension est un processus créatif qui repose sur quatre compétences appelées phonologie, syntaxe, sémantique et pragmatique. Facteurs Affectant les Compétences en Compréhension de Lecture Certaines études ont indiqué que la compréhension en lecture des étudiants est affectée par plusieurs facteurs. Selon Dennis (2008), l'un des facteurs qui influent sur la compréhension en lecture des apprenants est la complexité des textes. Un tel facteur est Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 111 GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. affecté par la force et la maîtrise de la langue du lecteur et sa compréhension de ses applications et des différentes significations. Les conditions environnementales influencent également les apprenants qui essaient de lire un passage. Si les apprenants se trouvent dans un endroit dangereux, ils ont du mal à se concentrer sur leur lecture. Les lecteurs peuvent perdre leur concentration à comprendre un texte lorsqu'il y a des bruits comme la télévision ou la radio Dennis 2008. L'anxiété est également un facteur qui affecte la compréhension en lecture des apprenants. Les examens, les travaux en classe ou les devoirs peuvent exercer une pression sur la lecture des apprenants pour le plaisir. Les apprenants diffèrent dans leur réaction face aux examens, car certains réagissent positivement tandis que d'autres sont submergés par la pression d'effectuer une activité de lecture (Dennis, 2008). Gilakjani & Sabouri (2016) indiquent que le décodage ou la reconnaissance de mots a un impact sur la compréhension en lecture des apprenants. Les lecteurs qui ont des problèmes de décodage et de reconnaissance des mots ont du mal à comprendre la signification des passages que ceux qui n'ont pas de problèmes de décodage. Les apprenants qui ont suffisamment de vocabulaire peuvent clarifier la signification des passages plus rapidement que ceux qui devraient deviner la signification de mots inconnus en fonction des indices de contexte. Stratégies Efficaces pour Améliorer les Compétences de Compréhension en Lecture L’application de stratégies de compréhension efficaces est très importante lorsque les apprenants souhaitent améliorer leurs compétences en compréhension de la lecture. Ces stratégies fournissent des instructions particulières pour développer les compétences de compréhension Cain et Oakhill 2009. Chaque type de texte nécessite l'utilisation de stratégies de lecture différentes. Certains lecteurs interagissent avec les textes en utilisant des stratégies telles que prédire, prévoir, déduire, résumer, examiner et évaluer Gilakjani et Sabouri 2016. Il existe des stratégies utiles pour améliorer les compétences en compréhension de la lecture. Il s'agit de l'enseignement réciproque, des conversations pédagogiques, de la visualisation, de la lecture par un partenaire, de plusieurs stratégies de lecture, de l'évaluation et de la gestion des enregistrements. Selon l'enseignement réciproque, les enseignants apprennent aux apprenants à prédire, résumer, clarifier et poser des questions pour des parties d'un texte. Ces stratégies ont été considérées comme des stratégies efficaces pour développer les compétences de compréhension en lecture des apprenants. Autrement dit, les apprenants développeront par eux-mêmes des compétences de compréhension de lecture plus solides si les enseignants leur donnent des instruments mentaux pour comprendre un texte Pressley 2002. Participants à l'étude Les participants à cette étude étaient 20 étudiants du department des Langues de la première au troisième année (10 hommes et 10 femmes) ā l’université de Samuel Adegboyega. Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 112 GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. Fréquence Tableau Pourcentage Pourcentage Pourcentage cumulé Valide Valide HOMMES 11 55.0 55.0 55.0 FEMMES 09 45.0 45.0 45.0 TOTAL 20 100 100 100 Ce tableau montre que 55% de l'échantillon étaient des hommes et que 45% étaient des femmes. Instruments de l'étude Un questionnaire a été préparé pour cette recherche. Le questionnaire abordait les domaines potentiels de défis pour les élèves tels que le problème de la compréhension de la signification de certains nouveaux mots, la difficulté à deviner la signification de certains nouveaux mots, la distinction entre les idées principales et les idées de soutien dans le texte de lecture, la taille de la classe qui dissuade la compréhension des textes de lecture, la rareté de l'utilisation du dictionnaire pour rechercher la signification des mots difficiles dans le texte de lecture, le manque de formation sur la compréhension du texte, la lecture du texte pour réussir l'examen au lieu de l'apprécier et d'interagir avec lui. Un total de 20 questionnaires ont été distribués et collectés, les données des 20 questionnaires étaient aptes à être testées. Likert Scale a été utilisée afin de répondre aux items du questionnaire sur la base de ces valeurs: - Totalement d'accord: (5) points - D'accord: (4) points - Neutre: (3) points - Pas d'accord: (2) points - Totalement en désaccord: (1) point Fiabilité de l'instrument Après l'application du questionnaire et la collecte des données pertinentes de l'échantillon de l'étude en cours, un plus petit nombre de 7 étudiants de l'échantillon de cette étude a reçu le questionnaire auquel répondre afin d'établir le coefficient de fiabilité. Les valeurs alpha de Cronbach étaient supérieures à 74,2%, ce qui indique que le questionnaire a une fiabilité appropriée pour analyser ses données. Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 113 GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. La méthode de correction du questionnaire Le chercheurs a utilisé les catégories suivantes pour juger de l'estimation des donnés: 1. Moyenne (1-moins de 2,33) estimation faible 2. Moyenne (2,34-moins de 3,66) estimation moyenne 3. Moyenne (3,67-5) estimation élevée. Procédures la collecte de données a été menée en 2020. Une fois les questionnaires remplis, les données quantitatives seront analysées à l'aide du progiciel statistique pour les sciences sociales. L'outil d'analyse de données spécifique utilisé est la régression, à la fois des régressions simples et multiples. Résultats et discussion Quels sont les principaux problèmes rencontrés par les étudiants de l'Université de Samuel Adegboyega? Problèmes de Compréhension de Lecture 1 J'ai rencontré un problème pour comprendre la signification de certains nouveaux mots dans le texte de lecture. 3.9500 et .99868 2 J'ai rencontré un problème pour identifier le but de la compréhension en lecture. 3,6500 et 0,81273 3 J'ai eu du mal à deviner la signification de certains nouveaux mots 3.8500 et 1.08942 4 J'ai rencontré un problème pour distinguer les idées principales des idées de soutien dans le texte de lecture 3.5500 et 1.27630 5 J'ai rencontré des difficultés pour extraire des idées principales et détaillées dans le texte 3.9500 et 1.09904 6 J'ai rencontré des difficultés pour reconnaître la signification totale du texte 3.9000 et 1.02084 7 Je traduis habituellement mot par mot lorsque je lis 3,9500 et 0,88704 8 Je me sens stressé en lisant un texte en anglais en classe. 3,7500 et 1,01955 9 Je ne me sens jamais vraiment sûr de moi quand je lis en classe. 3,5500 et 0,68633 10 Je n'arrête pas de penser que les autres étudiants sont meilleurs que moi en langue. 3.9500 et 0.99868 11 Même si je suis bien préparé pour le cours de langue, cela m'inquiète. 3,3500 et 1,13671 12 Je me sens bouleversé quand je ne comprends pas ce que je lis en anglais 3.2500 et 96655 13 J'ai rencontré des difficultés pour résumer les idées principales du texte 3.5000 et 76089 Moyenne Totale nous donne 3,704 et 0,989 les attitudes des participants à l'égard des énoncés du questionnaire; Moyenne moyenne (3.704), écart-type (0.989). On note que Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 114 GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. la moyenne moyenne était supérieure à (3.500), ce qui indique qu'il y a un degré élevé d'estimation pour les variables de l'étude. L'analyse du questionnaire révèle que ces élèves souffrent de problèmes, tels que la détermination de la signification de certains nouveaux mots dans le texte de lecture, l'extraction d'idées principales et détaillées dans le texte, en continuant à penser que les autres élèves sont meilleurs en langage que lui, elle est, reconnaissant la signification totale du texte, devinant la signification de certains nouveaux mots, se sentant stressée lors de la lecture d'un texte en français en classe. Les participants de cette étude traduisent généralement mot par mot lorsqu'ils lisent un texte. Les participants de cette étude ont une estimation modérée du problème d'identification de l'objectif derrière la compréhension de la lecture, des difficultés à distinguer les idées principales des idées de soutien dans le texte de lecture, des difficultés à résumer les idées principales du texte. Même s'ils sont bien préparés pour le cours de langue, ils en sont inquiets. Ils se sentent bouleversés lorsqu'ils ne comprennent pas ce qu'ils lisent en français. Ils ne se sentent jamais tout à fait sûrs d'eux-mêmes lorsqu'ils lisent en classe. Par ailleurs, le sexe masculin et féminin dans leurs difficultés de compréhension en lecture. (les femmes ont plus de problèmes de compréhension en lecture que les hommes.) Les résultats indiquent qu'il existe des différences statistiquement significatives dues au sexe (homme et femme) dans leurs difficultés de compréhension de lecture en faveur des hommes. (Les hommes ont plus de problèmes de compréhension en lecture que les femmes). Le questionnaire n'inclut pas de questions dont les réponses pourraient être en corrélation avec ces résultats, mais le chercheur suggère que le niveau plus élevé des hommes en compréhension de lecture peut être associé aux facteurs du système scolaire, car les hommes peuvent avoir des leçons de langue plus régulières, et cela peut contribuent à leur niveau supérieur de compréhension en lecture. De plus, les hommes utilisent davantage les stratégies de lecture que les femmes; les hommes ont également plus de compétences en lecture que les femmes. Conclusion Cette étude a tenté d'examiner les problèmes de compréhension de lecture rencontrés par les étudiants de l’Université de Samuel Adegboyega.. Les résultats de l'étude indiquent que les élèves ont une estimation élevée de certains problèmes qu'ils rencontrent dans la compréhension de la lecture en raison de la complexité des textes, de l'anxiété et de la reconnaissance des mots (décodage). Les résultats de l'étude ont également révélé qu'il existe des différences statistiquement significatives entre les élèves de sexe masculin et féminin en ce qui concerne leurs problèmes de compréhension en lecture. Lorsque les apprenants de sexe masculin et féminin ont été comparés, les apprenants de sexe masculin ont également montré une meilleure performance de compréhension et ont indiqué un niveau plus élevé de maîtrise français. Recommandations 1- Il est important de faire une enquête plus approfondie sur l’impact du niveau des apprenants et des connaissances de base sur la compréhension de la lecture. Le chercheur recommande donc aux chercheurs de mener des études similaires dans d’autres universités. Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 115 GVU JOURNAL OF LANGUAGE, LITERATURE AND AFRICAN STUDIES (GJOLLAAS) - VOLUME 1, NUMBE 1, DECEMBER, 2021. 2- Il est important d'encourager les élèves à lire autant que possible, car ils trouveront ainsi une aide précieuse dans leur processus d'apprentissage des langues. 3- Les enseignants devraient encourager les élèves à relire les passages. Ils pourront lire avec une vitesse et une fluidité accrues, ce qui leur permettra d'extraire plus de connaissances de ce qu'ils lisent. Références Cain, K. et Oakhill, J. (2011). Matthew Effects in Young Readers: La compréhension de la lecture et l'expérience de lecture aident au développement du vocabulaire. Journal of Learning Disabilities, 44 (5), 431-443. Dennis, D. V. (2008). Les données d'évaluation motivent-elles vraiment l'enseignement de la lecture au collège? Ce que nous pouvons apprendre de l’expérience d’un élève. Journal of Adolescent and Adult Literacy, 5(1) 578-587. Extrait de: http://www.ehow.com/info_8423949_factors-poor-readingcomprehensionskills.html Grabe, W. et Stoller, F. L. (2002). Enseigner et rechercher la lecture. Londres: Pearson Education Longman. Hollowell, K. (2013). Facteurs affectant une mauvaise compréhension de la lecture. Contributeur e-How. Magazine en ligne. Koda, K. (2005). Aperçu de la lecture en langue seconde: une approche interlinguistique. New York: Cambridge University Press. Medjahdi, W. B. (2015). Difficultés de compréhension en lecture chez les apprenants EFL: le cas des apprenants de troisième année à l'école secondaire Nehali Mohamed, mémoire de maîtrise. Université de Tlemcen, Algérie. Published by the Department of Languages, Glorious Vision University, P.M.B. 001, Ogwa, Edo State, Nigeria © 2021. 116
https://openalex.org/W3096069104	https://hal.archives-ouvertes.fr/hal-03167275/document	English	null	Wake redirection at higher axial induction	Wind energy science	2,021	cc-by	9,947	To cite this version: C. Cossu. Wake redirection at higher axial induction. Wind Energy Science, 2021, 6 (2), pp.377-388. 10.5194/wes-6-377-2021. hal-03167275 HAL Id: hal-03167275 https://hal.science/hal-03167275v1 Submitted on 11 Mar 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 This work is distributed under the Creative Commons Attribution 4.0 License. Wake redirection at higher axial induction Carlo Cossu Laboratoire d’Hydrodynamique Énergetique et Environnement Atmosphèrique (LHEEA) CNRS – Centrale Nantes, 1 rue de la Noë, 44300 Nantes, France Correspondence: Carlo Cossu (carlo.cossu@ec-nantes.fr) Received: 20 October 2020 – Discussion started: 26 October 2020 Revised: 13 January 2021 – Accepted: 4 February 2021 – Published: 11 March 2021 Carlo Cossu Carlo Cossu Laboratoire d’Hydrodynamique Énergetique et Environnement Atmosphèrique (LHEEA) CNRS – Centrale Nantes, 1 rue de la Noë, 44300 Nantes, France Correspondence: Carlo Cossu (carlo.cossu@ec-nantes.fr) Received: 20 October 2020 – Discussion started: 26 October 2020 Revised: 13 January 2021 – Accepted: 4 February 2021 – Published: 11 March 2021 Abstract. The energy produced by wind plants can be increased by mitigating the negative effects of turbine– wake interactions. In this context, axial-induction control and wake redirection control, obtained by intentionally yawing or tilting the rotor axis away from the mean wind direction, have been the subject of extensive research but only very few investigations have considered their combined effect. In this study we compute power gains that are obtained by operating tilted and yawed rotors at higher axial induction by means of large-eddy simulations using the realistic native National Renewable Energy Laboratory (NREL) 5 MW actuator disk model imple- mented in the Simulator for On/Offshore Wind Farm Applications (SOWFA). We show that, for the considered two-row wind-aligned array of wind turbines, the power gains of approximately 5 % obtained by standard wake redirection at optimal tilt or yaw angles and reference axial induction can be more than tripled, to above 15 %, by operating the tilted or yawed turbines at higher axial induction. It is also shown that signiﬁcant enhancements in the power gains are obtained even for moderate overinduction. These ﬁndings conﬁrm the potential of overin- ductive wake redirection highlighted by previous investigations based on more simpliﬁed turbine models that neglected wake rotation effects. The results also complement previous research on dynamic overinductive yaw control by showing that it leads to large power gain enhancements also in the case where both the yaw and the overinduction controls are static, hopefully easing the rapid testing and implementation of this combined-control approach. Published by Copernicus Publications on behalf of the European Academy of Wind Energy e.V. 1 Introduction sen et al., 2015, and Boersma et al., 2017, for a review). Among the many proposed approaches, two have received particular attention: axial-induction control and wake redi- rection control, which can be static (the control is steady if the incoming wind conditions are) or dynamic (the control can be unsteady even for steady incoming wind conditions). In wind farms, wind turbines shadowed by the wakes of other upwind turbines experience a decrease in the mean available wind speed and an increase in turbulent ﬂuctua- tions, resulting in decreased extracted wind power and in- creased fatigue loads (see Stevens and Meneveau, 2017, and Porté-Agel et al., 2019, for a review). In currently installed wind farms, however, each turbine is typically operated in “greedy” mode, maximizing its own individual power pro- duction. As the greedy operation mode does not generally lead to the global optimum, where the energy production of the whole wind farm is maximized (see, e.g., Steinbuch et al., 1988), a number of different approaches have been proposed where the collective control of all turbines is used to increase the power production of the whole wind farm by mitigating the negative effects of turbine–wake interactions (see Knud- In axial-induction control the induction factors of selected (usually upwind) turbines are steered away from the greedy operation mode in order to increase the power production of other (usually downwind) turbines. While static axial- induction control has not demonstrated signiﬁcant power gains in realistic settings (Knudsen et al., 2015; Annoni et al., 2016), dynamic axial-induction control has shown promise for signiﬁcant power gains (Goit and Meyers, 2015; Munters and Meyers, 2017). In wake redirection control the inten- tional misalignment of rotor axes from the wind direction is used to deﬂect turbine wakes in the horizontal or in the Published by Copernicus Publications on behalf of the European Academy of Wind Energy e.V. C. Cossu: Wake redirection at higher induction 378 they do not analyze the respective effects of yaw and induc- tion; furthermore, their optimal solutions in the aligned case converge to an underinductive operation mode for yawed tur- bines. 1 Introduction Munters and Meyers (2018a, b) show, by means of ad- joint methods with full-state information and an actuator disk turbine model where wake rotation is neglected, that high power gains result from the combination of dynamic yaw and axial-induction controls, with Munters and Meyers (2018b) highlighting the potential of quasi-static yaw control in the (dynamic) overinductive regime. Thus, from these previous studies, it is not clear if signiﬁcant power gains could be re- alized in the overinductive regime when both the yaw and the axial-induction control are static, nor is it clear to what extent the neglected wake rotation effects are important. vertical direction by acting on yaw or tilt angles, respec- tively, with a documented increase in the global power pro- duced by the wind farm (Dahlberg and Medici, 2003; Medici and Alfredsson, 2006; Jiménez et al., 2010; Fleming et al., 2014, 2015; Campagnolo et al., 2016; Howland et al., 2016; Bastankhah and Porté-Agel, 2016). In two recent studies (Cossu, 2020a, b) we have shown that an appropriate combination of (static) tilt and (static) axial-induction control results in a signiﬁcant enhancement in the global power gains obtained in spanwise-periodic wind turbine arrays. In these studies, for the considered three-row turbine arrays, power gains were observed to be highly en- hanced (up to a factor of 2 or 3) when the turbines with the rotor tilted by the optimal angle (ϕ ≈30◦) were operated with a disk-based thrust coefﬁcient C′ T = 3 higher than in the baseline case (C′ T = 1.5). The second objective of the present study is therefore to ascertain if signiﬁcant power gains can be obtained with a combination of static yaw control and static axial-induction control by operating yawed turbines at higher axial induc- tion and including the effect of wake rotation in the turbine model. An afﬁrmative answer would allow the isolation of the mean wake redirection as the most relevant physical ef- fect at play (instead of, e.g., the dynamical adaptation to the incoming wind) and indicate that it is robust with re- spect to the inclusion of wake rotation effects. Furthermore, if successful, static overinductive yaw control could be eas- ily implemented by simply updating existing yaw-control protocols with a prescription of the suitable turbine rotor- collective blade-pitch angle (controlling the axial induction and the thrust coefﬁcient) for each accessible yaw angle. 1 Introduction ( T ) The results reported in these previous studies (Cossu, 2020a, b) were obtained with an actuator disk model, where wake rotation and the radial distribution of actuator disk forces were neglected, and the turbines were assumed to op- erate at constant given C′ T. This highly idealized setting, used in many previous investigations (e.g., Calaf et al., 2010; Goit and Meyers, 2015; Munters and Meyers, 2017), has been instrumental in obtaining general results not depending on the speciﬁc turbine-control law and blade design, but it calls for conﬁrmation by means of more realistic turbine models. Hence, a ﬁrst goal of this study is to determine the power gains that can be obtained with high-induction (overinduc- tive) tilt control when realistic turbine models are used that take into due account blade design, wake rotation and the controller speciﬁcity. This goal is addressed in the ﬁrst part of this study by making use of the native actuator disk model of the Simulator for On/Offshore Wind Farm Applications (SOWFA; Churchﬁeld et al., 2012) for the National Renew- able Energy Laboratory (NREL) 5 MW turbine. In this im- plementation of the turbine model the radial dependence of the actuator disk force as well as wake rotation and C′ T is computed from turbine blade properties by means of a blade- element approach, and the NREL 5 MW’s ﬁve-region realis- tic controller (Jonkman et al., 2009) is used. ) y g The potential of static overinductive wake redirection is investigated by computing power gains that can be obtained in a wind turbine array composed of two spanwise-periodic rows of wind-aligned turbines where the same control is ap- plied to all upwind-row turbines, while downwind-row tur- bines are left in default operation mode. This idealized con- ﬁguration, which is an extension to the spanwise-periodic case of the two-turbine conﬁguration considered by Fleming et al. (2015), is chosen in order to keep the physical interpre- tation of the results simple by isolating the effects of tilt or yaw angle and axial induction of the upwind turbines without entering the problem of the optimization of these parameters encountered in more realistic conﬁgurations with more rows. As such, this approach is a necessary ﬁrst step needed to iso- late the main trends at play before considering more realistic settings. Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 2 Problem formulation Higher axial inductions are obtained by enforcing negative values of β (see Appendix A), resulting in higher local thrust coefﬁcients C′ T = 2T/(ρu2 nA), where T is the thrust magnitude, ρ is the ﬂuid density, and un is the disk- averaged wind velocity component normal to the rotor disk of area A = πD2/4. For all the considered cases the local power coefﬁcient C′ P = 2P/(ρu3 nA) (where P is the power) is well approximated as C′ P = χC′ T, with χ = 0.9; results of C′ P trends are therefore not shown in the following. The in- coming ﬂow, generated by means of a precursor simulation in a 3km × 3 km domain in the absence of turbines, has a 100 m thick capping-inversion layer centered at H = 750m, separating the neutral boundary layer with constant potential temperature (θ = 300 K) from the geostrophic region above where the vertical potential-temperature gradient is positive (dθ/dz)G = 0.03Km−1. In the capping-inversion layer this gradient is (dθ/dz)CI = 0.03Km−1. In the precursor simu- lation, the ABL is driven by a pressure gradient adjusted to maintain a horizontally averaged mean of 8 ms−1 from the west at z = 100m (a few meters above hub height zh = 89 m). In the region spanned by the turbines (z < 152 m) the streamwise mean velocity is well approximated by the log- arithmic law, and the vertical wind veer is less than 4◦(see In each (spanwise-periodic) row, turbines are spaced by 4D in the spanwise direction (where D = 126 m is the rotor diameter), and the two rows are spaced by 7D in the stream- wise direction, with corresponding turbines of each row aligned with respect to the mean wind direction (see Fig. 2, where the full computational domain is shown). Downwind- row turbines are always operated in default mode with the rotor axis at yaw angle γ = 0◦(aligned with the mean wind at z = 100 m), tilt angle ϕ = −5◦to prevent the blades from hitting the tower (see Fig. 1 for a deﬁnition of ϕ and γ ) and rotor-collective blade-pitch angle β = 0◦. In the base- line (reference) case upwind-row turbines are also operated in default mode. 2 Problem formulation We address the case of two spanwise-periodic rows of wind turbines immersed in a neutral atmospheric bound- ary layer (ABL) at latitude 41◦N. The ﬂow is simulated by means of large-eddy simulations with SOWFA (the Simula- tor for On/Offshore Wind Farm Applications developed at the NREL; see Churchﬁeld et al., 2012), which solves the ﬁl- tered Navier–Stokes equations including the Coriolis accel- eration associated with Earth’s rotation and the compressibil- ity effects modeled by means of the Boussinesq approxima- tion (see Appendix A for more details and Churchﬁeld et al., 2012, for the explicit expression of the solved equations and a full description of the used formulation and modeling as- sumptions used in SOWFA). Figure 1. Deﬁnition of the positive rotor tilt and yaw angles ϕ and γ used in the present study. Positive tilt angles can be obtained for downwind-oriented rotors to avoid blade–tower hits. Cossu, 2020b, where the same ABL has been already consid- ered). The streamwise turbulence intensity of the incoming wind at hub height is 5.7 % for the enforced low roughness length (z0 = 0.001 m) typical of offshore conditions. Simulations in the presence of wind turbines are repeated in the same 3km × 3 km domain starting from the solution of the precursor simulation at t0 = 20000 s, corresponding to a well-developed ABL, up to t1 = 30000 s. Statistics are computed starting from t = 24000 s, when turbine wakes are fully developed. The pressure gradient issued from the precursor simulation is enforced during the simulation with turbines, and the (previously stored) ABL solution at x = 0 (west boundary) is used as an inﬂow boundary condition. NREL 5 MW turbines (Jonkman et al., 2009) are consid- ered, which are modeled with SOWFA’s native actuator disk method, where wake rotation, the radial distribution of aero- dynamic forces and the thrust coefﬁcient are all computed from blade properties, providing a reliable description of the wake structure except in the near-wake region. We also make use of SOWFA’s native implementation of the NREL 5 MW’s realistic ﬁve-region turbine controller based on generator torque control in the Region II regime corresponding to the mean wind speeds considered in the following; in this regime we modify axial induction by changing the rotor-collective blade-pitch angle β. 1 Introduction Importantly, the relevance of these power gains will be tested without excessive assumptions by means of large- eddy simulations in the atmospheric boundary layer using a turbine model which includes the effects of wake rotation, radial-force distribution and a realistic turbine controller. In the second part of the study we address the case of yaw control. Indeed, the increased power gains obtained by operating tilted turbines at higher thrust coefﬁcients mostly result from the increase in wake deviations obtained with- out a penalization of the power production of the tilted tur- bine. Overinductive wake deﬂection could therefore be ben- eﬁcial also in the case of yaw control, where it is known that higher thrust coefﬁcients also result in larger wake de- viations (Jiménez et al., 2010; Howland et al., 2016; Shapiro et al., 2018). Surprisingly, however, only very few studies have investigated the potential beneﬁts of combining axial- induction control and yaw control. Park and Law (2015), based on simpliﬁed wake models and advanced optimiza- tion techniques, show that signiﬁcant power gains can be ob- tained by combining static yaw and induction control, but We anticipate that substantial enhancements (up to a fac- tor of 3) in the power gains induced by wake redirection are found when operating the tilted or yawed turbines at higher axial induction. The formulation of the problem at hand is introduced in Sect. 2. Results are reported in Sect. 3 and further discussed Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 C. Cossu: Wake redirection at higher induction 379 Figure 1. Deﬁnition of the positive rotor tilt and yaw angles ϕ and γ used in the present study. Positive tilt angles can be obtained for downwind-oriented rotors to avoid blade–tower hits. in Sect. 4. Additional details on used methods are provided in Appendix A, and additional results about the effect of using a less realistic turbine model, where wake rotation effects are neglected, are reported in Appendix B. in Sect. 4. Additional details on used methods are provided in Appendix A, and additional results about the effect of using a less realistic turbine model, where wake rotation effects are neglected, are reported in Appendix B. 2 Problem formulation The baseline case is then compared to a set of controlled cases where all the turbines of the upwind row are operated at the same non-zero tilt or yaw angle and, pos- sibly, non-zero rotor-collective blade-pitch angle. 3 Results 3.1 Effect of overinduction on tilt control 3.1 Effect of overinduction on tilt control In the baseline case (all turbines operated with γ = 0◦, ϕ = −5◦, β = 0◦), the usual situation is found where the turbines of the downwind row see a strongly reduced mean wind (see Figs. 2a and 3b), therefore producing only ≈30 % of the total power, i.e., ≈40 % of that produced by the upwind row of turbines (see Fig. 4b). The effect of wake rotation https://doi.org/10.5194/wes-6-377-2021 Wind Energ. Sci., 6, 377–388, 2021 380 C. Cossu: Wake redirection at higher induction Figure 2. Tilt control: mean (temporally averaged) streamwise velocity ﬁeld in the horizontal plane at hub height obtained (a) in the baseline case, where all turbines are operated in default mode; (b) with upwind turbines tilted by ϕ = 30◦and operated at the default rotor-collective blade-pitch angle β = 0◦; and (c) with upwind turbines tilted by ϕ = 30◦and operated at higher induction (β = −5◦). The mean wind is from the west (from the left, parallel to the x axis). Note that the entire 3km × 3 km computational domain is shown in the ﬁgure and that periodic boundary conditions are applied on the north and south boundaries. C. Cossu: Wake redirection at higher induction 380 Figure 2. Tilt control: mean (temporally averaged) streamwise velocity ﬁeld in the horizontal plane at hub height obtained (a) in the baseline case, where all turbines are operated in default mode; (b) with upwind turbines tilted by ϕ = 30◦and operated at the default rotor-collective blade-pitch angle β = 0◦; and (c) with upwind turbines tilted by ϕ = 30◦and operated at higher induction (β = −5◦). The mean wind is from the west (from the left, parallel to the x axis). Note that the entire 3km × 3 km computational domain is shown in the ﬁgure and that periodic boundary conditions are applied on the north and south boundaries. Figure 3. Tilt control: cross-stream view of the mean streamwise vorticity and velocity ﬁelds in the baseline case (a, b) and with upwind turbines tilted by ϕ = 30◦and operated at β = −5◦(c, d). 3.1 Effect of overinduction on tilt control From the streamwise vorticity ﬁelds (a, c), extracted 3D downstream of the ﬁrst turbine row, the negative streamwise vorticity in the wake core associated with wake rotation can be clearly seen in the baseline case (a) as well as its combination with the two counter-rotating streamwise vortices forced by the tilted rotor (c). Streamwise (color scale) and cross-stream (arrows) velocity ﬁelds (b, d) are extracted D/2 upstream of the second row of turbines; to improve readability only the ﬁelds of the two central turbine columns (between y = 1000 and 2000m) are shown. The circles in black represent the perimeter of downstream rotors. Figure 3. Tilt control: cross-stream view of the mean streamwise vorticity and velocity ﬁelds in the baseline case (a, b) and with upwind turbines tilted by ϕ = 30◦and operated at β = −5◦(c, d). From the streamwise vorticity ﬁelds (a, c), extracted 3D downstream of the ﬁrst turbine row, the negative streamwise vorticity in the wake core associated with wake rotation can be clearly seen in the baseline case (a) as well as its combination with the two counter-rotating streamwise vortices forced by the tilted rotor (c). Streamwise (color scale) and cross-stream (arrows) velocity ﬁelds (b, d) are extracted D/2 upstream of the second row of turbines; to improve readability only the ﬁelds of the two central turbine columns (between y = 1000 and 2000m) are shown. The circles in black represent the perimeter of downstream rotors. is clearly discernible in the mean streamwise vorticity ﬁeld (see Fig. 3a). In the following, power gains will be computed with respect to the mean power PRef produced in this base- line case. is compensated by the increase in the power produced by downwind-row turbines, resulting in global power gains of ≈5 % for ϕ = 30◦(see Fig. 4b). In a further step, the rotor-collective blade-pitch angle of the tilted upwind-row turbines is changed. Enforcing increas- ingly negative values of β (i.e., increasing the mean angle of attack of all rotor blades, as explained in Appendix A) results in increased thrust coefﬁcients (increased axial induction) which, starting from C′ T = 1.5 in the baseline case (β = 0◦), attain C′ T = 3 for β = −5◦in turbines tilted by ϕ = 30◦(see Fig. 4a). Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 3.2 Effect of overinduction on yaw control Figure 5. Effect of the tilt angle ϕ on (a) the local thrust coefﬁcients C′ T of upwind-row turbines when they are operated with β = 0◦ (default axial induction) or with β = −5◦(strongly overinductive regime), (b) the total power gain (P −PRef)/PRef for selected val- ues of β. We now evaluate the beneﬁts of combining static yaw con- trol with static overinduction. We proceed similarly to the tilt-control case by using the same precursor simulation and the same baseline case, where all turbines operate at default values γ = 0◦, ϕ = −5◦and β = 0◦. We ﬁrst simulate the standard yaw control where the yaw angle γ of upwind-row turbines is changed (while keeping the other parameters, ϕ = −5◦and β = 0◦, unchanged), re- sulting in the well-known horizontal deviation in upwind- row turbine wakes and the increase in the mean wind speed seen by downwind rotors (see Fig. 6b). From Fig. 8b it is seen that the increase in the power produced by downwind-row turbines compensates the reduction in the power produced by the yawed turbines (upwind-row), resulting in maximum power gains of ≈5 % obtained for γ ≈30◦, similar to the values found by Fleming et al. (2015) for the two-turbine case. vortices is reinforced (see Fig. 3c and d), which increases the mean wind speed seen by downstream rotors (see Figs. 2c and 3d) and their extracted power despite the higher wake deﬁcit of upwind turbines (compare Fig. 2c to b), and (b) the power produced by tilted turbines is also (slightly) increased1 (see Fig. 4b). The combination of these two effects results in optimal power gains which are highly enhanced (almost tripled) with respect to those obtained by tilt without overin- duction. Finally, a full set of ϕ–β combinations is considered. For these simulations we observe that, for turbines operated at constant β, the increase in C′ T with ϕ is noticeable only for ϕ ≳30◦, as shown in Fig. 5a (we have veriﬁed that this incre- Increasing the local thrust coefﬁcient C′ T by means of increasingly negative blade-pitch angles in yawed turbines (see Fig. 8a) has effects similar to those observed for the tilt-control case: an increase in velocity deﬁcits in upwind- row turbine wakes but also their higher deviation away from downwind turbines (see Figs. 1This might be related to blockage effects which induce an in- crease with C′ T in the power produced by an (upwind) spanwise- periodic row of turbines as shown by Strickland and Stevens (2020), and it is not surprising given that, for the NREL5 turbine, β = 0◦ corresponds by design to the maximum CP (at the optimal tip speed ratio) for an isolated non-tilted turbine but not necessarily so when ϕ = 30◦. C. Cossu: Wake redirection at higher induction 381 Figure 4. Effect of enforcing negative rotor-collective blade-pitch angles β on upwind-row turbines tilted by ϕ = 30◦. (a) Temporally averaged local thrust coefﬁcient C′ T of the individual turbines of the upwind row. (b) Wind power extracted by the upwind (hatched red) and downwind (cross-hatched green) rows of turbines normalized by the total power PRef produced in the baseline case (Ref). ment is consistent with the effects of changing the tilt angle and the associated change in the induction factor). Consid- ering the (P −PRef)/PRef power gains with respect to the baseline case, from Fig. 5b it can be seen that the max- imum power gains are reached for ϕ ≈30◦, with optimal values obtained with signiﬁcant overinduction (power gains larger than 15 % for β ≈−5◦), which are almost 3 times those (≈5 %) obtained with tilt control at reference induc- tion rates (β = 0◦). This effect of overinduction in tilt con- trol is very strong: from Fig. 5b it is indeed also seen that at ϕ = 30◦, even with the moderate rotor-collective blade- pitch angle β = −2◦, power gains have already almost dou- bled with respect to standard tilt control with β = 0◦. Figure 4. Effect of enforcing negative rotor-collective blade-pitch Figure 4. Effect of enforcing negative rotor-collective blade-pitch angles β on upwind-row turbines tilted by ϕ = 30◦. (a) Temporally averaged local thrust coefﬁcient C′ T of the individual turbines of the upwind row. (b) Wind power extracted by the upwind (hatched red) and downwind (cross-hatched green) rows of turbines normalized by the total power PRef produced in the baseline case (Ref). The high enhancement in power gains obtained by com- bining overinduction with tilt control with respect to those obtained by standard tilt control at baseline induction is consistent with that found in our previous studies (Cossu, 2020a, b), therefore conﬁrming the robustness of this trend. The absolute levels of power gains are, however, smaller than those reported by Cossu (2020a, b) both because two-row ar- rays are considered here instead of the previously considered three-row arrays (which have higher power gains; see, e.g., Annoni et al., 2017) and because wake rotation effects, ne- glected in the previous studies, are here taken into account (see Appendix B for further details). Figure 5. C. Cossu: Wake redirection at higher induction Effect of the tilt angle ϕ on (a) the local thrust coefﬁcients C′ T of upwind-row turbines when they are operated with β = 0◦ (default axial induction) or with β = −5◦(strongly overinductive regime), (b) the total power gain (P −PRef)/PRef for selected val- ues of β. https://doi.org/10.5194/wes-6-377-2021 3.1 Effect of overinduction on tilt control We then consider the case where upwind-row turbines are tilted by ϕ = 30◦, an angle in the range where the best power gains have been found in previous studies (Fleming et al., 2014, 2015; Cossu, 2020a, b), while keeping their rotor-collective blade-pitch angle at the default value β = 0◦. In this case, the wakes of the upwind turbines are pushed down by the tilt-induced downwash, increasing the mean wind available to downwind turbines (see Fig. 2b). The tilt- induced decrease in power produced by upwind-row turbines The effect of the increased thrust is twofold: (a) the down- wash associated with the stronger tilt-induced streamwise Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 C. Cossu: Wake redirection at higher induction 3.2 Effect of overinduction on yaw control 6c and 7b) induced by the stronger yaw-induced, vertically staked, counter-rotating streamwise vortices (see Fig. 7a), resulting in an increase in the mean power produced by all turbines with respect to the standard yaw-control case with β = 0◦(Fig. 8b). https://doi.org/10.5194/wes-6-377-2021 Wind Energ. Sci., 6, 377–388, 2021 382 C. Cossu: Wake redirection at higher induction Figure 6. Yaw control: mean streamwise velocity ﬁeld in the horizontal plane at hub height obtained (a) in the baseline case, where all turbines are operated in default mode (γ = 0◦, β = 0◦; same as Fig. 2a, reproduced here to ease the comparison); (b) in the case with upwind turbines yawed by γ = 30◦and operated at the default β = 0◦; and (c) with upwind turbines yawed by γ = 30◦and operated at higher induction (β = −4◦). C. Cossu: Wake redirection at higher induction 382 Figure 6. Yaw control: mean streamwise velocity ﬁeld in the horizontal plane at hub height obtained (a) in the baseline case, where all turbines are operated in default mode (γ = 0◦, β = 0◦; same as Fig. 2a, reproduced here to ease the comparison); (b) in the case with upwind turbines yawed by γ = 30◦and operated at the default β = 0◦; and (c) with upwind turbines yawed by γ = 30◦and operated at higher induction (β = −4◦). Figure 7. Yaw control: cross-stream view of the mean streamwise vorticity and velocity ﬁelds with upwind turbines yawed by γ = 30◦and operated at β = −4◦. The signature of the two vertically staked counter-rotating streamwise vortices forced by the yawed rotor combined with wake rotation is clearly visible in the streamwise vorticity ﬁeld (a) extracted 3D downstream of the ﬁrst turbine row. Their effect on the lateral displacement of the wake is clearly discernible in the streamwise (color scale) and cross-stream (arrows) velocity ﬁelds (b) extracted D/2 upstream of the second row of turbines. Only the ﬁelds of the two central turbine columns (between y = 1000 and 2000m) are shown. Figure 7. Yaw control: cross-stream view of the mean streamwise vorticity and velocity ﬁelds with upwind turbines yawed by γ = 30◦and operated at β = −4◦. The signature of the two vertically staked counter-rotating streamwise vortices forced by the yawed rotor combined with wake rotation is clearly visible in the streamwise vorticity ﬁeld (a) extracted 3D downstream of the ﬁrst turbine row. 3.2 Effect of overinduction on yaw control Their effect on the lateral displacement of the wake is clearly discernible in the streamwise (color scale) and cross-stream (arrows) velocity ﬁelds (b) extracted D/2 upstream of the second row of turbines. Only the ﬁelds of the two central turbine columns (between y = 1000 and 2000m) are shown. Figure 8. Effect of changing the rotor-collective blade-pitch angle β of turbines yawed by γ = 30◦. (a) Local thrust coefﬁcient C′ T of the turbines of the upwind row. (b) Wind power extracted by the upwind (hatched red) and downwind (cross-hatched green) rows of turbines normalized by the total power PRef extracted in the base- line case. Figure 9. Effect of the yaw angle γ on (a) the local thrust coefﬁ- cients C′ T of upwind-row turbines when they are operated at β = 0◦ or at β = −5◦and (b) power gains for selected values of rotor- collective blade-pitch angle β. hi hl h d h d bi d hi h Figure 8. Effect of changing the rotor-collective blade-pitch angle β of turbines yawed by γ = 30◦. (a) Local thrust coefﬁcient C′ T of the turbines of the upwind row. (b) Wind power extracted by the upwind (hatched red) and downwind (cross-hatched green) rows of turbines normalized by the total power PRef extracted in the base- line case. The analysis of a full range of γ –β combinations leads to results similar to those obtained for the tilt-control case. A non-negligible increase in C′ T is observed for large yaw angles γ ≳30◦when operating at constant β, as reported in Fig. 9a, and global power gains obtained by yaw control are Figure 9. Effect of the yaw angle γ on (a) the local thrust coefﬁ- cients C′ T of upwind-row turbines when they are operated at β = 0◦ or at β = −5◦and (b) power gains for selected values of rotor- collective blade-pitch angle β. highly enhanced when yawed turbines are operated at higher induction (more negative values of the rotor-collective blade- pitch angle β). Also similarly to the tilt-control case, max- imum power gains are obtained for γ ≈30◦regardless of the β value. Overall optimal power gains (above 15 %) are reached for relatively high overinduction (β ≈−4◦). Also in this case, power gains obtained by γ = 30◦yaw control are Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 Figure 9. 4 Conclusions The main goal of this study was to assess the magnitude of global power gains that can be obtained in wind turbine ar- rays by combining static wake redirection control and static axial-induction control operating tilted or yawed turbines at higher axial induction (overinduction). Results have been ob- tained by means of large-eddy simulations of a two-row array of NREL 5 MW turbines in a neutral atmospheric boundary layer. Another important result, obtained for both tilt and yaw overinductive controls, is that while maximum power gains (≈15 %) are obtained for relatively large rotor-collective blade-pitch angle (β = −5◦) for the optimal large tilt and yaw angles (ϕ,γ ≈30◦), signiﬁcant power gains (≈10 %) are already obtained for smaller values β = −2◦, showing the robust beneﬁcial effect of even moderately overinductive turbine operation. In the ﬁrst part of the study we consider the effect of higher induction on tilt control by using an actuator disk model less idealized than the one used in our previous studies of this ap- proach. The results conﬁrm that, also with this more realistic turbine model, power gains can be highly increased by oper- ating tilted turbines at higher induction (power gains above 15 % are found for the considered set of parameters com- pared to ≈5 % obtained with default induction). This sub- stantial enhancement in power gains due to the use of overin- duction in tilt control is consistent with those found in our previous studies, but the absolute levels of the power gains are smaller because of the differences in array conﬁgurations and in the used turbine models. Indeed, when included in the turbine model, wake rotation results in an inclination of the formerly vertical downwash, which displaces higher-altitude, higher-speed ﬂuid towards downstream rotors, and, as a con- sequence, and also results in a decrease in tilt-induced power gains. It is also to be noted that here we have considered only two rows of turbines and a single conﬁguration, with a small value of the D/δ ratio of rotor diameter to the ABL thick- ness, but that higher power gains can be expected for a larger number of turbine rows (Park and Law, 2015; Annoni et al., 2017; Cossu, 2020a) and for larger values of D/δ (Cossu, 2020a, b). Additional investigations are, however, necessary to fur- ther reﬁne, in many directions, the conclusions of the present study. 3.2 Effect of overinduction on yaw control These results conﬁrm the ﬁrst intuition that, also in the static yaw-control case, static overinduction leads to a sub- stantial improvement of the power gains which is based on the same mechanisms discussed for the tilt-control case con- ﬁrming, that these mechanisms are quite robust. with NREL 5 MW turbines simulated with SOWFA) where wake rotation effects are fully taken into account. They also probably isolate the main physical mechanisms underlying the signiﬁcant power gains found by Munters and Mey- ers (2018a, b) by means of combined (dynamic and static) yaw and (dynamic) induction control using adjoint meth- ods with full-state information on large-eddy simulations where the turbines were modeled with a simpliﬁed actuator disk method neglecting wake rotation effects. Furthermore, static overinductive yaw control is suitable for immediate experimental testing with most existing standard horizontal- axis wind turbines unlike tilt control which is promising for speciﬁcally designed future generation, downwind-oriented and/or ﬂoating turbines (Bay et al., 2019; Nanos et al., 2020). more than doubled already for β = −2◦and almost tripled for the optimal value β = −4◦with respect to the standard operation mode (β = 0◦) at the same yaw angle γ = 30◦. These results conﬁrm the ﬁrst intuition that, also in the static yaw-control case, static overinduction leads to a sub- stantial improvement of the power gains which is based on the same mechanisms discussed for the tilt-control case con- ﬁrming, that these mechanisms are quite robust. https://doi.org/10.5194/wes-6-377-2021 Wind Energ. Sci., 6, 377–388, 2021 3.2 Effect of overinduction on yaw control Effect of the yaw angle γ on (a) the local thrust coefﬁ- cients C′ T of upwind-row turbines when they are operated at β = 0◦ or at β = −5◦and (b) power gains for selected values of rotor- collective blade-pitch angle β. Figure 9. Effect of the yaw angle γ on (a) the local thrust coefﬁ- cients C′ T of upwind-row turbines when they are operated at β = 0◦ or at β = −5◦and (b) power gains for selected values of rotor- collective blade-pitch angle β. Figure 8. Effect of changing the rotor-collective blade-pitch angle β of turbines yawed by γ = 30◦. (a) Local thrust coefﬁcient C′ T of the turbines of the upwind row. (b) Wind power extracted by the upwind (hatched red) and downwind (cross-hatched green) rows of turbines normalized by the total power PRef extracted in the base- line case. Figure 8. Effect of changing the rotor-collective blade-pitch angle Figure 8. Effect of changing the rotor collective blade pitch angle β of turbines yawed by γ = 30◦. (a) Local thrust coefﬁcient C′ T of the turbines of the upwind row. (b) Wind power extracted by the upwind (hatched red) and downwind (cross-hatched green) rows of turbines normalized by the total power PRef extracted in the base- line case. highly enhanced when yawed turbines are operated at higher induction (more negative values of the rotor-collective blade- pitch angle β). Also similarly to the tilt-control case, max- imum power gains are obtained for γ ≈30◦regardless of the β value. Overall optimal power gains (above 15 %) are reached for relatively high overinduction (β ≈−4◦). Also in this case, power gains obtained by γ = 30◦yaw control are The analysis of a full range of γ –β combinations leads to results similar to those obtained for the tilt-control case. A non-negligible increase in C′ T is observed for large yaw angles γ ≳30◦when operating at constant β, as reported in Fig. 9a, and global power gains obtained by yaw control are Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 C. Cossu: Wake redirection at higher induction 383 more than doubled already for β = −2◦and almost tripled for the optimal value β = −4◦with respect to the standard operation mode (β = 0◦) at the same yaw angle γ = 30◦. 4 Conclusions A ﬁrst important issue is to understand what the ef- fects of overinduction are on the static and dynamic structural loads experienced by the blades of tilted and yawed turbines. A complete aeroelastic analysis based on higher-ﬁdelity sim- ulations making use of the actuator line method, requiring more reﬁned grids and time steps and larger computational resources, is highly desirable, especially for the largest con- sidered values of the yaw, tilt and pitch angles, where the near- and middle-wake structures are probably more sensi- tive to details of the turbine model. In the second part of the study we ascertain if similar power gain enhancements can be obtained by combining static overinduction with static yaw control. To this end, we have ﬁrst considered the standard case where yawed turbines are operated at the reference rotor-collective blade-pitch an- gle β = 0, ﬁnding power gains of the order of 5 %, similar to those found in many previous studies (e.g., Fleming et al., 2015, for the two-turbine case). We then show that a very signiﬁcant increase in power gains (almost threefold, up to ≈15 % for the cases considered) is obtained by operating yawed turbines at higher induction, similarly to what was found for tilt control. Other issues are wind direction and array conﬁguration. The present study is limited to a two-row array in the wind- aligned case, but it is, of course, important to evaluate power gains in arrays with many more rows also in non-aligned con- ﬁgurations. This kind of analysis, where the optimal combi- nation of tilt, yaw and pitch angles of all turbines has to be computed for a high number of wind directions and intensi- ties, would be too computationally demanding if performed by means of large-eddy simulations and is customarily based on less computationally demanding simpliﬁed sets of equa- tions where the accurate modeling of the controlled wakes is of primary importance (see, e.g., Boersma et al., 2017). In this context, the results presented in the present study could be used to help in the improvement and validation of simpli- The ﬁndings concerning the static overinductive yaw con- trol are probably the most relevant of this study for short- term applications because they show that signiﬁcant power gains can be realized with a simple static overinductive yaw control in a realistic model (the atmospheric boundary layer Wind Energ. Sci., 6, 377–388, 2021 384 Wind Energ. Sci., 6, 377–388, 2021 C. Cossu: Wake redirection at higher induction 385 Appendix A: Methods NREL 5 MW blade proﬁles and the local angle of attack α = φ −(θ +β), computed as the difference between the an- gle φ formed by the relative wind seen by the blades with the rotor plane and the local pitch angle, which is the sum of the local twist angle θ of the blades and the rotor-collective blade-pitch angle β (the reader is referred to, e.g., Burton et al., 2001, and Sørensen, 2011, for a detailed discussion of turbine modeling in general and of the BEM in particu- lar). The Gaussian projection of the discretized body forces proposed by Sørensen and Shen (2002) is also used with a smoothing parameter ε = 20m to avoid numerical instabili- ties (Martínez-Tossas and Leonardi, 2013). NREL 5 MW blade proﬁles and the local angle of attack α = φ −(θ +β), computed as the difference between the an- gle φ formed by the relative wind seen by the blades with the rotor plane and the local pitch angle, which is the sum of the local twist angle θ of the blades and the rotor-collective blade-pitch angle β (the reader is referred to, e.g., Burton et al., 2001, and Sørensen, 2011, for a detailed discussion of turbine modeling in general and of the BEM in particu- lar). The Gaussian projection of the discretized body forces proposed by Sørensen and Shen (2002) is also used with a smoothing parameter ε = 20m to avoid numerical instabili- ties (Martínez-Tossas and Leonardi, 2013). The large-eddy simulations presented in this study are per- formed with SOWFA, a set of libraries and codes able to simulate atmospheric ﬂows over wind turbines (Churchﬁeld et al., 2012) that is based on the OpenFOAM software en- vironment designed to solve partial differential equations by means of ﬁnite-volume spatial discretization on unstructured meshes (Jasak, 2009; OpenCFD, 2011). The ﬁltered Navier– Stokes equations are solved using the Smagorinsky (1963) model to approximate subgrid-scale stresses, with compress- ibility effects accounted for by means of the Boussinesq ap- proximation and Earth’s rotation effects accounted for by the Coriolis acceleration term in the equations (see Church- ﬁeld et al., 2012, for all details on the used formulation and for a validation of the code in the atmospheric boundary layer). C. Cossu: Wake redirection at higher induction Schumann (1975) stress boundary conditions, mod- eling the effect of ground roughness, are applied near the ground, and slip boundary conditions are enforced at the top of the solution domain. The solutions are advanced in time using the PIMPLE scheme, which is a combination of the PISO (Pressure-Implicit with Splitting of Operators) and SIMPLE (Semi-Implicit Method for Pressure-Linked Equa- tions) schemes. The NREL 5 MW ﬁve-region controller implemented in SOWFA is used to control the turbine’s rotational speed and axial induction. In the Region II regime, the one accessed in the presented simulation, the turbine is driven to the de- sign point (tip-speed ratio and thrust coefﬁcient correspond- ing to the maximum power coefﬁcient for an isolated non- tilted, non-yawed turbine) by means of generator torque con- trol at the default rotor-collective blade-pitch angle β = 0◦. In this regime, we enforce the axial-induction control by changing the rotor-collective blade-pitch angle β while leav- ing the other parameters of the generator torque controller unchanged. Periodic boundary conditions are applied in the x (west– east) direction for the preliminary “precursor” simulations, where the atmospheric boundary layer ﬂow is computed in the absence of wind turbines in order to generate realistic in- ﬂow wind conditions (Keating et al., 2004; Tabor and Baba- Ahmadi, 2010; Churchﬁeld et al., 2012). The mean pressure gradient is adapted in order to maintain (horizontally av- eraged) mean westerly winds of 8 ms−1 at z = 100m. The time history of the mean pressure gradient and of the so- lution at x = 0 is stored and then used in the simulations with wind turbines, which are run in the same domain with the same grid but removing the periodicity constraint in the streamwise direction and replacing it with an inﬂow condi- tion enforcing the solution found at x = 0 in the precursor simulation. Periodic boundary conditions are applied in the y (south–north) direction for both precursor simulations and simulations with turbines. The local thrust coefﬁcient is retrieved from the com- puted turbine thrust magnitude and rotor-averaged normal mean wind speed un by making use of its deﬁnition C′ T = 8T/πρu2 nD2. C. Cossu: Wake redirection at higher induction ﬁed wake models in regimes of moderate to high tilt or yaw, particularly in the case of signiﬁcant overinduction. Such im- proved models would allow for more reliable predictions of annual energy production gains obtained with overinductive yaw or tilt control for realistic wind roses and wind farm con- ﬁgurations by using advanced optimization methods such as those used by Park and Law (2015). ﬁed wake models in regimes of moderate to high tilt or yaw, particularly in the case of signiﬁcant overinduction. Such im- proved models would allow for more reliable predictions of annual energy production gains obtained with overinductive yaw or tilt control for realistic wind roses and wind farm con- ﬁgurations by using advanced optimization methods such as those used by Park and Law (2015). Finally, it would be very interesting to ascertain if addi- tional power gain enhancements could come from the simul- taneous activation of tilt, yaw and axial-induction control. It might indeed be possible that, as a consequence of the symmetry breaking associated with wake rotation effects and Coriolis acceleration, optimal power gains are obtained with “hybrid” yaw–tilt rotor-axis rotations even in wind-aligned conﬁgurations. This is the subject of current intense research effort. Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 https://doi.org/10.5194/wes-6-377-2021 Appendix B: Effect of the used turbine model on tilt control Tilt control: cross-stream view of the mean streamwise vorticity and velocity ﬁelds obtained by using the ADMC turbine model in the baseline case, where all turbines are operated at C′ T = 1.5 with no tilt or yaw (a, b) and with upwind turbines tilted by ϕ = 30◦and operated at C′ T = 3 (c, d). The streamwise vorticity ﬁelds (a, c) are extracted 3D downstream of the ﬁrst turbine row, while the streamwise velocity ﬁelds (b, d) are extracted D/2 upstream of the second row of turbines. C. Cossu: Wake redirection at higher induction 386 Figure B1. Tilt control: cross-stream view of the mean streamwise vorticity and velocity ﬁelds obtained by using the ADMC turbine model in the baseline case, where all turbines are operated at C′ T = 1.5 with no tilt or yaw (a, b) and with upwind turbines tilted by ϕ = 30◦and operated at C′ T = 3 (c, d). The streamwise vorticity ﬁelds (a, c) are extracted 3D downstream of the ﬁrst turbine row, while the streamwise velocity ﬁelds (b, d) are extracted D/2 upstream of the second row of turbines. For the two-row-array layout, therefore, the ADMC model also predicts that power gains obtained by overinductive tilt control are much larger than those obtained by standard tilt control (by ∼240 % for the ADMC turbine model and by ∼330 % with SOWFA’s ADM for ϕ = 30◦). However, the absolute levels of power gains computed with the ADMC model are higher than those computed with SOWFA’s ADM turbine model. In this context, the effect of wake rotation appears to be important. In the ADMC model, which ap- plies a uniformly distributed force purely normal to the ro- tor disk, wake rotation effects are indeed neglected, resulting in a negligible mean axial vorticity in the rotor wake in the baseline case and in almost-symmetric counter-rotating vor- tices in the tilted case (see Fig. B1a and c). In the ADMC tilted case, therefore, the downwash associated with the tilt- induced streamwise vortices is purely vertical, resulting in a highly efﬁcient displacement of higher-altitude higher- momentum ﬂuid towards the downstream-rotor-swept area (see Fig. B1d). Appendix B: Effect of the used turbine model on tilt control A quantitative analysis of the effect of the improved actu- ator disk method (ADM) model used in the present study by means of a direct comparison with the results obtained in Cossu (2020b) is not possible due to the difference in the con- sidered array conﬁgurations (two arrays here, three in Cossu, 2020b). Additional simulations of tilt control have therefore been performed by using the same turbine model (which we denote as ADMC) used in Cossu (2020b) for the same ar- ray conﬁguration used in the present study. We recall that, contrary to SOWFA’s ADM used in the present study, in the ADMC model wake rotation effects are neglected, and a uni- form load is assumed over the rotor disk that is assumed to operate at constant C′ T. The solution domain extends 1 km in the vertical direction and 3km × 3 km along the x and y axes and is discretized with cells extending 15m × 15 m in the x and y directions and 7m (near the ground) to 21m (near the top boundary) in the vertical direction; 1t = 0.8s time steps are used to advance the solution. These parameters keep the number of data stored in the precursor simulation manageable. First a baseline case has been simulated, with all turbines operated at the reference values C′ T = 1.5, ϕ = −5◦and γ = 0◦. Then, a standard tilt-control case has been considered, with upwind-row turbines operated at C′ T = 1.5, ϕ = 30◦ (and γ = 0◦), obtaining a power gain 1P/PRef ≈11 %. Fi- nally, overinductive tilt control has been tested by operating the upwind-row turbines at C′ T = 3 tilted by ϕ = 30◦, obtain- ing a power gain of ≈27 %. The aerodynamic forces developing on NREL 5 MW tur- bines, having a D = 126 m rotor diameter and zh = 89 m hub height (Jonkman et al., 2009), are modeled with SOWFA’s native actuator disk method based on the blade-element method (BEM). The forces exerted on the ﬂuid are com- puted for each radial blade section by using the lift and drag coefﬁcients cL(α) and cD(α) associated with the local Wind Energ. Sci., 6, 377–388, 2021 386 C. Cossu: Wake redirection at higher induction Figure B1. https://doi.org/10.5194/wes-6-377-2021 Wind Energ. Sci., 6, 377–388, 2021 References Goit, J. P. and Meyers, J.: Optimal Control of Energy Extraction in Wind-Farm Boundary Layers, J. Fluid Mech., 768, 5–50, https://doi.org/10.1017/jfm.2015.70, 2015. Annoni, J., Gebraad, P. M. O., Scholbrock, A. K., Flem- ing, P. A., and van Wingerden, J.-W.: Analysis of Axial- Induction-Based Wind Plant Control Using an Engineering and a High-Order Wind Plant Model, Wind Energy, 19, 1135–1150, https://doi.org/10.1002/we.1891, 2016. Howland, M. F., Bossuyt, J., Martínez-Tossas, L. A., Meyers, J., and Meneveau, C.: Wake Structure in Actuator Disk Models of Wind Turbines in Yaw under Uniform Inﬂow Conditions, J. Renew. Sustain. Energ., 8, 043301, https://doi.org/10.1063/1.4955091, 2016. Annoni, J., Scholbrock, A., Churchﬁeld, M., and Fleming, P. A.: Evaluating Tilt for Wind Plants, in: IEEE 2017 Ameri- can Control Conference (ACC), Seattle, WA, USA, 717–722, https://doi.org/10.23919/ACC.2017.7963037, 2017. Jasak, H.: OpenFOAM: open source CFD in research and industry, Int. J. Nav. Arch. Ocean, 1, 89–94, 2009. Bastankhah, M. and Porté-Agel, F.: Experimental and Theoretical Study of Wind Turbine Wakes in Yawed Conditions, J. Fluid Mech., 806, 506–541, https://doi.org/10.1017/jfm.2016.595, 2016. Jiménez, A., Crespo, A., and Migoya, E.: Application of a LES Technique to Characterize the Wake Deﬂection of a Wind Turbine in Yaw, Wind Energy, 13, 559–572, https://doi.org/10.1002/we.380, 2010. Bay, C. J., Annoni, J., Martínez-Tossas, L. A., Pao, L. Y., and Johnson, K. E.: Flow Control Leveraging Downwind Rotors for Improved Wind Power Plant Operation, in: IEEE 2019 Ameri- can Control Conference (ACC), 10–12 July 2019, Philadelphia, Pennsylvania, USA, 2843–2848, 2019. Jonkman, J., Butterﬁeld, S., Musial, W., and Scott, G.: Deﬁnition of a 5-MW reference wind turbine for offshore system develop- ment, Technical Paper NREL/TP-500-38060, NREL – National Renewable Energy Lab., Golden, CO, USA, 2009. Keating, A., Piomelli, U., Balaras, E., and Kaltenbach, H.- J.: A priori and a posteriori tests of inﬂow conditions for large-eddy simulation, Phys. Fluids, 16, 4696–4712, https://doi.org/10.1063/1.1811672, 2004. Boersma, S., Doekemeijer, B., Gebraad, P., Fleming, P., An- noni, J., Scholbrock, A., Frederik, J., and van Wingerden, J.- W.: A tutorial on control-oriented modeling and control of wind farms, in: IEEE 2017 American Control Conference (ACC), 24–26 May 2017, Seattle, Washington, D.C., USA, 1–18, https://doi.org/10.23919/ACC.2017.7962923, 2017. Knudsen, T., Bak, T., and Svenstrup, M.: Survey of wind farm control-power and fatigue optimization: Survey of wind farm control, Wind Energy, 18, 1333–1351, https://doi.org/10.1002/we.1760, 2015. Burton, T., Jenkins, N., Sharpe, D., and Bossanyi, E.: Wind energy handbook, John Wiley and Sons, Chichester, UK, 2001. Martínez-Tossas, L. Competing interests. The author declares that there is no con- ﬂict of interest. Competing interests. The author declares that there is no con- ﬂict of interest. Dahlberg, J. Å. and Medici, D.: Potential improvement of wind tur- bine array efﬁciency by active wake control (AWC), in: Proc. European Wind Energy Conference, European Wind Energy As- sociation, Madrid, Spain, 2003. Acknowledgements. I gratefully acknowledge the use of the Simulator for On/Offshore Wind Farm Applications (SOWFA) de- veloped at the NREL (Churchﬁeld et al., 2012) based on the Open- FOAM ﬁnite volume framework (Jasak, 2009; OpenCFD, 2011). Fleming, P., Gebraad, P. M., Lee, S., van Wingerden, J.-W., John- son, K., Churchﬁeld, M., Michalakes, J., Spalart, P., and Mo- riarty, P.: Simulation Comparison of Wake Mitigation Control Strategies for a Two-Turbine Case, Wind Energy, 18, 2135–2143, https://doi.org/10.1002/we.1810, 2015. Review statement. This paper was edited by Johan Meyers and reviewed by Wim Munters and one anonymous referee. Fleming, P. A., Gebraad, P. M., Lee, S., van Wingerden, J.- W., Johnson, K., Churchﬁeld, M., Michalakes, J., Spalart, P., and Moriarty, P.: Evaluating Techniques for Redirecting Tur- bine Wakes Using SOWFA, Renew. Energ., 70, 211–218, https://doi.org/10.1016/j.renene.2014.02.015, 2014. Appendix B: Effect of the used turbine model on tilt control In the case of the more realistic SOWFA ADM turbine model, in contrast, wake rotation effects are fully taken into account, resulting in non-negligible mean axial vorticity in the rotor wake in the baseline case and in strongly non-symmetric counter-rotating vortices in the tilted case (see Fig. 3a and c). In the more realistic case, there- fore, the tilt-induced streamwise vortices are associated with an oblique downwash which is less efﬁcient in displacing high-momentum ﬂuid towards the downstream rotors (see Fig. 3d). This explains that lower absolute values of tilt- induced power gains are obtained when wake rotation effects are taken into due account. Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 C. Cossu: Wake redirection at higher induction C. Cossu: Wake redirection at higher induction 387 Data availability. Data can be obtained from the author upon re- quest. Cossu, C.: Replacing wakes with streaks in wind turbine arrays, Wind Energy, https://doi.org/10.1002/we.2577, in press, 2020a. Cossu, C.: Evaluation of tilt control for wind-turbine arrays in the atmospheric boundary layer, Wind Energ. Sci. Dis- cuss. [preprint], https://doi.org/10.5194/wes-2020-106, in re- view, 2020b. Wind Energ. Sci., 6, 377–388, 2021 References and Leonardi, S.: Wind Turbine Mod- eling for Computational Fluid Dynamics, Subcontract Re- port NREL/SR-5000-55054, US National Renewable Energy Laboratory, Golden, CO, USA, 2013. Calaf, M., Meneveau, C., and Meyers, J.: Large Eddy Simulation Study of Fully Developed Wind-Turbine Array Boundary Lay- ers, Phys. Fluids, 22, 015110, https://doi.org/10.1063/1.3291077, 2010. Medici, D. and Alfredsson, P. H.: Measurements on a Wind Tur- bine Wake: 3D Effects and Bluff Body Vortex Shedding, Wind Energy, 9, 219–236, https://doi.org/10.1002/we.156, 2006. Campagnolo, F., Petrovi´c, V., Schreiber, J., Nanos, E. M., Croce, A., and Bottasso, C. L.: Wind Tunnel Testing of a Closed-Loop Wake Deﬂection Controller for Wind Farm Power Maximization, J. Phys. Conf. Ser., 753, 032006, https://doi.org/10.1088/1742- 6596/753/3/032006, 2016. Munters, W. and Meyers, J.: An Optimal Control Framework for Dynamic Induction Control of Wind Farms and Their Interac- tion with the Atmospheric Boundary Layer, Philos. T. Roy. Soc. A, 375, 20160100, https://doi.org/10.1098/rsta.2016.0100, 2017. Churchﬁeld, M. J., Lee, S., Michalakes, J., and Moriarty, P. J.: A Numerical Study of the Effects of Atmospheric and Wake Turbulence on Wind Turbine Dynamics, J. Turbul., 13, N14, https://doi.org/10.1080/14685248.2012.668191, 2012. Munters, W. and Meyers, J.: Optimal Dynamic Induction and Yaw Control of Wind Farms: Effects of Turbine Spacing and Layout, Wind Energ. Sci., 6, 377–388, 2021 https://doi.org/10.5194/wes-6-377-2021 C. Cossu: Wake redirection at higher induction C. Cossu: Wake redirection at higher induction 388 J. Phys. Conf. Ser., 1037, 032015, https://doi.org/10.1088/1742- 6596/1037/3/032015, 2018a. Shapiro, C. R., Gayme, D. F., and Meneveau, C.: Modelling Yawed Wind Turbine Wakes: A Lifting Line Approach, J. Fluid Mech., 841, R1, https://doi.org/10.1017/jfm.2018.75, 2018. Munters, W. and Meyers, J.: Dynamic Strategies for Yaw and Induction Control of Wind Farms Based on Large- Eddy Simulation and Optimization, Energies, 11, 177, https://doi.org/10.3390/en11010177, 2018b. Smagorinsky, J.: General circulation experiments with the primitive equations: I. The basic experiment, Mon. Weather Rev., 91, 99–164, https://doi.org/10.1175/1520- 0493(1963)091<0099:GCEWTP>2.3.CO;2, 1963. Nanos, E. M., Letizia, S., Barreiro, D. J., Wang, C., Rotea, M., Iungo, V. I., and Bottasso, C. L.: Vertical wake deﬂection for offshore ﬂoating wind turbines by differential ballast control, J. Phys. Conf. 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J. and Meneveau, C.: Flow Structure and Turbu- lence in Wind Farms, Annu. Rev. Fluid Mech., 49, 311–339, https://doi.org/10.1146/annurev-ﬂuid-010816-060206, 2017. Porté-Agel, F., Bastankhah, M., and Shamsoddin, S.: Wind-Turbine and Wind-Farm Flows: A Review, Bound.-Lay. Meteorol., 174, 1–59, https://doi.org/10.1007/s10546-019-00473-0, 2019. Strickland, J. M. I. and Stevens, R. J. A. M.: Effect of Thrust Coefﬁ- cient on the Flow Blockage Effects in Closely-Spaced Spanwise- Inﬁnite Turbine Arrays, J. Phys. Conf. Ser., 1618, 062069, https://doi.org/10.1088/1742-6596/1618/6/062069, 2020. Schumann, U.: Subgrid scale model for ﬁnite difference sim- ulations of turbulent ﬂows in plane channels and annuli, J. Comput. Phys., 18, 376–404, https://doi.org/10.1016/0021- 9991(75)90093-5, 1975. Tabor, G. R. and Baba-Ahmadi, M. H.: Inlet conditions for large eddy simulation: A review, Comput. Fluids, 39, 553–567, https://doi.org/10.1016/j.compﬂuid.2009.10.007, 2010. Wind Energ. 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https://openalex.org/W2587024474	https://www.nature.com/articles/srep42524.pdf	English	null	Plasmon-Enhanced Photocurrent using Gold Nanoparticles on a Three-Dimensional TiO2 Nanowire-Web Electrode	Scientific reports	2,017	cc-by	5,957	Plasmon-Enhanced Photocurrent using Gold Nanoparticles on a Three-Dimensional TiO2 Nanowire- Web Electrode received: 16 September 2016 accepted: 11 January 2017 Published: 10 February 2017 received: 16 September 2016 accepted: 11 January 2017 Published: 10 February 2017 Yin-Cheng Yen, Jau-An Chen, Sheng Ou, Yi-Shin Chen & Kuan-Jiuh Lin In this study, an anatase/rutile mixed-phase titanium dioxide (TiO2) hierarchical network deposited with Au nanoparticles (Au/TiO2 ARHN) was synthesized using a facile hydrothermal method followed by a simple calcination step. Such a unique structure was designed for improving the light harvest, charge transportation/separation, and the performance of photo-electro-chemical (PEC) cells. The properties of the as-synthesized Au/TiO2 ARHN in PEC cells were investigated by electrochemical measurements using a three-electrode system in a 1 M NaOH electrolyte. Remarkably, a 4.5-folds enhancement of the photocurrent for Au/TiO2 ARHN was observed as compared to that for TiO2 nanowire (NW), under AM1.5G solar illumination, suggesting its potential application in PEC cells. A mechanism has been proposed to explain the high photocurrent of Au/TiO2 ARHN in PEC water splitting. Honda and Fujishima elucidated the possibility of water splitting using TiO2 as electrode1. Since then, various TiO2 nano-architectonic topographies have been greatly desired for enhancing the performance of PEC cells2–8. In general, a predominant PEC cell relies on two factors: the efficient usage of solar energy and the instant trans- portation/separation of charges9. Hence, the development of nano-sized photo-active semiconductors to satisfy the requirements has been a long-standing objective in the research of PEC cells, especially for one-dimensional TiO2 due to its superior charge transport property10. To date, many hierarchical TiO2 nanostructures based on nanowires (NWs) and nanotubes (NTs) have been synthesized for enhanced photo-electric efficiency in solar energy harvesting, conversion, and pollutant purification11–13. Such a heterojunction nanostructure would stag- ger energy levels and scatter incident light to enlarge light absorption in the UV region14. However, the strategies for fabricating hierarchical TiO2 nanostructures have the disadvantages of an extremely time-consuming pro- cess, specific/highly expensive fabricating apparatus and back-side illumination, thus making it economically non-competitive15–25. p Recently, to expand the TiO2 optical adsorption spectrum from the UV into the visible region, plasmonic electrodes composed of Au/TiO2 nano-architectonic topographies have been developed with localized surface plasmon resonance (LSPR) property26–35. However, most of these reports focus on the discussion of the relation- ship between particle size/shape/distance/concentration and the photo-electrochemical performance28–30,32–35. Especially, one novel example of demonstrating the influence of TiO2 nanostructure on the LSPR property was reported by Wang et al.36. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China orrespondence and requests for materials should be addressed to K.-J.L. (email: kjlin@dragon.nchu.edu.tw) Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 Plasmon-Enhanced Photocurrent using Gold Nanoparticles on a Three-Dimensional TiO2 Nanowire- Web Electrode An Au/TiO2/Au nanostructure with a 5-nm-thick TiO2 middle layer was synthe- sized which resulted in a maximum 38-fold enhancement of the electric field density of LSPR and about 3-fold improvement of the photocurrent in a wavelength range of 400–650 nm. The enhanced performance is mainly arising from the thickness of TiO2 satisfying the requirement for generating the coupling effect between the oppositely aligned and nearly touching Au NPs on TiO2 nanosheet. However, the longer time (4 days) required to synthesize the TiO2 nanostructures and the non-transparency of the Au/TiO2/Au film limit their application. Furthermore, there is limited knowledge on how to design and synthesize a TiO2 nanostructured film on a trans- parent substrate by a simple yet effective method. Therefore, the aim of this study is to provide a novel strategy for building a new TiO2 nanostructure to intensify the coupling effect between Au NPs that significantly enhances the photoelectric conversion. p Herein, a three-dimensional (3D) web constructed by Au plasmonic NPs on TiO2 anatase/rutile hierarchi- cal network (Au/TiO2 ARHN) is proposed, which is schematically shown in Fig. 1. In order to strengthen the Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China orrespondence and requests for materials should be addressed to K.-J.L. (email: kjlin@dragon.nchu.edu.tw) Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 1 www.nature.com/scientificreports/ Figure 1. Schematic representation of Au/TiO2 ARHN on FTO substrates. Figure 1. Schematic representation of Au/TiO2 ARHN on FTO substrates. Figure 2. Schematic description of the synthesis process of TiO2 NWs, ARHN and Au/TiO2 ARHN on FTO substrates. Figure 2. Schematic description of the synthesis process of TiO2 NWs, ARHN and Au/TiO2 ARHN on FTO substrates. electromagnetic coupling of the Au NPs, the solid support―TiO2 NWs connected with TiO2 threads― was synthesized by a two-step hydrothermal process. When tested in the PEC experiment, the TiO2 ARHN and Au/ TiO2 ARHN exhibited 1.5 times and 4.5 times higher photocurrent than TiO2 NWs. electromagnetic coupling of the Au NPs, the solid support―TiO2 NWs connected with TiO2 threads― was synthesized by a two-step hydrothermal process. When tested in the PEC experiment, the TiO2 ARHN and Au/ TiO2 ARHN exhibited 1.5 times and 4.5 times higher photocurrent than TiO2 NWs. Results and Discussion Figure 2 schematically depicts the three-step fabrication process of reproducible Au/TiO2 ARHN. Figure 3a represents a top-view SEM image of the TiO2 NWs. The light-gray needle-like regions in the SEM image rep- resent the TiO2 NWs and the dark regions are the underlying FTO substrate. A cross-sectional SEM image of the NWs (Fig. 3b) shows that the thickness of TiO2 layer is ~1 μm and the NWs have an average diameter of 40 nm. XRD patterns of the TiO2 NWs show predominantly rutile phase with preferential orientation of (110) (Supplementary Fig. 1). The top-view and cross-sectional SEM image of the TiO2 ARHN (Fig. 3c,d) shows that the TiO2 threads are bridged with the TiO2 NWs to form a 3D hierarchical network. Nest-like porous cavities with diameters of a few hundred nanometers are clearly observed and the diameters of the threads are ~10 nm. The XRD patterns and Raman spectra show that the threads belong to the anatase phase (Supplementary Figs 2 and 3). The top-view SEM image of the Au/TiO2 ARHN is shown in Fig. 3e. The white dot regions in the SEM represent the Au NPs. The size distribution histograms of Au NPs show an average particle size of 15 nm (Fig. 3f). T i i h f i h i f TiO ARHN i f i f d Fi l h To investigate the formation mechanism of TiO2 ARHN, a series of experiments were performed. Firstly, we failed to obtain TiO2 ARHN without TiCl4 treatment. We found that TiO2 threads cannot grow on TiO2 NWs with a smooth surface. Secondly, in the absence of the Ti film in step 2 (in Fig. 2), only TiO2 NWs were observed. These findings suggest that both TiCl4 treatment and the formation of the Ti layer for the formation of TiO2 ARHN are indispensable. Therefore, we propose that small TiO2 seed crystals grow on the surface of TiO2 NWs after TiCl4 treatment, which leads to a rough surface for the growth of TiO2 threads. Moreover, during alkali Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 2 www.nature.com/scientificreports/ Figure 3. FESEM images. (a,c,e) top images of top TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN, respectively; (b,d) cross-sectional images of TiO2 NW and TiO2 ARHN; (f) the size distribution of Au NPs. Figure 3. FESEM images. Results and Discussion UV-Vis spectra of desorbed N719 solution. Figure 5. UV-Vis spectra of desorbed N719 solution. We suggest the enhanced photocurrent of TiO2 ARHN electrode could be attributed to better photocatalytic activity, due to increased surface area, or better light harvest efficiency, due to the hierarchical network structure. Therefore, the dye absorption/desorption experiment and UV-visible spectrum measurement were perform to verify it. Here, dye N719 was choose as an adsorbate to execute dye absorption/desorption experiment due to it could be monolayer absorbed on the surface of TiO2. Therefore, we can evaluate the related surface area via measuring the absorption of N719 dye which detach from TiO2 structure. As shown in Fig. 5, there are three absorbed peak of N719 located on 310 nm, 370 nm and 505 nm, respectively42. It is observed that the absorption of detached N719 solution based on TiO2 ARHN is obviously large than TiO2 NW on entire spectrum. It represents the surface area of TiO2 ARHN is related large than TiO2 NW due to there are more dye absorbed on TiO2 ARHN. Based on the result, we make a sure that the high surface area of TiO2 ARHN is a reasonable reason which bring to a high photo activity on PEC measurement. g p y Furthermore, the UV-visible absorption spectra of the TiO2 NW and TiO2 ARHN with/without Au NPs are shown in Fig. 6. TiO2 NW exhibits a stronger absorption at the wavelengths below 400 nm due to electron transitions of TiO2 from the valence band to the conduction band. In addition, the absorption spectra of TiO2 ARHN showed an enhanced absorption in the entire spectral range as compared with TiO2 NW, which is attrib- uted to the scattering effect in the ARHN structure; this also explains the increased photocurrent in the TiO2 ARHN. With deposited Au NPs, the absorption show a significantly enhancement on visible range which is driven by the LSPR absorption. From incident photon-to-electron conversion efficiency (IPCE) measurement (Supplementary Fig. 4), it demonstrates that such an absorption successfully boosts the PEC performance in the region from 400 nm to 700 nm. In this work, a LSPR peak for the Au NPs with average size of 15 nm centered at around 540 nm. For Au NPs of size 10–20 nm, the absorption peak of plasmon resonance is usually located at 520–525 nm43,44. Results and Discussion (a,c,e) top images of top TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN, respectively; (b,d) cross-sectional images of TiO2 NW and TiO2 ARHN; (f) the size distribution of Au NPs. hydrothermal process, the Ti layers can generate large amounts of Ti-containing species37,38 as precursors that can deposit on the TiO2 seeds and produce a network structure. To evaluate the enhanced PEC performance of the designed Au/TiO2 ARHN, the linear sweep voltammo- grams and the photocurrent-versus-time (I-t curve) of TiO2 NW and TiO2 ARHN with/without Au NPs were conducted under AM 1.5 G simulated solar illumination, as shown in Fig. 4. The measured photocurrent was nor- malized to the sample area to obtain the photocurrent density for comparison. As presented in Fig. 4a, the TiO2 NW electrode produced a photocurrent density of 4 × 10−5 A cm−2 at 0.23 V vs. Ag/AgCl, which is the potential often chosen as a metric to evaluate the performance of photoanodes as it corresponds to the water oxidation potential4. The low photocurrent density is attributed to the limit of wide band-gap characteristics of TiO2 (3.2 eV for anatase39 and 3.0 eV for rutile40), due to which only UV light can be used in the PEC water splitting system. The photocurrent was enhanced for the TiO2 ARHN (6 × 10−5 A cm−2) when compared with TiO2 NW, with an enhancement factor of 1.5. As expected, a significant photocurrent density enhancement was clearly observed on the Au/TiO2 ARHN, having a photocurrent density of 1.8 × 10−4 A cm−2. As compared to TiO2 NW, a photo- current enhancement higher than 4.5 times was achieved. From Fig. 4b, all electrode represent a good reproduc- ibility and stability as the illumination was turned on and off. Furthermore, the sharp spike in the photocurrent during the on/off illumination cycles demonstrates the predominant transport of photogenerated electrons in the designed TiO2 structure41. Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 3 www.nature.com/scientificreports/ Figure 4. (a) Linear sweep voltammograms and (b) amperometric I―t curves of TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN photoelectrode. Figure 4. (a) Linear sweep voltammograms and (b) amperometric I―t curves of TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN photoelectrode. Figure 5. UV-Vis spectra of desorbed N719 solution. Figure 4. (a) Linear sweep voltammograms and (b) amperometric I―t curves of TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN photoelectrode. Figure 5. UV-Vis spectra of desorbed N719 solution. Figure 5. Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 Results and Discussion A redshift of 15–20 nm of the plasmon resonance peak was observed as compared to previous reports. This may be attributed to the TiO2 changing the surrounding dielectric property of Au NPs (Au NPs well deposited and in contact withTiO2 surface) and the enhancement of electromagnetic field of LSPR30,45,46. It has been reported that the high electromagnetic field of LSPR and strong coupling between Au NPs and TiO2 will benefit the plasmon-induced charge transportation and separation, enabling SPR-enhanced photocatalysis. Typically, the LSPR-induced charge separation at the interface between the Au NPs and TiO2 can occur by transferring the energy contained in the oscillating electrons or local plasmonic field from Au NPs to TiO2 through direct electron transfer, also known Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 4 www.nature.com/scientificreports/ Figure 6. UV-Vis spectra of TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN. Figure 6. UV-Vis spectra of TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN. Figure 6. UV-Vis spectra of TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN. Figure 6. UV Vis spectra of TiO2 NW, TiO2 ARHN and Au/TiO2 ARHN. Figure 7. I − t curves collected at 0 V versus Ag/AgCl for Au/TiO2 NW and Au/TiO2 ARHN electrode under visible light illumination (using a cutoff filter with a wavelength of 400 nm). Figure 7. I − t curves collected at 0 V versus Ag/AgCl for Au/TiO2 NW and Au/TiO2 ARHN electrode under visible light illumination (using a cutoff filter with a wavelength of 400 nm). as hot electron injection47,48. Higher electromagnetic field generates more hot electron49,50. In order to verify our assumption, the design of TiO2 ARHN is helpful to improve electromagnetic field as compared to NWs, the PEC measurement was performed to check the hot electron effect. Figure 7 shows the I-t curve measured under visible-light illumination. From Fig. 7, it is obvious that the photocurrent of Au/TiO2 ARHN is two times higher than Au/TiO2 NW. It means that the high plasmon electromagnetic field of Au/TiO2 ARHN results in a high hot-electron current. Therefore, we confirm that the design of TiO2 ARHN successfully provides a model for strengthening LSPR ability and demonstrates a remarkable enhancement on PEC performance.fff as hot electron injection47,48. Higher electromagnetic field generates more hot electron49,50. In order to verify our assumption, the design of TiO2 ARHN is helpful to improve electromagnetic field as compared to NWs, the PEC measurement was performed to check the hot electron effect. Results and Discussion Figure 7 shows the I-t curve measured under visible-light illumination. From Fig. 7, it is obvious that the photocurrent of Au/TiO2 ARHN is two times higher than Au/TiO2 NW. It means that the high plasmon electromagnetic field of Au/TiO2 ARHN results in a high hot-electron current. Therefore, we confirm that the design of TiO2 ARHN successfully provides a model for strengthening LSPR ability and demonstrates a remarkable enhancement on PEC performance.fff g g y p In this study, we are further interested the effectiveness of density effect and size effect on the performance of photo electrochemical property. The related data and discussion were shown in Supplementary Information. In addition, the stability test and Faradaic efficiency were obtained. Under continuously illumination for 10800 seconds (equal to 3 hr), the photocurrent density was decrease from 1.8 × 10−4 A/cm2 to 1.5 × 10−4 A/cm2 in the case of Au/ TiO2 ARHN, as shown in Fig. 8a. This photocurrent decay is similar to previous report51. We suggest it could be attributed to photo induced corrosion which competes with water oxidation reaction51. However, such a corrosion could be suppress by surface treatment of TiO2 nanostructure or use of sacrificial reagent/catalyst for longstand- ing application51. From Fig. 8b, the calculated Faradaic efficiency exceed 90% and 85% for TiO2 ARHN and Au/ TiO2 ARHN, respectively. The high value of Faradaic efficiency of oxygen gas during 10 hr demonstrated that the photo generated current indeed utilized for water oxidation. Also, we could observe hydrogen gas from real pic- ture as shown inset diagram in Fig. 8c. Therefore, we propose the electron transfer mechanism in Au/TiO2 ARHN system as shown in Fig. 8c. Under illumination, Au NPs absorb visible light, generating the energetic hot elec- trons from the process of SP excitation, and injecting them into the conduction band of the adjacent TiO2 (green arrow). Simultaneously, the UV light is absorbed by TiO2, producing a photo-excited electron and a hole (black arrow). The plasmon-induced electromagnetic field promotes the separation of photogenerated electrons and holes. Furthermore, as illustrated, the energy bands of anatase and rutile are different which provides a driving force to pro- mote electron transfer from anatase to rutile (blue arrow). Finally, the electrons transferred to the cathode (Pt) react with H+ ions and produce H2 (pink arrow) whereas the holes present in the anode oxidize H2O and generate O2. Results and Discussion Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 5 www.nature.com/scientificreports/ p / Figure 8. (a) Current―time curves of Au/TiO2 ARHN collected in 1 M NaOH at 0.23 V vs Ag/AgCl under 100 mW/cm2 for 3 h. (b) The calculated Faradaic efficiency for O2 gas evolution. (c) Charge transfer mechanism of Au/TiO2 ARHN under solar illumination. Figure 8. (a) Current―time curves of Au/TiO2 ARHN collected in 1 M NaOH at 0.23 V vs Ag/AgCl under 100mW/cm2 for 3h (b) The calculated Faradaic efficiency for O2 gas evolution (c) Charge transfer mechanism Figure 8. (a) Current―time curves of Au/TiO2 ARHN collected in 1 M NaOH at 0.23 V vs Ag/AgCl under 100 mW/cm2 for 3 h. (b) The calculated Faradaic efficiency for O2 gas evolution. (c) Charge transfer mechanism of Au/TiO2 ARHN under solar illumination. In conclusion, this work demonstrates a plasmon-induced effect on a designed 3D web architecture con- structed from rutile TiO2 NWs, anatase TiO2 threads and Au NPs. Such a nanostructure was achieved for the first time through a simple and inexpensive hydrothermal procedure followed by calcination. The PEC performance tests, reveal that the photocurrent of Au/TiO2 ARHN was 4.5 times greater than that of the TiO2 NW photoelec- trode. The observed optical properties and dark current measurements confirm that the excellent PEC perfor- mance of Au/TiO2 ARHN was due to three reasons: (1) the high surface area of TiO2 ARHN that increase the photoactive center, (2) the scattering effect in the TiO2 ARHN and the LSPR properties of Au NPs that enhanced the light harvest, (3) the strength coupling effect between Au NPs and TiO2 nanostructure that accelerated the charge transportation and separation. The mechanism of charge transportation in the Au/TiO2 ARHN was pro- posed based on our findings. Practical use of the Au/TiO2 ARHN was demonstrated to indicate their significant potential for use in photoelectric conversion system. Methods M i l Materials. F:SnO2 (FTO) (1.5 cm × 3 cm, TEC-7, 7 ohm/sq., 2.2 mm thick) was used as the substrate for growth of the TiO2 film. All chemicals were used without further purification. Sodium hydroxide (NaOH, 99%), titanium tetrachloride (TiCl4, 99%), 2-butanone (C4H8O, >99%), hydrochloric acid (HCl, 12 M) and nitric acid (HNO3, 65%) were purchased from Merck. Tetrabutyl titanate (C16H36O4Ti, >97%) and ruthenium 535 bis-TBA (N719 dye) were obtained from Aldrich and Solaronix, respectively. Hydrothermal synthesis of TiO2 NWs on FTO substrates. First, FTO was cleaned by ultrasonic agita- tion in a mixture of ethanol, acetone and deionized water (volume ratio of 1:1:1) for 15 min. The FTO substrates were immersed in an aqueous of 0.5 M TiCl4 at 80 °C for 30 min, followed by heat treatment at 500 °C for 30 min to yield a thin TiO2 layer. The TiCl4-treated substrates were then suspended in a reagent solution containing 6 mL HCl, 6 mL 2-butanone and 0.6 mL tetrabutyl titanate in a Teflon vessel. The Teflon vessel was sealed in an auto- clave and heated at 200 °C for 1.5 h. Further annealing at 500 °C for 30 min resulted in the growth of crystalline TiO2 NWs on FTO substrates52. Hydrothermal synthesis of TiO2 ARHN on FTO substrates. The TiO2 NW substrate (without cal- cination) was first treated with TiCl4 solution as mentioned above. A 200-nm-thick Ti layer was sputtered on the TiCl4-treated TiO2 NW using a magnetic sputter (K575X, Quorum Technologies). The substrates were then transferred to a Teflon vessel with the addition of a 5 M aqueous NaOH solution and were encapsulated in a stainless-steel autoclave. Then, the autoclave was heated at 80 °C for 30 min. After the low-temperature hydro- thermal process, the substrate was rinsed with 0.1 M HNO3 followed by deionized water, and was finally calcined at 500 °C for 30 min to obtain hierarchical nanostructures. 6 Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 www.nature.com/scientificreports/ Synthesis of Au/TiO2 ARHN. A 5-nm Au layer was sputtered on the TiO2 NW and ARHN using a magnetic sputter. The Au deposited TiO2 were subsequently calcined at 500 °C for 1 h to obtain Au/TiO2 ARHN. Synthesis of Au/TiO2 ARHN. A 5-nm Au layer was sputtered on the TiO2 NW and ARHN using a magnetic sputter. The Au deposited TiO2 were subsequently calcined at 500 °C for 1 h to obtain Au/TiO2 ARHN. Synthesis of Au/TiO2 ARHN. References Acta Physico-Chimica Sinica 28, 1368–1372 (2012). y 5. Wang, H. et al. Rutile TiO2 Nano-Branched Arrays on FTO for Dye-Sensitized Solar Cells. Physical Chemistry Chemical Physics 13 7008–7013 (2011). 6. Liao, W.-P. & Wu, J.-J. Wet Chemical Route to Hierarchical TiO2 Nanodendrite/Nanoparticle Composite Anodes for Dye-Sensitized Solar Cells. Journal of Materials Chemistry 21, 9255–9262 (2011). f y 7. Hu, A., Li, H., Jia, Z. & Xia, Z. TiO2 Nanorods Branched on Fast-Synthesized Large Clearance TiO2 Nanotube Arrays for Dye Sensitized Solar Cells. 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Methods M i l A 5-nm Au layer was sputtered on the TiO2 NW and ARHN using a magnetic sputter. The Au deposited TiO2 were subsequently calcined at 500 °C for 1 h to obtain Au/TiO2 ARHN. Characterizations and measurements. The surface and cross-section morphologies of samples were examined by field-emission scanning electron microscopy (FE-SEM, Zeiss Ultraplus). The SigmaScan Pro 5 soft- ware was used to calculate particle size (300 particles were counted). The crystal structure was characterized by X-ray diffraction (XRD, PANalytical X’Pert Pro MRD) and Raman spectroscopy (Tokyo Instruments, INC). A UV/Vis spectrometer (Perkin Elmer/Lambda 900) was used to obtain the absorption spectra. The dye absorption/ desorption experiment was performed by desorbing a dye-sensitized TiO2 electrode in a 0.1 M NaOH solution in 1:1 H2O/EtOH53. Subsequently, a UV–vis spectrometer was introduced to measure the absorbance of the desorb- ing solution. The electrochemical measurement was carried out using three-electrode system. TiO2 electrode with or without Au NPs was the working electrode; an Ag/AgCl (3 M KCl) electrode in saturated KCl was the reference electrode; the Pt plate was used as the counter electrode. All PEC cells were examined in 1 M NaOH solution with a PARSTAT 2263 Advanced Electrochemical System under illumination by Newport solar simulator with AM 1.5 G (100 mW/cm2). The incident photon-to-current conversion efficiency (IPCE) was measured with an action spectrum measurement setup (Peccell, PEC-S20). References 1. Fujishima, A. & Honda, K. Electrochemical Photolysis of Water at a Semiconductor Electrode. Nature 238, 37–38 (1972). Author Contributions Y.C.Y. and K.J.L. conceived and design the project. J.A.C., S.O. and Y.S.C. conducted experimental work. Y.C.Y. and J.A.C. analyzed the data. Y.C.Y. wrote the main manuscript text. 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Acknowledgementsi g We greatly acknowledge financial support from the Ministry of Science and Technology of Taiwan (104-2113- M-005 -011 -MY3; 105-2811-M-005-020). We would like to thank Prof. Hao-Ming Chen from National Taiwan University for his cooperation in the measurement and calculation of Faradaic efficiency and Prof. Chen-Yu Yeh from National Chung Hsing University for his kindly help in the IPCE measurement. We greatly acknowledge financial support from the Ministry of Science and Technology of Taiwan (104-2113- M-005 -011 -MY3; 105-2811-M-005-020). We would like to thank Prof. Hao-Ming Chen from National Taiwan University for his cooperation in the measurement and calculation of Faradaic efficiency and Prof. Chen-Yu Yeh from National Chung Hsing University for his kindly help in the IPCE measurement. www.nature.com/scientificreports/ Tunable Plasmonic Response from Alkanethiolate-Stabilized Gold Nanoparticle Superlattices: Evidence of Near-Field Coupling. 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Plasmonic Gold Nanocrystals Coupled with Photonic Crystal Seamlessly on TiO2 Nanotube Photoelectrodes for Efficient Visible Light Photoelectrochemical Water Splitting. Nano Letters 13, 14–20 (2013) Scientific REPOrTS \| 7:42524 \| DOI: 10.1038/srep42524 7 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Yen, Y.-C. et al. Plasmon-Enhanced Photocurrent using Gold Nanoparticles on a Three-Dimensional TiO2 Nanowire-Web Electrode. Sci. Rep. 7, 42524; doi: 10.1038/srep42524 (2017). How to cite this article: Yen, Y.-C. et al. Plasmon-Enhanced Photocurrent using Gold Nanoparticles on a Three-Dimensional TiO2 Nanowire-Web Electrode. Sci. Rep. 7, 42524; doi: 10.1038/srep42524 (2017). How to cite this article: Yen, Y.-C. et al. Plasmon-Enhanced Photocurrent using Gold Nanoparticles on a Three-Dimensional TiO2 Nanowire-Web Electrode. Sci. Rep. 7, 42524; doi: 10.1038/srep42524 (2017). ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. 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https://openalex.org/W2734941977	https://pureadmin.qub.ac.uk/ws/files/132319953/9217912.pdf	English	null	Antidiabetic Micro-/Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro	BioMed research international	2,017	cc-by	8,729	Antidiabetic Micro-/Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro Lin, D., Du, Q., Wang, H., Gao, G., Zhou, J., Ke, L., Chen, T., Shaw, C., & Rao, P. (2017). Antidiabetic Micro- /Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro. BioMed Research International, 2017, 1-8. Article 9217912. https://doi.org/10.1155/2017/9217912 Antidiabetic Micro-/Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro Lin, D., Du, Q., Wang, H., Gao, G., Zhou, J., Ke, L., Chen, T., Shaw, C., & Rao, P. (2017). Antidiabetic Micro- /Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro. BioMed Research International, 2017, 1-8. Article 9217912. https://doi.org/10.1155/2017/9217912 Antidiabetic Micro-/Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro Lin, D., Du, Q., Wang, H., Gao, G., Zhou, J., Ke, L., Chen, T., Shaw, C., & Rao, P. (2017). Antidiabetic Micro- /Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro. BioMed Research International, 2017, 1-8. 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Oct. 2024 Hindawi BioMed Research International Volume 2017, Article ID 9217912, 8 pages https://doi.org/10.1155/2017/9217912 Dai Lin,1 Qian Du,2 Huiqin Wang,1 Guanzhen Gao,1 Jianwu Zhou,1 Lijing Ke,1 Tianbao Chen,3 Chris Shaw,3 and Pingfan Rao1 1Food Nutrition Research Centre, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou Zhejiang 310012, China 2Institute of Biotechnology, Fuzhou University, Fuzhou, Fujian 350002, China 3Natural Drug Discovery Group, School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast, UK Correspondence should be addressed to Lijing Ke; lijingke@zjgsu.edu.cn Received 14 March 2017; Accepted 25 May 2017; Published 17 July 2017 Academic Editor: Sung-Hoon Kim Copyright © 2017 Dai Lin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The antidiabetic effects of Ge-Gen-Qin-Lian-Tang decoction (GQD) have been proven clinically. In a pharmacological study conducted on STZ-induced diabetic rats, the constitutive aggregates/sediments of Ge-Gen-Qin-Lian-Tang decoction exhibited stronger hypoglycemic and antioxidant activities compared to the soluble compositions. This study aims to demonstrate the pharmacological properties of aggregates derived from GQD by measuring permeability of the active monomer phytochemicals (e.g., baicalin) in a Caco-2 cell monolayer and determine the cellular viability, intracellular redox status (MDA and SOD), and insulin secretion of pancreatic 𝛽-cell line, INS-1, following STZ-induced oxidative stress. The aggregates were separated into three fractions, namely, “MA (microaggregates),” “400 g supernatant,” and “MNA (micro-/nanoaggregates),” by centrifugation at 400 ×g and 15000 ×g, respectively. Aggregates in the sediment increased baicalin absorption, showed little toxicity to 𝛽-cells, elevated intracellular SOD levels, and significantly suppressed oxidative damage effects on cellular viability and functions. The “MA” fraction had a larger particle size and provided higher antioxidant cellular protection than “MNA” in vitro, implying that the sediments may be the active components in the herbal decoction. The actions of these micro-/nanoaggregates may provide a new perspective for understanding the antidiabetic effects of herbal decoctions and aid in interpretation of synergistic actions between the multiple components. 1. Introduction GQD significantly reduced glycated hemoglobin (HbA1c) and fasting blood glucose (FBG) in streptozotocin- (STZ-) and high-fat-diet-induced diabetic SD rats and enhanced glucose consumption in 3T3-L1 adipocytes [13]. In T2D patients, GQD significantly reduced HbA1c, while regulating the ecological structure of the gut microbiota by enriching the amounts of beneficial bacteria, indicating that the gut is among the important biological targets of this herbal tonic [12]. GQD contains several well-known active phytochemicals, that is, baicalin [14, 15], berberine [16, 17], puerarin [18], glycyrrhizic acid, and liquiritin [19], which are correlated to the antidiabetic, antioxidant, and immunoregulative effects. Apart from its antihyperglycemic and antioxidant activities, baicalin is also known as a prolyl endopeptidase inhibitor which induces apoptosis in pancreatic cancer cells [20, 21] and it also induces apoptosis of lymphoma cells by down- regulation of the PI3K/Akt signaling pathway [22]. However, taking the plant-derived flavone baicalin as an example, poor solubility and bioavailability are a common problem of these phytochemicals when applied clinically, resulting in the efficacy and pharmaceutical mechanisms of TCM decoctions being often questioned. 2.2. Preparation of GQD, MA, and MNA. Ge-Gen-Qin-Lian- Tang decoction (GQD) was prepared by soaking Radix Pue- rariae Lobatae 72 g, Radix Scutellariae 27 g, Rhizoma Cop- tidis 27 g, Radix Glycyrrhizae 18 g, and ginger 4.5 g in 1.2 L deionized water for 30 min at 25∘C and then boiled for 40 min, cooled to room temperature, and filtered through two layers of cotton gauze. The filtrate was named GQD. After being centrifuged at 400 ×g for 5 min, the sediment of GQD was collected and resuspended with cell culture medium (the same volume as GQD) and named “microaggregates (MA),” while the supernatant was collected and named “400 g super- natant.” Part of the supernatant was centrifuged at 15000 ×g for 15 min. The sediment was collected and resuspended to obtain the sample named “micro-/nanoaggregates (MNA).” In our previous studies, GQD was found to exert antihy- perglycemic effects on streptozocin- (STZ-) induced diabetic rats, while the micro-/nanoaggregates (sediments produced after high speed centrifugation) showed stronger activities than the supernatant on lowering blood glucose levels, ele- vating fasting blood insulin and insulin secretion index and increasing SOD activity of liver and pancreas [23]. These data implied that the micro-/nanoaggregates of TCM decoctions may account for their therapeutic effects, being at least as effective as the soluble components. 1. Introduction Besides the assumption that multiple components may be able to regulate several molecular pathways simultaneously, the rich content of colloidal particles and aggregates formed by various components in a decoction has been demonstrated in TCM (Ma-Xing-Shi-Gan-Tang). In one such instance, ephedrine and pseudoephedrine were mostly found to be bound to colloidal nanoparticles which changed their bioac- tivities [10] and this effect may provide the supramolecular structures for these synergistic actions. Besides the assumption that multiple components may be able to regulate several molecular pathways simultaneously, the rich content of colloidal particles and aggregates formed by various components in a decoction has been demonstrated in TCM (Ma-Xing-Shi-Gan-Tang). In one such instance, ephedrine and pseudoephedrine were mostly found to be bound to colloidal nanoparticles which changed their bioac- tivities [10] and this effect may provide the supramolecular structures for these synergistic actions. Herbal decoctions from traditional Chinese medicine (TCM) have always been an option for treating oxidative stress- related chronic diseases [1–3], including diabetes. Many active components have been identified from various medic- inal plants, for example, flavonoids [4], polyphenols [5], and organic acids and alkaloids, polypeptides, and polysaccha- rides [1, 6]. These components may work alone as a single chemical compound or, in many instances, may deliver much more potent therapeutic effects in synergy with other com- ponents from the same plant or in a combination of different herbs, as demonstrated in many herbal TCM studies [7–9]. As a classic herbal TCM dating back to the East Han Dynasty, Ge-Gen-Qin-Lian-Tang decoction (GQD) is pre- pared from a formula composed of Radix Puerariae Lobatae, Radix Scutellariae, Rhizoma Coptidis (Chinese goldthread), 2 BioMed Research International BioMed Research International Cell culture medium (RPMI-1640, DMEM), HBSS buffer, penicillin-streptomycin solution, and NEAA were from HyClone (Xiamen, China); Fetal Bovine Serum (FBS) was from Biological Industries (BIOIND); HEHPE, 0.25% Trypsin-EDTA, L-glutathione (reduced), and MTT were from AMRESCO Co. Ltd. (USA); streptozocin (STZ), sodium bicarbonate, and sodium pyruvate were from Sigma (USA). Baicalin was purchased from the Fujian Institute for Food and Drug Quality Control. Malondialdehyde (MDA), super- oxide dismutase (SOD), and bicinchoninic acid (BCA) kits were from Nanjing Jiancheng Biotechnology Institute; ELISA kits for insulin measurements were purchased from Yanyu Biotech (Shanghai) Co. Ltd. Radix Glycyrrhizae (licorice, honey-processed), and Zingiber officinale Roscoe (ginger) and has been used for the clinical treatments of type 2 diabetes and intestinal inflammation [11, 12]. 1. Introduction The rich content of insol- uble small molecules and self-assembled colloidal particles in these decoctions may account for the formation of micro-/ nanometer scale aggregates by secondary aggregation. The properties and functions of these aggregates warrant careful and systematic study. 2.3. Determination of Particle Size. The hydrodynamic diam- eter of particles was determined by Dynamic Light Scattering (DLS) analysis on a Zetasizer Nano device (Malvern Instru- ments, Worcestershire, UK). 2.4. Cell Culture and MTT Assay. Human colonic epithelial cells, Caco-2 (8 × 104 cells/mL, 200 𝜇L/well), and rat pancre- atic 𝛽-cell line, INS-1 (3 × 105 cells/mL, 200 𝜇L/well), were cultured at 37∘C under 5% CO2 and 95% relative humidity for 24 h and used to evaluate the influence of GQD and aggregates on cellular viability and proliferation using the MTT assay. Samples were adjusted to the universal serial concentrations in terms of dry weight of herbs (0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, and 125 mg/mL), added to the cells in 96-well plates (200 𝜇L/well), and cultured for 12 h with 5 replicates for each concentration. The test was repeated 3 times. The cell survival rate was calculated with the following equation (mean ± SD, 𝑛= 5): To understand the cellular functions and antihyper- glycemic mechanisms of aggregates in GQD, their particu- late characteristics and effects on cell proliferation, insulin secretion, and redox status of pancreatic 𝛽-cells were assessed together with their impact on absorption of baicalin across Caco-2 colonic epithelial cell monolayers in vitro. survival rate% = 𝐴570 nm sample 𝐴570 nm control × 100%. (1) survival rate% = 𝐴570 nm sample 𝐴570 nm control × 100%. (1) (1) 2. Materials and Methods 2.5. Permeability Tests on Caco-2 Cell Monolayers. Baicalin concentrations were determined by an RPLC method as pre- viously reported [24]. An RPLC column, Daisogel-C18 (5 𝜇m, 4.6 × 250 mm), was used with methanol-water-phosphoric acid (47 : 53 : 0.2) as eluting buffer, flow rate of 1.0 mL/min, monitored at 280 nm; column temperature was set to 40∘C. 2.1. Materials. The herbs used in this study, for example, Radix Puerariae Lobatae, Radix Scutellariae, Rhizoma Cop- tidis (Chinese goldthread), Radix Glycyrrhizae (licorice, honey-processed), and sun-dried roots of Zingiber officinale Roscoe (ginger) were purchased from Beijing Yanjing Chi- nese Medicinal Herbs Co. Ltd. and authenticated by Professor Chengzi Yang from Fujian University of Traditional Chinese Medicine. Permeability of GQD and its fractions was determined following a previously described protocol [25]. Briefly, the BioMed Research International 3 Table 1: Average diameter of aggregates in centrifuged sediments of GQD. Sample 𝑍-Average d (nm) Derived count rate (kcps) Microaggregates (MA) 2775 ± 712 331 ± 17 Micro-/nanoaggregates (MNA) 531 ± 23 241 ± 2 𝑛= 3; 𝑍-Average d (nm) is the average diameter of particles. Derived count rate (kcps) partially indicates the particle concentration. MA: the resuspended sediment of GQD after low speed centrifugation (400 ×g); MNA: the resuspended sediment of GQD obtained by high speed centrifugation (15,000 ×g). Derived count rate: the intensity of light scattered by particles, presented as “thousand counts per second (kcps).” Table 1: Average diameter of aggregates in centrifuged sediments of GQD. at 4∘C and 200 ×g for 10 min and then stored at −20∘C. The remaining KRBB was removed from cells before 16.7 mM glucose (dissolved in KRBB) was gently added and incubated for 1 h. The supernatant collection was then repeated as above. The cells were digested with pancreatin until 500 𝜇L of culture medium was added to stop the digestion. The cell suspension was moved to 1.5 mL tubes and centrifuged (4∘C, 200 ×g, 10 min) to collect cells for MDA, SOD, insulin (INS), and protein (bicinchoninic acid) assays. cells were seeded onto Transwell plates and allowed to form a confluent monolayer over 20 days prior to the experiment. On day 21, the test samples (0.4 mL), namely, GQD, MA, and MNA, were added to the apical side of the membrane and 0.6 mL HBSS buffer was added to the basolateral side. 2. Materials and Methods The transport of baicalin across the monolayer was monitored over a 3-hour time period at 37∘C under 5% CO2. Samples (100 𝜇L) were collected from the BL side at 30, 60, 90, 120, and 180 min. HBSS buffer (100 𝜇L) was added to the BL side each time the sample was collected. MA and MNA were dispersed evenly in cell culture medium by vortexing. 2.8. Statistical Analysis. The raw data were processed with EXCEL (Microsoft, Inc.) and SigmaPlot (Systat Software, Inc.), and significance levels were determined by a one-way ANOVA and indicated as 𝑃< 0.05 or 𝑃< 0.01. The permeability coefficient (𝑃app) and absorption rate (𝐴%) were calculated from the following equations (𝑛= 3): 𝑃app = (𝑑𝑄/𝑑𝑡) 𝐴× 𝐶0 𝐴% = 100 × 𝑄 (𝐶0 × 𝑉), (2) 3. Results and Discussion (2) 3.1. Particle Size Distribution of GQD Aggregates. GQD was separated into three fractions by centrifugation. As shown in Figure 1, the aggregates in resuspended sediment produced after low speed centrifugation (400 ×g) had an average diameter of 2∼3 𝜇m. The remaining particles in the super- natant were further separated with high speed centrifugation (15,000 ×g) and resuspended to obtain a colloidal suspension with particles having an average diameter around 530 nm (Table 1) and a major size distribution from 300 nm to 1000 nm. This centrifugation primarily separated the aggre- gates according to their average size and relative density, although some small colloidal particles may still remain in the supernatant of the 15,000 ×g centrifugation. where the unit of 𝑃app is cm/s, 𝑑𝑄/𝑑𝑡is the rate of permeation of the drug across the cells (𝜇g/s), 𝐴is the area of cell monolayer, 𝐶0 is the donor compartment concentration at time zero (𝜇g/mL), and 𝑄is the total concentration of drug transfer across the cell monolayer (𝜇g). 𝐶0 is obtained from analysis of the dosing solution at the start of the experiment. 𝑉is the volume of donor compartment. 2.6. Effects of GQD, MA, and MNA on STZ-Induced Cellular Damage. INS-1 cells were seeded into 96-well plates, grown for 24 h to form a confluent monolayer, and washed with PBS. GQD, MA, and MNA (each 100 𝜇L) were added with 100 𝜇L STZ (IC50) and incubated for 12 h prior to MTT assay. Five duplicates were used for each sample. The test was repeated 3 times. The cell survival rate was calculated with (1) and the protection rate was calculated with the following equation (means ± SD, 𝑛= 5): 3.2. Increased Baicalin Absorption. As demonstrated by Lin et al. [28], hydrophobic phytochemicals, that is, baicalin, puerarin, and berberine hydrochloride, are dispersed with the assistance of components from the constituent herbs, result- ing in elevated solubility. In combined use with berberine, puerarin, glycyrrhizic acid, and liquiritin, the solubility and absorption of baicalin were improved [29, 30]. As a purified component, baicalin is barely soluble in aqueous solution and has a very poor absorption rate of only 1% [26, 27]. 3.2. Increased Baicalin Absorption. As demonstrated by Lin et al. [28], hydrophobic phytochemicals, that is, baicalin, puerarin, and berberine hydrochloride, are dispersed with the assistance of components from the constituent herbs, result- ing in elevated solubility. 3. Results and Discussion 𝑃app > 5×10−6 cm/s: high absorption; 𝑃ap 1 10 100 1000 10000 0 10 20 30 40 Intensity (%) Size (d (nm)) (a) 1 10 100 1000 10000 0 5 10 15 20 Intensity (%) 531.2 d (nm) Mean = 13.5% Min = 11.2% Max = 15.8% Size (d (nm)) (b) Figure 1: The particle size distribution of aggregates in GQD. (a) Particle size distribution of MA; (b) particle size distribution of MNA. Three duplicates were performed for each sample. 1 10 100 1000 10000 0 10 20 30 40 Intensity (%) Size (d (nm)) (a) (a) 1 10 100 1000 10000 0 5 10 15 20 Intensity (%) 531.2 d (nm) Mean = 13.5% Min = 11.2% Max = 15.8% Size (d (nm)) (b) Figure 2: Absorption rate (𝐴%) of baicalin in GQD aggregates on monolayers of Caco-2 cells. Baicalin concentrations in the basolateral side solutions were determined by HPLC at different time points (𝑛= 4). It is well known that the glycyrrhizic acid (a licorice- derived glycoside) is capable of forming intermolecular complexes to increase the solubility of poorly soluble drugs [31]. Our earlier work has also shown that even aqueous soluble plant-derived alkaloids (ephedrine) were mainly carried by colloidal nanoparticles self-assembled in another TCM herbal decoction and therefore exhibited different pharmacological characteristics from own monomer of Ge- Gen-Qin-Lian-Tang decoction (GQD) [10]. As demonstrated above, higher 𝑃app and absorption rates of GQD aggregates indicate that the inclusion of baicalin in the higher order structures (i.e., supermolecular complexes and aggregates) changes pharmacokinetics of Ge-Gen-Qin-Lian-Tang decoc- tion (GQD) and may be essential for its synergistic actions in the herbal decoction. Such complexes could be formed with flavonoids (such as puerarin and liquiritin), alkaloids (such as berberine), glycosides (such as baicalin and glycyrrhizic acid), polysaccharides, and glycated proteins. (b) Figure 1: The particle size distribution of aggregates in GQD. (a) Particle size distribution of MA; (b) particle size distribution of MNA. Three duplicates were performed for each sample. highest 𝑃app and absorption rate (Table 2). It is quite clear that the inclusion of baicalin in the sediments, even in terms of micrometer-scaled aggregates, strongly assisted transportation of Ge-Gen-Qin-Lian-Tang decoction (GQD) across the Caco-2 cell monolayers, significantly increasing its absorption. 3. Results and Discussion In combined use with berberine, puerarin, glycyrrhizic acid, and liquiritin, the solubility and absorption of baicalin were improved [29, 30]. As a purified component, baicalin is barely soluble in aqueous solution and has a very poor absorption rate of only 1% [26, 27]. protection rate% = (𝐴570sample −𝐴570STZ) (𝐴570Normal −𝐴570STZ) × 100%. (3) (3) In this study, the intestinal absorption of baicalin from GQD was assessed in a Caco-2 cell monolayer model for evaluating whether formation of aggregates altered the bioavailability of Ge-Gen-Qin-Lian-Tang decoction (GQD). The baicalin-containing GQD showed 5-fold higher 𝑃app than baicalin alone (Table 2). MA contained 48% of decocting baicalin, exhibiting nearly twice 𝑃app and absorption rate in comparison to GQD. Meanwhile, MNA representing approx. 46% of total baicalin in the decoction exhibited 2.7. Effects of GQD, MA, and MNA on MDA, SOD, and Insulin Secretion. INS-1 cells (5.5 × 106 cells) were seeded into 12- well plates and grown for 24 h to form a confluent monolayer. GQD, MA, and MNA (each 500 𝜇L) were added with 500 𝜇L STZ (IC50 = 46.4 mM) and incubated for 12 h. Cells were then washed with 1 mL KRBB prior to the addition of 3.3 mM glucose (dissolved in KRBB) and incubated for 1 h. The culture supernatants (500 𝜇L) were collected and centrifuged BioMed Research International 4 Table 2: The apparent permeability (𝑃app) and absorption rate of baicalin across Caco-2 cell monolayers. Sample Apical side baicalin concentration (𝜇g/mL) 𝑃app (×10−6 cm/s) Absorption rate in 3 h Absorption level Pure baicalin∗ — 0.66 ± 0.10 ∼1% Low GQD 27.6 3.40 ± 0.21 35% Medium MA 12.9 6.60 ± 0.18 67% High 400 g supernatant 14.2 6.59 ± 0.29 66% High MNA 12.8 7.30 ± 0.17 74% High ∗Data is cited from [26, 27]. 𝑛= 4. 𝑃app > 5×10−6 cm/s: high absorption; 𝑃app = 1∼5 × 10−6 cm/s: medium absorption; 𝑃app < 1×10−6 nm/s: low absorption. 1 10 100 1000 10000 0 10 20 30 40 Intensity (%) Size (d (nm)) (a) 1 10 100 1000 10000 0 5 10 15 20 Intensity (%) 531.2 d (nm) Mean = 13.5% Min = 11.2% Max = 15.8% Size (d (nm)) (b) Figure 1: The particle size distribution of aggregates in GQD. (a) Particle size distribution of MA; (b) particle size distribution of MNA. Three duplicates were performed for each sample. 3. Results and Discussion MA MNA 0 10 20 30 40 50 60 70 80 A (%) 50 100 150 200 0 t (min) Figure 2: Absorption rate (𝐴%) of baicalin in GQD aggregates on monolayers of Caco-2 cells. Baicalin concentrations in the basolateral side solutions were determined by HPLC at different time points (𝑛= 4). It is well known that the glycyrrhizic acid (a licorice- derived glycoside) is capable of forming intermolecular complexes to increase the solubility of poorly soluble drugs [31]. Our earlier work has also shown that even aqueous soluble plant-derived alkaloids (ephedrine) were mainly carried by colloidal nanoparticles self-assembled in another TCM herbal decoction and therefore exhibited different pharmacological characteristics from own monomer of Ge- Table 2: The apparent permeability (𝑃app) and absorption rate of baicalin across Caco-2 cell monolayers. ption rate of baicalin across Caco-2 cell monolayers. 𝑃app (×10−6 cm/s) Absorption rate in 3 h Absorption level 0.66 ± 0.10 ∼1% Low 3.40 ± 0.21 35% Medium 6.60 ± 0.18 67% High 6.59 ± 0.29 66% High 7.30 ± 0.17 74% High = 1∼5 × 10−6 cm/s: medium absorption; 𝑃app < 1×10−6 nm/s: low absorption. MA MNA 0 10 20 30 40 50 60 70 80 A (%) 50 100 150 200 0 t (min) Figure 2: Absorption rate (𝐴%) of baicalin in GQD aggregates on monolayers of Caco-2 cells. Baicalin concentrations in the basolateral side solutions were determined by HPLC at different time points (𝑛= 4). Table 2: The apparent permeability (𝑃app) and absorption rate of baicalin across Caco-2 cell monolayers. Table 2: The apparent permeability (𝑃app) and absorption rate of baicalin across Caco-2 cell monolayers. Sample Apical side baicalin concentration (𝜇g/mL) 𝑃app (×10−6 cm/s) Absorption rate in 3 h Absorption level Pure baicalin∗ — 0.66 ± 0.10 ∼1% Low GQD 27.6 3.40 ± 0.21 35% Medium MA 12.9 6.60 ± 0.18 67% High 400 g supernatant 14.2 6.59 ± 0.29 66% High MNA 12.8 7.30 ± 0.17 74% High ∗Data is cited from [26, 27]. 𝑛= 4. 𝑃app > 5×10−6 cm/s: high absorption; 𝑃app = 1∼5 × 10−6 cm/s: medium absorption; 𝑃app < 1×10−6 nm/s: low absorption. Pure baicalin GQD 27.6 MA 12.9 400 g supernatant 14.2 MNA 12.8 ∗Data is cited from [26, 27]. 𝑛= 4. 3. Results and Discussion Differences are significant according to a one- way ANOVA indicated with an asterisk (𝑃< 0.05; 𝑛= 4) or double asterisks (𝑃< 0.01; 𝑛= 4). supernatant. Meanwhile, the larger sized aggregates generally showed significantly higher protection rates (𝑃 < 0.01) than the smaller sized aggregates. Notably, the protection rates of MA were irrelevant to dosage, while those of MNA were dose-dependent, indicating that these two groups of aggregates may work via different mechanisms to inhibit cellular oxidative damage. that most of the cytotoxic compositions of GQD are in the supernatant after high speed centrifugation, which contains the majority of aqueous solutes. At medium concentrations (7.81∼62.5 mg/mL), both MA and MNA mildly promoted cell proliferation, while the larger sized aggregates (MA) exhibited slightly higher proliferation rates, that is, 28% at 31.25 mg/mL. The proliferation promoting activities of GQD and its aggregates may be attributed to their intracellular antioxidant capacities, since the pancreatic 𝛽-cells are sensi- tive to oxidative stress.hf that most of the cytotoxic compositions of GQD are in the supernatant after high speed centrifugation, which contains the majority of aqueous solutes. At medium concentrations (7.81∼62.5 mg/mL), both MA and MNA mildly promoted cell proliferation, while the larger sized aggregates (MA) exhibited slightly higher proliferation rates, that is, 28% at 31.25 mg/mL. The proliferation promoting activities of GQD and its aggregates may be attributed to their intracellular antioxidant capacities, since the pancreatic 𝛽-cells are sensi- tive to oxidative stress.hf The significantly elevated MDA level (Table 3) and reduced SOD level (Table 4) indicated that INS-1 cells had been damaged by STZ-induced oxidation. The aggregates (both MA and MNA) significantly restored the cellular SOD activity and reduced the MDA level, whereas GQD only exhibited significant antioxidant effects at 7.81 mg/mL. This is consistent with the different performance of the aggregates and GQD on regulating cellular viability (Figure 4). By com- paring the MAD and SOD levels of GQD and its fractions, their antioxidant activities were ranked in sequence: MA > 400 g supernatant > MNA > GQD. The effective concentration of GQD and its fractions appeared to be very high (in milligrams). It is because the concentration was presented in terms of the total dry weights of herbal materials used in preparing GQD. Given that the decocting only extracts a small portion of herbal materials, the dry weight of actual GQD dispersion and its aggregate fractions would be many times lower. 3. Results and Discussion The absorption rate (𝐴%) of baicalin in GQD aggregates across the Caco-2 cells monolayer (from apical side to basolateral side) was determined at 30, 60, 90, 120, and 180 min of incubation, as shown in Figure 2. Within the first 90 min, the baicalin absorption rates of MA and MNA were the same. After incubation for a longer time (2 h and 3 h), the MNA exhibited an 8% higher absorption than the MA, indicating that smaller particles may act as the more efficient vehicle for baicalin. Meanwhile, 𝐴% of baicalin in GQD were 19% at 30 min and 35% at 3 h, which were lower than those of aggregates but higher than those of baicalin alone, implying a significantly improved absorption in the herbal suspension compared to the pure baicalin solution. 3.3. Influence of GQD and Its Aggregates on INS-1 Pancreatic 𝛽-Cell Proliferation. As shown in Figure 3, GQD suppressed the growth of INS-1 pancreatic 𝛽-cells at 31.25∼62.50 mg/mL, implying a significant cytotoxicity (𝑃< 0.01). However, at lower concentrations (15.63 mg/mL and lower), GQD showed no inhibition on the cell proliferation but rather promotion of such (max. 60% at 7.81 mg/mL). In contrast, the aggregates, both of MA and MNA, showed no cytotoxicity on INS-1 cells at concentrations as high as 125 mg/mL. This indicates 5 31.25 15.63 7.81 3.91 1.95 Concentration (mg/ml) −60 −40 −20 0 20 40 60 80 100 Protecction rate (%) GQD MA MNA ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗∗ Figure 4: Protection of GQD and its aggregates against STZ- induced oxidative suppression of the growth of INS-1 𝛽-cells. 𝑛= 5. Oxidative damage was induced with STZ at its IC50 (46.4 mM). GQD: compared with STZ controls, 31.25 mg/mL (0.01 < 𝑃< 0.05, labelled “∗”) and others (𝑃> 0.05); MA: 400 g sediment, compared with STZ controls, at all concentrations, 𝑃 < 0.01 (“∗∗”); MNA: 15000 g sediment, compared with STZ controls, at all concentrations, 𝑃< 0.01 (“∗∗”). BioMed Research International 5 125 62.5 31.25 15.63 7.81 3.91 1.95 0.98 Concentration (mg/ml) GQD MA MNA 0 20 40 60 80 100 120 140 160 180 Survival rate (%) ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗ ∗ ∗ Figure 3: Effects of GQD and its aggregates on proliferation of INS-1 pancreatic 𝛽-cells. 𝑛= 5. 3. Results and Discussion GQD (in blue): compared with normal controls, 0.98∼1.95 mg/mL (𝑃> 0.05), 15.63 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃< 0.01, “∗∗”); MA (in orange): 400 g sediment, compared with normal controls, 125.0 mg/mL and 1.95 mg/mL (𝑃> 0.05), 62.50 and 3.91 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃< 0.01, “∗∗”); MNA (in grey): 15000 g sediment, compared with normal controls, 125.0 mg/mL (𝑃< 0.01, “∗∗”), 31.25 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃> 0.05). Error bars + SEM. Differences are significant according to a one- way ANOVA indicated with an asterisk (𝑃< 0.05; 𝑛= 4) or double asterisks (𝑃< 0.01; 𝑛= 4). 31.25 15.63 7.81 3.91 1.95 Concentration (mg/ml) −60 −40 −20 0 20 40 60 80 100 Protecction rate (%) GQD MA MNA ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗∗ Figure 4: Protection of GQD and its aggregates against STZ- induced oxidative suppression of the growth of INS-1 𝛽-cells. 𝑛= 5. Oxidative damage was induced with STZ at its IC50 (46.4 mM). GQD: compared with STZ controls, 31.25 mg/mL (0.01 < 𝑃< 0.05, labelled “∗”) and others (𝑃> 0.05); MA: 400 g sediment, compared with STZ controls, at all concentrations, 𝑃 < 0.01 (“∗∗”); MNA: 15000 g sediment, compared with STZ controls, at all concentrations, 𝑃< 0.01 (“∗∗”). supernatant. Meanwhile, the larger sized aggregates generally showed significantly higher protection rates (𝑃 < 0.01) than the smaller sized aggregates. Notably, the protection BioMed Research International 5 31.25 15.63 7.81 3.91 1.95 Concentration (mg/ml) −60 −40 −20 0 20 40 60 80 100 Protecction rate (%) GQD MA MNA ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗∗ Figure 4: Protection of GQD and its aggregates against STZ- induced oxidative suppression of the growth of INS-1 𝛽-cells. 𝑛= 5. Oxidative damage was induced with STZ at its IC50 (46.4 mM). GQD: compared with STZ controls, 31.25 mg/mL (0.01 < 𝑃< 0.05, labelled “∗”) and others (𝑃> 0.05); MA: 400 g sediment, compared with STZ controls, at all concentrations, 𝑃 < 0.01 (“∗∗”); MNA: 15000 g sediment, compared with STZ controls, at all concentrations, 𝑃< 0.01 (“∗∗”). 3. Results and Discussion 125 62.5 31.25 15.63 7.81 3.91 1.95 0.98 Concentration (mg/ml) 0 20 40 60 80 100 120 140 160 180 Survival rate (%) ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗ ∗ ∗ 31.25 15.63 7.81 3.91 1.95 Concentration (mg/ml) −20 0 20 40 60 80 100 Protecction rate (%) ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ 31.25 15.63 7.81 3.91 1.95 Concentration (mg/ml) −60 −40 −20 0 20 40 60 80 100 Protecction rate (%) GQD MA MNA ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗∗ f d Figure 4: Protection of GQD and its aggregates against STZ- induced oxidative suppression of the growth of INS-1 𝛽-cells. 𝑛= 5. Oxidative damage was induced with STZ at its IC50 (46.4 mM). GQD: compared with STZ controls, 31.25 mg/mL (0.01 < 𝑃< 0.05, labelled “∗”) and others (𝑃> 0.05); MA: 400 g sediment, compared with STZ controls, at all concentrations, 𝑃 < 0.01 (“∗∗”); MNA: 15000 g sediment, compared with STZ controls, at all concentrations, 𝑃< 0.01 (“∗∗”). Figure 3: Effects of GQD and its aggregates on proliferation of INS-1 pancreatic 𝛽-cells. 𝑛= 5. GQD (in blue): compared with normal controls, 0.98∼1.95 mg/mL (𝑃> 0.05), 15.63 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃< 0.01, “∗∗”); MA (in orange): 400 g sediment, compared with normal controls, 125.0 mg/mL and 1.95 mg/mL (𝑃> 0.05), 62.50 and 3.91 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃< 0.01, “∗∗”); MNA (in grey): 15000 g sediment, compared with normal controls, 125.0 mg/mL (𝑃< 0.01, “∗∗”), 31.25 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃> 0.05). Error bars + SEM. Differences are significant according to a one- way ANOVA indicated with an asterisk (𝑃< 0.05; 𝑛= 4) or double asterisks (𝑃< 0.01; 𝑛= 4). Figure 3: Effects of GQD and its aggregates on proliferation of INS-1 pancreatic 𝛽-cells. 𝑛= 5. GQD (in blue): compared with normal controls, 0.98∼1.95 mg/mL (𝑃> 0.05), 15.63 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃< 0.01, “∗∗”); MA (in orange): 400 g sediment, compared with normal controls, 125.0 mg/mL and 1.95 mg/mL (𝑃> 0.05), 62.50 and 3.91 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃< 0.01, “∗∗”); MNA (in grey): 15000 g sediment, compared with normal controls, 125.0 mg/mL (𝑃< 0.01, “∗∗”), 31.25 mg/mL (0.01 < 𝑃< 0.05, “∗”), and others (𝑃> 0.05). Error bars + SEM. 3. Results and Discussion The supernatant is rather high in antioxidant activity but is toxic to the cells, implying that, at high GQD concen- trations, the cytotoxic components overrule the antioxidant (cytoprotective) components and therefore kill the cells. 3.4. Inhibition of STZ-Induced Cellular Oxidation. As shown in Figure 4, GQD protected INS-1 cells from STZ-induced oxidative damage by 23% at 7.81 mg/mL but showed no protection at higher or lower concentrations. In comparison, both MA and MNA significantly protected the cells at a much wider range of concentrations (1.95 to 31.25 mg/mL) and achieved much stronger protection (MA, 78% at 7.81 mg/mL). It indicates that antioxidants or components capable of elevating the cellular antioxidant capacity are embedded in the aggregates but not in the soluble fraction. Given that neither MA nor MNA exhibited cytotoxicity (Figure 3), the cytotoxic components of GQD are most likely to be in the 3.5. Restoration of Insulin Secretion. The impacts of STZ- induced oxidation and GQD samples on insulin secretion from pancreatic 𝛽-cells were evaluated at either baseline levels (3.3 mM) or stimulated levels (16.7 mM) of glucose, as shown in Figure 5. The insulin secretion index (ISI) was calculated as a ratio of glucose-stimulated insulin secretion (GSIS)/basal insulin secretion (BIS) and data are shown in Figure 6. STZ-induced oxidation reduced the expression and secre- tion of insulin, causing cells to be irresponsive to the glucose BioMed Research International 6 Table 3: GQD and components reduced STZ-induced MDA in pancreatic 𝛽-cell line, INS-1. Group MDA (nmol/mL) GQD MA 400 g supernatant MNA 7.81 mg/mL herbs 9.40 ± 0.47ab 7.13 ± 1.71ab 8.29 ± 1.07ab 8.85 ± 0.77ab 3.91 mg/mL herbs 9.81 ± 0.24ab 7.41 ± 1.55ab 8.78 ± 0.81ab 9.17 ± 0.60ab 1.95 mg/mL herbs 10.06 ± 0.12a 7.49 ± 1.51ab 9.04 ± 0.67ab 9.37 ± 0.50ab STZ control 10.25 ± 0.24a Normal 5.89 ± 0.17b 𝑛= 5; acompared with normal cells (𝑃< 0.05); bcompared with STZ controls (𝑃< 0.05). Table 3: GQD and components reduced STZ-induced MDA in pancreatic 𝛽-cell line, INS-1. Table 4: GQD and components restored SOD activity in STZ-treated pancreatic 𝛽-cell line, INS-1. 3. Results and Discussion Group SOD (U/mg⋅protein) GQD MA 400 g supernatant MNA 7.81 mg/mL herbs 83.95 ± 5.32ab 133.33 ± 8.02b 106.44 ± 4.36ab 93.77 ± 8.87ab 3.91 mg/mL herbs 66.22 ± 7.60a 127.40 ± 8.40ab 94.97 ± 5.35ab 85.63 ± 3.69ab 1.95 mg/mL herbs 62.00 ± 6.56a 125.33 ± 5.51ab 87.93 ± 6.76ab 80.60 ± 4.23ab STZ Control 60.75 ± 4.07a Normal 155.88 ± 6.80b 𝑛= 5; acompared with normal cells (𝑃< 0.05); bcompared with STZ controls (𝑃< 0.05). Table 4: GQD and components restored SOD activity in STZ-treated pancreatic 𝛽-cell line, INS-1. could be compensated by the richer contents of particles in MA indicated by their higher scattering light intensity (kcps). stimulus. The baseline insulin secretion of normal INS- 1 𝛽-cells was 67 pg/mL, which was dramatically increased threefold to the stimulated level of 214 pg/mL (ISI = 3.23). In contrast, STZ-damaged cells did not respond to such a stimulus. The presence of GQD did not improve the baseline insulin secretion of STZ-damaged cells but doubled the insulin secretion (max. 97 pg/mL, 𝑃< 0.01) at corresponding elevated glucose levels. Although the higher absorption rate and antioxidant and cellular protective activities in vitro do not necessarily mean better therapeutic effectiveness in vivo, it is reasonable to anticipate that the micro-/nanoscale aggregates may have a vital contribution to the overall antidiabetic effects of the herbal decoction (GQD), noting that the herbal components would eventually interact with the mammalian digestive tract in the form of multiple-order aggregates, such as chyle. Aggregates from GQD, namely MA and MNA, signif- icantly improved both the baseline and stimulated insulin secretions (𝑃 < 0.01, Figure 5). Notably, the larger size aggregates (MA) showed much stronger restorative power than the MNA, wherein the BIS was almost fully restored and the GSIS (max. 174 pg/mL, ISI = 2.7) was about twofold higher than that of MNA. The MNA significantly improved the GSIS (max. ISI = 1.9) in a dose-dependent manner. Its overall effects were rather like GQD, except that the effective concentration of MNA was lower than that of GQD. 4. Conclusions The antidiabetic herbal tonic, GQD, contains micro- and nanoscale aggregates which improve the bioavailability of insoluble phytochemicals, that is, baicalin, and possess little cytotoxicity on colonic epithelial cells and pancreatic 𝛽- cells (INS-1) in vitro. It also elevates cellular antioxidant enzymes and protects 𝛽-cells from STZ-induced oxidation and restores their insulin secretion capability. The centrifugal separation results in two different size distribution fractions of aggregates (centrifuge sediments), and the larger size aggregates (MA) possessed stronger protection on cellular viability and function of 𝛽-cells in vitro. These data are consistent with an earlier antihyperglycemic study of GQD aggregates on STZ-induced diabetic rats. The aggregates from the TCM decoction, for the first time, have been found to contain active components that contribute to the antidiabetic activity of the herbal tonic by exhibiting antioxidant effects on the endocrine cells and the carrying of insoluble compounds across the intestinal mucosal barrier. These data also imply that the aggregates and sediments in the herbal decoction should be handled with greater care for both TCM herbal medicine production and pharmacological studies. All the insulin secretion results were consistent with the cellular protection and antioxidant effects of GQD and its constitutive aggregates. The significant higher antioxidant activity of aggregates on cells was in good agreement with their antioxidant effects in vivo [23], wherein the aggregates elevated SOD levels in pancreas, kidney, and liver of STZ- induced diabetic rats. Despite the higher bioavailability of baicalin in MNA demonstrated earlier in this study, the aggregates in MA showed more potent protective effects against STZ-induced oxidative stress upon cells. Although it remains unclear why larger size aggregation particles exhibited stronger antioxidant activity, one can anticipate that such aggregates may have a higher content of free radical scavenging compounds, such as berberine, puerarin, liquiritin, and glycyrrhizin acid [15–19, 32]. Besides, the MA is still a high absorption drug, whose absorption rate was only 7% lower than that of MNA. Conflicts of Interest All the authors declare that there are no financial conflicts of interest. 4. Conclusions The slightly lower absorption rate 7 BioMed Research International 7.81 3.91 1.95 STZ Normal Concentration (mg/ml) 0.0 50.0 100.0 150.0 200.0 250.0 Insulin secretion (pg/ml) 3.3 mM glucose induced 16.7 mM glucose induced 0.0 50.0 100.0 150.0 200.0 250.0 Insulin secretion (pg/ml) 7.81 3.91 1.95 STZ Normal Concentration (mg/ml) ∗∗ ∗∗ ∗∗ ## ∗∗ ## ∗∗ ## ## ## ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ∗∗ ∗∗ ## 7.81 3.91 1.95 STZ Normal Concentration (mg/ml) 0.0 50.0 100.0 150.0 200.0 250.0 Insulin secretion (pg/ml) GQD MA MNA 3.3 mM glucose induced 16.7 mM glucose induced 0.0 50.0 100.0 150.0 200.0 250.0 Insulin secretion (pg/ml) 7.81 3.91 1.95 STZ Normal Concentration (mg/ml) GQD MA MNA ∗∗ ∗∗ ∗∗ ## ∗∗ ## ∗∗ ## ## ## ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ## ∗∗ ∗∗ ∗∗ ## Figure 5: GQD and its aggregates restoration of insulin secretion in STZ-damaged INS-1 cells. “∗∗”: compared with normal cells, 𝑃< 0.01, 𝑛= 3; “##”: compared with STZ controls, 𝑃< 0.01, 𝑛= 3. Figure 5: GQD and its aggregates restoration of insulin secretion in STZ-damaged INS-1 cells. “∗∗”: compared with normal cells, 𝑃< 0.01, 𝑛= 3; “##”: compared with STZ controls, 𝑃< 0.01, 𝑛= 3. 7.81 3.91 1.95 STZ Normal Concentration (mg/ml) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 GISI/BIS GQD MA MNA ∗∗ ## ## ∗∗ ## ∗∗ ∗∗ ∗∗ ∗∗ ## ∗∗ ## ## ## Figure 6: GQD and its constitutive aggregates elevation of insulin secretion index (ISI) of STZ-damaged INS-1 𝛽-cells. “∗∗”: compared with normal cells, 𝑃< 0.01, 𝑛= 3; “##”: compared with STZ controls, 𝑃< 0.01, 𝑛= 3; ISI: GSIS/BIS. 7.81 3.91 1.95 STZ Normal Concentration (mg/ml) 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 GISI/BIS ∗∗ ## ## ∗∗ ## ∗∗ ∗∗ ∗∗ ∗∗ ## ∗∗ ## ## ## more comprehensive studies on these colloidal micro-/ nanoparticles and their constituent compounds are war- ranted to fully understand their pharmacological character- istics and chemical natures, which may inspire and lead to the development of active supramolecular complexes for the treatment of oxidative diseases. 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https://openalex.org/W2151799331	https://zenodo.org/records/2455799/files/article.pdf	German	null	Neue Bücher<b>Thermodynamik und Kinetik der Körper</b>Von Prof. Dr. B. Weinstein. I. Rand. Allgemeine Thermodynamik und Kinetik und Theorie der idealen und wirklichen Gase und Dämpfe. Verlag von Friedrich Vieweg & Sohn, Braunschweig. 1901 XVIII und 484 Seiten. Geheftet 12 mk	Zeitschrift für Elektrochemie	1,901	public-domain	941	N E U E B U C H E R . wiirischen iibrig, ein Uiristaiid, der deli Verfasser zu sehr heherzigenswerten Ikuierkungeii iiher die Art der Veroffentlichung voii Reobachtungsergebuissen ver- anlasst. Die Ausfiilluiig der Mlngel iiiiserer Erfahrung, die eine durchgreifende Vergleicliung vnn Theorie uutl Erfahrung erfordern wiirde, erhofft Weiiistein niir voii der ph3,sikaliscli - technische~~ Reiclisanstalt. Dcr Inhalt des Theorie und Erfahruiig gleich sorgfaltig behaiirlelnden ersten Batides umfasst in acht Kapiteln 1V:iiriiie u i i d WUrnirerscheinutlgeii, Grundlagen der \1’aruielehre, Zustandsgleicliung der Korper, ins- besondere tler Gase und Pliissigkeiteii , Gleichuiigeli u i i d 1)arstellungen der Therniodyiiarnik , Zustands- glrichung und Kiuetik der idealen Gase, thermisches Vcrhalten der idealen Gase, Bewegung, Keibung und W5ririeleitung in idealen Gaseii, M a x w ellsche Theorie tler G ~ s e uncl schliesslich die wirklicheii Gase. Thermodynamik und Kinetik der K6rper. Von Prof. Dr. B.U7einsteiii. I. Rand. Allgeiiieine Thernio- - dynariiik u n d K i i i e t i k uud T h e o r i e d e r i d e a l e n u n d w i r k l i c h e n G a s e u n d Ddinpfe. Verlag voii Friedrich Vieweg Sz Sohn, Braunschweig. igor. XVIII uud 484 Seiten. Ckheftet 12 Mk. Der Verfasser liefert uns hier den ersteii Batid eines v o l l s t a u d i g e n Lehrbuclies tler neueren Tliernio- dynamik, das sicherlich eiiie Liicke in der einschllgigeii Litteratur auszufiiilen hernfeii ist. Die Aulage des Werkes geht aeit iiber deli Kahiiieii der vorziiglichen kleinen l’lanc kschen Therrnodyiianiik Iiiiiaus. Ohne die grossartigen Erfolge der tieiiercii Therniodyiiamik zii verkenneti, ist W e i n s t e i n der Ansicht, dass aucli die Kinetik 1x4 Hehandlung der Tlierniodynaniik iiicht enthehrt werdeu kanu, und hat clalier beide Wisseti- schaften eug durcheinander gearbeitet. Die tlieoretische Rehandlung wird iiach Illiiglichkeit durch zahlreiche Tabellen und uunierische Rechuungcii an die Erfahruiig angeschlossen , nanieutlicli gilt dies voii der Disknssioii tler Ztistandsgleicliungeii. IIier ldsst allerdings das Beobachtungsniaterial trotz seiner Fiille sehr riel zu Das niit grosster Liehe zur Sache gesc1iriel)eiie Werk wird sich iu deni nach Breite und Tiefe uiigeheuer angewachseneii Gebiete sicherlicli als ein guter Fiihrer c.rr.eiseii. E n i i l nose. E n i i l nose. 160 Amp. bei etwa 4 Volt braucht man 500 g Quecksilber pro Sekunde, fur Platinannden redu- ziert sich die Zahl auf IOO bis 150 g pro Sekunde. Diese Angabe ist nicht ganz vershnd- lich , die Erklarungsversuche des Verf. scheinen drm Ref. mindestens nicht erschdpfend zu sein, so dass wir auf eine Wiedergabe verzichten konnen. Die Installation einer Anlage wird kurz beschrieben, doch ist nicht angegeben, ob und wo eine solche Verwendung findet. H. D. durch den Elektrolyten fliessenden Quecksilber- fadens als Kathode (vergl. diese Zeitschr. 3, 309; vergl. auch Jahl-buch ftir Elektrochemie 3, 281) und stellt folgende Arbeitsbedingungen auf. Wenn ein Quecksilberstrahl, dei- z g Hg pro Sekunde bef6rdert und 6 bis 8 cin lang init den1 Elektrolyten in Beriihrung ist , zwischen Kohlenanoden (Entfernung 3 cm) elektrolysiert wird, erhalt man 0,7 Amp. bci 4 Volt (Platin- anoden I his I,Z Amp.). Fiir einen Stroni yon H. D. V E R E I N S N A C k l K I C H T E N . die Geschaftsstelle - VOII Adresseiiaiiderungeii zu bv- iiachrichtigen. Etaaige aeitere Mitteiluugeti iiher ~ ~ d ~ ~ ~ , fchlende Hefte, inshesondere voni .4uslande, silltl an die Geschaftsstelle zu richten. die Geschaftsstelle - VOII Adresseiiaiiderungeii zu bv- iiachrichtigen. Etaaige aeitere Mitteiluugeti iiher ~ ~ d ~ ~ ~ , fchlende Hefte, inshesondere voni .4uslande, silltl an die Geschaftsstelle zu richten. Deutsche Elektrochemische Gesellschaft. ~ l l ~ l ~ ~ t ~ u I I g e l l zlIr b1itglieclscliaft sitld satzllllgs- gealass an den ersten ~ ~ ~ ~ i t ~ ~ H~~~~~ professor Dr. vaii’t Hoff, C I i a r l o t t e n l , u r g , Uhlalldstr. 2, zu richtea ; die Aniiieldtingeii miissen voti eineni Mit- glied der Gesellschaft hefiirwortet sein. Au die neu eintretenden Herreri Mitglieder wird die Vereinszeitschrift erst nacli Zahlung des britglied- IJeitrages geliefert. Zahlungen wertletl ausschliesslich an deu Schatz- nieister, H e r r 11 M a r q u a r t , 13 e t t e n h a u s e n - Casse I , erbeten. 1) r. Uer Verband Lkutscher Elektrotechuiker hat unsere Gesellschaft zu seiner voni 27. bis 30. Juni in 1)resdeii stattfitidenden Jahresversaniiiiluiig freundliclist ein- geladeii. r)ie Tagesordiiung kaiiii vom Geiieralsekrctariat tles Verbandes, Berlin N. 24, erheteii werden. an die G es gftss t el (1 D tS e n El e k t r o - C. e s e 11 s cl1 a f t , ~ e i p z i g , b~ 0 z a r t s tr. 7, richten. Alle alldrrell gescll~ftlicllerl ~~itteilullgetl wolle i1 n1is c 11 e Die Verseiiilung tler Vereinszeitscliritt geschielit durch die Verlagshuclihandlunfi. unter deren Ver- antwortlichkeit, utid zwar Iiach dein Auslande uiiter Kreuzband, nach deiii Inlatide ohlie solches, wie bei Tageszeituugen. Die Nachlieferung fehlender Nutnniern ist deshalb ini Inlande uiiiglichst bald bei der Bestell- Postanstalt nacliziisuchen , cl)eiiso ist (licse - wie auch Ausgetreten. ~ l ~ ~ ~ h , per 30. 6, rgor. xr, T42, Ausgetreten. ~ l ~ ~ ~ h , per 30. 6, rgor. xr, T42, ~ l ~ ~ ~ h , per 30. 6, rgor. xr, T42, Heft 48, S. 680, Spalte 2, Zeile 12 v. u. lies H II r g e r statt Horger.
https://openalex.org/W4386817496	https://bmcpalliatcare.biomedcentral.com/counter/pdf/10.1186/s12904-023-01250-0	English	null	Dissemination and Implementation of a Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC): a study protocol for a hybrid type 1 randomized controlled trial	BMC palliative care	2,023	cc-by	11,284	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Dissemination and Implementation of a Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC): a study protocol for a hybrid type 1 randomized controlled trial Amn Siddiqi1, Olivia Monton1,2, Alison Woods1, Taleaa Masroor1, Shannon Fuller3, Jill Owczarzak3, Gayane Yenokyan4, Lisa A. Cooper3,5,6, Karen M. Freund7, Thomas J. Smith8, Jean S. Kutner9, Kathryn L. Colborn10, Robert Joyner11, Ronit Elk12 and Fabian M. Johnston13* Abstract Background There are persistent racial and ethnic health disparities in end-of-life health outcomes in the United States. African American patients are less likely than White patients to access palliative care, enroll in hospice care, have documented goals of care discussions with their healthcare providers, receive adequate symptom control, or die at home. We developed Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC) to address these disparities. DeCIDE PC is an integrated community health worker (CHW) pallia- tive care intervention that uses community health workers (CHWs) as care team members to enhance the receipt of palliative care for African Americans with advanced cancer. The overall objectives of this study are to (1) assess the effectiveness of the DeCIDE PC intervention in improving palliative care outcomes amongst African American patients with advanced solid organ malignancy and their informal caregivers, and (2) develop generalizable knowl- edge on how contextual factors influence implementation to facilitate dissemination, uptake, and sustainability of the intervention. Methods We will conduct a multicenter, randomized, assessor-blind, parallel-group, pragmatic, hybrid type 1 effec- tiveness-implementation trial at three cancer centers across the United States. The DeCIDE PC intervention will be delivered over 6 months with CHW support tailored to the individual needs of the patient and caregiver. The primary outcome will be advance care planning. The treatment effect will be modeled using logistic regression. The secondary outcomes are quality of life, quality of communication, hospice care utilization, and patient symptoms. Discussion We expect the DeCIDE PC intervention to improve integration of palliative care, reduce multilevel barriers to care, enhance clinic and patient linkage to resources, and ultimately improve palliative care outcomes for African Correspondence: Fabian M. Johnston fjohnst4@jhmi.edu Full list of author information is available at the end of the article Correspondence: Fabian M. Johnston fjohnst4@jhmi.edu Full list of author information is available at the end of the article Siddiqi et al. BMC Palliative Care (2023) 22:139 https://doi.org/10.1186/s12904-023-01250-0 Siddiqi et al. BMC Palliative Care (2023) 22:139 https://doi.org/10.1186/s12904-023-01250-0 BMC Palliative Care Open Access Background and rationale Palliative care is an interdisciplinary approach that aims to improve the quality of life (QOL) of patients with advanced stage illnesses and their families [1]. The Amer- ican Society of Clinical Oncology (ASCO) recommends early integration of palliative care for all patients with advanced cancer [2]. This approach enables early iden- tification and management of distressing physical, psy- chosocial, and spiritual issues, facilitates communication and support throughout decision-making processes, and enhances the receipt of goal-concordant care [3, 4]. p We developed an integrated community health worker (CHW) palliative care intervention, Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC). The DeCIDE PC intervention is a theory- driven, stakeholder-informed palliative care intervention, which utilizes CHWs as care team members to enhance the receipt of palliative care for African Americans with advanced cancer [20, 21]. In this role, CHWs may help patients and their families overcome barriers in the adop- tion of palliative care services by improving provision of non-physician support services, enhancing palliative care education, helping patients navigate the healthcare sys- tem, advocating for patients and their families within the healthcare setting, and empowering patients to discuss goals of care and advance care planning (ACP) with their families and care teams. A pilot study demonstrated that this intervention was acceptable, feasible, and effective at mitigating the adverse impact of SDOH and improving palliative care outcomes, such as completion of advance directives. The collective experience of our study team highlights the potential benefit of the DeCIDE PC inter- vention and supports further investigation [20, 21]. There are persistent racial and ethnic health disparities in end-of-life health outcomes in the United States. Afri- can American patients are less likely than White patients to access or receive palliative care, receive adequate pain control, or enroll in hospice services, all of which results in unnecessary suffering at the end-of-life [5–7]. Physi- cian or patient-initiated referral to palliative care is the standard of cancer care for patients with advanced cancer in the United States, but stark and worsening disparities in outcomes suggest this model may be inadequate for African Americans, especially when considering cultural influences and underlying social determinants of health (SDOH). The totality of these disparities results in Afri- can American patients failing to receive goal-concordant end-of-life care [5, 6, 8, 9]. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Siddiqi et al. BMC Palliative Care (2023) 22:139 Page 2 of 15 American patients with advanced cancer. If found to be effective, the DeCIDE PC intervention may be a transforma- tive model with the potential to guide large-scale adoption of promising strategies to improve palliative care use and decrease disparities in end-of-life care for African American patients with advanced cancer in the United States. Trial registration Registered on ClinicalTrials.gov (NCT05407844). First posted on June 7, 2022. American patients with advanced cancer. If found to be effective, the DeCIDE PC intervention may be a transforma- tive model with the potential to guide large-scale adoption of promising strategies to improve palliative care use and decrease disparities in end-of-life care for African American patients with advanced cancer in the United States. Trial registration Registered on ClinicalTrials.gov (NCT05407844). First posted on June 7, 2022. Keywords Palliative care, Cancer, Advanced cancer, Community health worker, CHW, Community, Health disparities, Racial disparities Keywords Palliative care, Cancer, Advanced cancer, Community health worker, CHW, Community, Health disparities, Racial disparities care, Cancer, Advanced cancer, Community health worker, CHW, Community, Health disparities, Background and rationale Multiple randomized controlled trials have demon- strated the benefit of diverse interventions to improve palliative care utilization; however, few have been imple- mented [10, 11]. Literature often fails to comprehensively account for multilevel barriers when evaluating existing palliative care programs. Moreover, the lack of racial and ethnic representation in existing studies fails to provide insight into the role of culture and underlying SDOH in palliative care utilization [8, 12]. Furthermore, the opin- ions and needs of stakeholders (patients, caregivers, oncologists, palliative care providers, and cancer center leaders) have not been adequately addressed and require further evaluation. Lastly, successful studies often fail to consider the role of implementation or dissemination of their findings, which perpetuates the lack of quality pal- liative and end-of-life care [13]. Introduction Community health workers (CHWs) are non-clinician public health workers who can improve care consist- ency by addressing SDOH and helping patients from underserved communities overcome barriers to health care. Numerous studies have demonstrated the abil- ity of CHWs to decrease care disparities across multiple levels, settings, and diseases [14–16]. CHWs bridge the gap between communities and the healthcare system by delivering culturally sensitive and contextually appro- priate care. This has been shown to improve healthcare engagement, self-management, self-efficacy, treatment plan adherence, and health outcomes [17–19]. 1. Assess the effectiveness of the DeCIDE PC interven- tion in improving palliative care outcomes amongst African American patients with advanced solid organ malignancy and their informal caregivers, and Objectivesh j The overall objectives of this study are to: 1. Assess the effectiveness of the DeCIDE PC interven- tion in improving palliative care outcomes amongst African American patients with advanced solid organ malignancy and their informal caregivers, and 1. Assess the effectiveness of the DeCIDE PC interven- tion in improving palliative care outcomes amongst African American patients with advanced solid organ malignancy and their informal caregivers, and Siddiqi et al. BMC Palliative Care (2023) 22:139 Page 3 of 15 2. Develop generalizable knowledge on how contextual factors influence implementation to facilitate dis- semination, implementation, and sustainability of the intervention. (Aim 2). This will be accomplished through a multi- center, randomized, assessor-blind, parallel-group, pragmatic, hybrid type 1 effectiveness-implementation trial with a 1:1 allocation ratio. Finally, we will use a mixed methods approach and employ the CFIR (Con- solidated Framework for Implementation Research) and RE-AIM (Reach, Effectiveness, Adoption, Imple- mentation, Maintenance) frameworks to evaluate implementation of the DeCIDE PC intervention (Aim 3). This will be achieved through multilevel analysis of intervention delivery and qualitative interviews. An overview of the study design is outlined in Fig. 1. This paper will focus on the protocol related to Aim 2, the randomized controlled trial. These objectives will be accomplished through three specific aims, which are to: 1. Refine the DeCIDE PC intervention to address multi- level implementation barriers,f 2. Compare the effectiveness of the DeCIDE PC inter- vention to enhanced standard of care in improving palliative care outcomes, and 3. Evaluate the implementation of the DeCIDE PC intervention. Eligibility criteria Th i i The intervention will target patients with advanced can- cer and their self-designated informal caregivers (patient- caregiver dyads). Eligibility criteria for patients: Adult (≥ 18 years old) patients who (1) self-identify as African American, (2) have advanced solid organ malignancy (AJCC stage III or IV), (3) are English speaking, (4) have intact cognition and an ability to provide informed con- sent, and (5) have not had any palliative care experience within the last year. Exclusion criteria for patients include: (1) age < 18 years old, (2) unable to read or comprehend English, (3) unable to provide informed consent, and (4) palliative care experience within the last year. Eligibility criteria for caregivers: Adult (≥ 18 years old) caregiv- ers who (1) provide informal (unpaid) care to an eligible African American cancer patient (related or unrelated), (2) are English speaking, and (3) have intact cognition and an ability to provide informed consent. Exclusion criteria for caregivers include: (1) age < 18 years old, (2) unable to read or comprehend English, and (3) unable to provide informed consent. In instances where no car- egiver is available or willing to participate, patients will remain eligible to participate in the study individually. The participant flowchart is outlined in Fig. 2. Study setting Thi t d i We hypothesize that the DeCIDE PC intervention will improve ACP (primary outcome), QOL (principal sec- ondary outcome), and other palliative care outcomes in African American patients with advanced cancer. Fur- ther, we anticipate that the findings from this project will inform wider implementation and scale-up of the DeCIDE PC intervention. This study will take place at three cancer centers across the United States: Johns Hopkins Hospital (Broadway and Bayview campuses, Baltimore, Maryland), the Uni- versity of Alabama at Birmingham Hospital (Birming- ham, Alabama), and TidalHealth Peninsula Regional (Salisbury, Maryland). These sites were strategically cho- sen as they represent different socioeconomic, cultural, and demographic features of the African American com- munity, and have established CHW programs. Methodsh This study protocol was written in accordance with the Standard Protocol Items: Recommendations for Inter- ventional Trials (SPIRIT) statement [22] (Tables 1 and 2). Study designi y We will first use a mixed methods approach to refine the DeCIDE PC intervention by addressing multi- level implementation barriers (Aim 1). This stage will involve gathering input from patients and caregivers through patient-caregiver dyad focus groups and elicit- ing input from stakeholders in oncology, palliative care, and the community through baseline key informant interviews. Additionally, we will initiate annual envi- ronmental scans and establish a Community Advisory Board (CAB) at each enrollment site. The site-specific CABs will be comprised of patients, family members, community members, and health system members. We will aim to recruit 8–10 members at each site. The CABs will meet quarterly and will be asked to review and provide feedback on recruitment and retention approaches, data collection procedures, intervention content and delivery, interpretation of results, and dis- semination of findings. Following Aim 1, we will evalu- ate the effectiveness of the DeCIDE PC intervention in improving palliative care outcomes in African Ameri- can patients with advanced cancer and their informal caregivers compared to enhanced standard of care Siddiqi et al. BMC Palliative Care (2023) 22:139 Page 4 of 15 Table 1 SPIRIT 2013 Checklist Section/Item Item no Description Item re Administrative information Title 1 Descriptive title identifying the study design, population, interventions, and, if applicable, trial acronym Yes Trial registration 2a Trial identifier and registry name. Study designi If not yet registered, name of intended registry Yes 2b All items from the World Health Organization Trial Registration Data Set Yes (Ta Protocol version 3 Date and version identifier Yes Funding 4 Sources and types of financial, material, and other support Yes Roles and responsibilities 5a Names, affiliations, and roles of protocol contributors Yes 5b Name and contact information for the trial sponsor Yes 5c Role of study sponsor and funders, if any, in study design; collection, management, analysis, and interpretation of data; writing of the report; and the decision to sub- mit the report for publication, including whether they will have ultimate authority over any of these activities Yes 5d Composition, roles, and responsibilities of the coordinating centre, steering com- mittee, endpoint adjudication committee, data management team, and other individuals or groups overseeing the trial, if applicable (see Item 21a for data Yes Introduction Background and rationale 6a Description of research question and justification for undertaking the trial, includ- ing summary of relevant studies (published and unpublished) examining benefits and harms for each intervention Yes 6b Explanation for choice of comparators Yes Objectives 7 Specific objectives or hypotheses Yes Trial design 8 Description of trial design including type of trial (eg, parallel group, crossover, fac- torial, single group), allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory) Yes Methods: Participants, interventions, and outcomes Study setting 9 Description of study settings (eg, community clinic, academic hospital) and list of countries where data will be collected. Reference to where list of study sites can be obtained Yes Eligibility criteria 10 Inclusion and exclusion criteria for participants. Study designi If applicable, eligibility criteria for study centres and individuals who will perform the interventions (eg, surgeons, psychotherapists) Yes Interventions 11a Interventions for each group with sufficient detail to allow replication, includ- ing how and when they will be administered Yes 11b Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose change in response to harms, participant request, or improving/worsening disease) Yes 11c Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence (eg, drug tablet return, laboratory tests) Yes 11d Relevant concomitant care and interventions that are permitted or prohibited during the trial Yes Outcomes 12 Primary, secondary, and other outcomes, including the specific measurement vari- able (eg, systolic blood pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation (eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen efficacy and harm outcomes is strongly recommended Yes Participant timeline 13 Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recom- mende Yes Sample size 14 Estimated number of participants needed to achieve study objectives and how it was determined, including clinical and statistical assumptions supporting any sample size calculations Yes Recruitment 15 Strategies for achieving adequate participant enrolment to reach target sample size Yes Item reported Siddiqi et al. BMC Palliative Care (2023) 22:139 Page 5 of 15 Table 1 (continued) Section/Item Item no Description Item re Methods: Assignment of interventions (for controlled trials) Allocation: Sequence generation 16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any factors for stratification. Study designi To reduce predictability of a ran- dom sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions Yes Allocation concealment mechanism 16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to con- ceal the sequence until interventions are assigned Yes Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions Yes Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts), and how Yes 17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant’s allocated intervention during the trial Yes Methods: Data collection, management, and analysis Data collection methods 18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measure- ments, training of assessors) and a description of study instruments (eg, question- naires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol Yes 18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols Yes Data management 19 Plans for data entry, coding, security, and storage, including any related processes to promote data quality (eg, double data entry; range checks for data values). Reference to where details of data management procedures can be found, if not in the protocol Yes Statistical methods 20a Statistical methods for analysing primary and secondary outcomes. Reference to where other details of the statistical analysis plan can be found, if not in the pro- tocol Yes 20b Methods for any additional analyses (eg, subgroup and adjusted analyses) Yes 20c Definition of analysis population relating to protocol non-adherence (eg, as ran- domised analysis), and any statistical methods to handle missing data (eg, multiple imputation) Yes Methods: Monitoring Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Study designi Alternatively, an explanation of why a DMC is not needed Yes 21b Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial Yes Harms 22 Plans for collecting, assessing, reporting, and managing solicited and spontane- ously reported adverse events and other unintended effects of trial interventions or trial conduct Yes Auditing 23 Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent from investigators and the sponsor Yes Ethics and dissemination Research ethics approval 24 Plans for seeking research ethics committee/institutional review board (REC/IRB) approval Yes Protocol amendments 25 Plans for communicating important protocol modifications (eg, changes to eligibil- ity criteria, outcomes, analyses) to relevant parties (eg, investigators, REC/IRBs, trial ti i t t i l i t i j l l t ) Yes Item reported Item reported Methods: Assignment of interventions (for controlled trials) Allocation: Methods: Data collection, management, and analysis Methods: Data collection, management, and analysis Siddiqi et al. BMC Palliative Care (2023) 22:139 Page 6 of 15 Table 1 (continued) Section/Item Item no Description Item reported Consent or assent 26a Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and how (see Item 32) Yes 26b Additional consent provisions for collection and use of participant data and bio- logical specimens in ancillary studies, if applicable Yes Confidentiality 27 How personal information about potential and enrolled participants will be col- lected, shared, and maintained in order to protect confidentiality before, during, and after the trial Yes Declaration of interests 28 Financial and other competing interests for principal investigators for the overall trial and each study site Yes Access to data 29 Statement of who will have access to the final trial dataset, and disclosure of con- tractual agreements that limit such access for investigators Yes Ancillary and post-trial care 30 Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from trial participation Yes Dissemination policy 31a Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals, the public, and other relevant groups (eg, via publication, reporting in results databases, or other data sharing arrangements), including any publication restrictions Yes 31b Authorship eligibility guidelines and any intended use of professional writers Yes 31c Plans, if any, for granting public access to the full protocol, participant-level dataset, and statistical code Yes Appendices Informed consent materials 32 Model consent form and other related documentation given to participants and authorised surrogates Not applicable Biological specimens 33 Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in the current trial and for future use in ancillary studies, if applicable Not applicable Informed consent medical record (EMR) at their respective sites to follow their patients’ course of care and communicate with the clinical team through patient notes and direct messaging. The CHW intervention activities will include educating patients on palliative and hospice care, facilitating pallia- tive and hospice care referrals and care team communi- cations, linking patients to community-based resources, empowering patients to discuss their goals of care with their oncologists and encouraging patients to discuss advance care planning with their caregivers and fami- lies, removing barriers to care by addressing SDOH, and providing instrumental, logistical, and emotional support [21, 23, 24]. This intervention will be refined based on the findings from Aim 1, which will include input from patients and caregivers, stakeholders, the CAB, and the baseline environmental scan. Study coordinators will approach potential participants in-person or via telephone to discuss the study goals, risks, and alternatives, and obtain oral and written informed consent. Participants will identify a caregiver and a separate informed consent will be obtained. Additional consent provisions for collection and use of participant data and biological specimens There are no additional consent provisions for this study. Intervention group – DeCIDE PC intervention Participants in the intervention group will receive the DeCIDE PC intervention in addition to standard of care. Following study enrollment, participants randomized to the DeCIDE PC intervention group will be connected with the CHW at their enrollment site. The CHWs will then meet with the patient-caregiver dyad or the patient alone to perform a baseline assessment of their SDOH and barriers to healthcare. Following the baseline assess- ment, CHWs will perform weekly check-ins and tailor ongoing support to the individual needs of the patient and caregiver. CHWs will have access to the electronic We will hire and train one CHW at each enrollment site in the first year of the study. All CHWs will undergo a three-month CHW training program, which will include a combination of synchronous (didactic and problem- based sessions, communication and motivational inter- viewing skills training), asynchronous, and experiential training components. For the purposes of program evalu- ation, all CHWs will complete pre- and post-training tests Siddiqi et al. Key Inclusion and Exclusion Criteria Intervention group – DeCIDE PC intervention BMC Palliative Care (2023) 22:139 Page 7 of 15 Page 7 of 15 Table 2 World Health Organization Trial Registration Data Set (Version 1.3.1) Data Item Information Primary Registry and Trial Identifying Number Registered on ClinicalTrials.gov, NCT05407844 Date of Registration in Primary Registry First posted on June 7, 2022 Secondary Identifying Numbers Not applicable Source(s) of Monetary or Material Support This study is funded by the National Cancer Institute (N Grant number: 1R01CA252101-01A1 Primary Sponsor The study sponsor is Johns Hopkins University Secondary Sponsor(s) Not applicable Contact for Public Queries OM (omonton1@jh.edu) TM (tmasroo1@jh.edu) Contact for Scientific Queries FJ (fjohnst4@jhmi.edu) Public Title Community Health Worker Intervention for Disparities i Scientific Title Dissemination and Implementation of a Community He tive Care (DeCIDE PC): a study protocol for a hybrid type Countries of Recruitment United States Health Condition(s) or Problem(s) Studied Disparities in the access to and utilization of palliative c with advanced solid organ malignancy Intervention(s) Intervention group: Community Health Worker Interven theory-driven, stakeholder-informed palliative care inte bers to enhance the receipt of palliative care for African Comparator group: Enhanced standard of care; standar Key Inclusion and Exclusion Criteria Inclusion criteria for patients: Adult (≥ 18 years old) pati (2) have advanced solid organ malignancy (AJCC stage cognition and an ability to provide informed consent, a ence within the last year Exclusion criteria for patients include: (1) age < 18 years (3) unable to provide informed consent, and (4) palliativ Inclusion criteria for caregivers: Adult (≥ 18 years old) ca to an eligible African American cancer patient (related o have intact cognition and an ability to provide informed Exclusion criteria for caregivers include: (1) age < 18 yea and (3) unable to provide informed consent Study Type Type of study: Interventional Study design: Multicenter, randomized, assessor-blind, p tiveness-implementation trial involving patients from th Date of First Enrollment September 2023 (anticipated) Sample Size Target sample size: 160 patient-caregiver dyads (total 32 Recruitment Status Pending: Participants are not yet being recruited or enro Primary Outcome(s) Outcome Name: Advance care planning Metric/method of measurement: Self-reported or docu discussion of care preferences between the patient and Timepoints: Baseline, 2 months, 6 months Key Secondary Outcomes Outcome Name: Quality of life Metric/method of measurement: Quality of Life measur Illness Therapy - Palliative Care Timepoints: Baseline, 2 months, 6 months Ethics Review Status: Approved Date of approval: March 31, 2022 Completion Date Not applicable Summary Results Not applicable IPD Sharing Statement Plan to share IPD: Yes Plan description: We plan to make the full protocol, deid code available from the corresponding author on reaso Table 2 World Health Organization Trial Registration Data Set (Version 1.3.1) Data Item Information Primary Registry and Trial Identifying Number Registered on ClinicalTrials.gov, NCT05407844 Date of Registration in Primary Registry First posted on June 7, 2022 Secondary Identifying Numbers Not applicable Source(s) of Monetary or Material Support This study is funded by the National Cancer Institute (NCI) Grant number: 1R01CA252101-01A1 Primary Sponsor The study sponsor is Johns Hopkins University Secondary Sponsor(s) Not applicable Contact for Public Queries OM (omonton1@jh.edu) TM (tmasroo1@jh.edu) Contact for Scientific Queries FJ (fjohnst4@jhmi.edu) Public Title Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC) Scientific Title Dissemination and Implementation of a Community Health Worker Intervention for Disparities in Pallia- tive Care (DeCIDE PC): a study protocol for a hybrid type 1 randomized controlled trial Countries of Recruitment United States Health Condition(s) or Problem(s) Studied Disparities in the access to and utilization of palliative care among African American patients with advanced solid organ malignancy Intervention(s) Intervention group: Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC); theory-driven, stakeholder-informed palliative care intervention, which utilizes CHWs as care team mem- bers to enhance the receipt of palliative care for African Americans with advanced cancer Comparator group: Enhanced standard of care; standard of care and a palliative care brochure Key Inclusion and Exclusion Criteria Inclusion criteria for patients: Adult (≥ 18 years old) patients who (1) self-identify as African American, (2) have advanced solid organ malignancy (AJCC stage III or IV), (3) are English speaking, (4) have intact cognition and an ability to provide informed consent, and (5) have not had any palliative care experi- ence within the last year Exclusion criteria for patients include: (1) age < 18 years old, (2) unable to read or comprehend English, (3) unable to provide informed consent, and (4) palliative care experience within the last year Inclusion criteria for caregivers: Adult (≥ 18 years old) caregivers who (1) provide informal (unpaid) care to an eligible African American cancer patient (related or unrelated), (2) are English speaking, and (3) have intact cognition and an ability to provide informed consent Exclusion criteria for caregivers include: (1) age < 18 years old, (2) unable to read or comprehend English, and (3) unable to provide informed consent Study Type Type of study: Interventional Study design: Multicenter, randomized, assessor-blind, parallel-groups, pragmatic, hybrid type 1 effec- tiveness-implementation trial involving patients from three oncology practices in the United States Date of First Enrollment September 2023 (anticipated) Sample Size Target sample size: 160 patient-caregiver dyads (total 320 participants) Recruitment Status Pending: Participants are not yet being recruited or enrolled at any site Primary Outcome(s) Outcome Name: Advance care planning Metric/method of measurement: Self-reported or documented Advance Directive or a documented discussion of care preferences between the patient and caregiver or healthcare team Timepoints: Baseline, 2 months, 6 months Key Secondary Outcomes Outcome Name: Quality of life Metric/method of measurement: Quality of Life measured by the Functional Assessment of Chronic Illness Therapy - Palliative Care Timepoints: Baseline, 2 months, 6 months Ethics Review Status: Approved Date of approval: March 31, 2022 Completion Date Not applicable Summary Results Not applicable IPD Sharing Statement Plan to share IPD: Yes Plan description: We plan to make the full protocol, deidentified participant-level data, and the statistical code available from the corresponding author on reasonable request Table 2 World Health Organization Trial Registration Data Set (Version 1.3.1) Registration Data Set (Version 1.3.1) Information Registered on ClinicalTrials.gov, NCT05407844 First posted on June 7, 2022 Not applicable This study is funded by the National Cancer Institute (NCI) Grant number: 1R01CA252101-01A1 The study sponsor is Johns Hopkins University Not applicable OM (omonton1@jh.edu) TM (tmasroo1@jh.edu) FJ (fjohnst4@jhmi.edu) Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC) Dissemination and Implementation of a Community Health Worker Intervention for Disparities in Pallia- tive Care (DeCIDE PC): a study protocol for a hybrid type 1 randomized controlled trial United States Primary Registry and Trial Identifying Number Date of Registration in Primary Registry Secondary Identifying Numbers Source(s) of Monetary or Material Support Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC) Dissemination and Implementation of a Community Health Worker Intervention for Disparities in Pallia- tive Care (DeCIDE PC): a study protocol for a hybrid type 1 randomized controlled trial Disparities in the access to and utilization of palliative care among African American patients with advanced solid organ malignancy Intervention group: Community Health Worker Intervention for Disparities in Palliative Care (DeCIDE PC); theory-driven, stakeholder-informed palliative care intervention, which utilizes CHWs as care team mem- bers to enhance the receipt of palliative care for African Americans with advanced cancer Comparator group: Enhanced standard of care; standard of care and a palliative care brochure Inclusion criteria for patients: Adult (≥ 18 years old) patients who (1) self-identify as African American, (2) have advanced solid organ malignancy (AJCC stage III or IV), (3) are English speaking, (4) have intact cognition and an ability to provide informed consent, and (5) have not had any palliative care experi- ence within the last year Exclusion criteria for patients include: (1) age < 18 years old, (2) unable to read or comprehend English, (3) unable to provide informed consent, and (4) palliative care experience within the last year Inclusion criteria for caregivers: Adult (≥ 18 years old) caregivers who (1) provide informal (unpaid) care to an eligible African American cancer patient (related or unrelated), (2) are English speaking, and (3) have intact cognition and an ability to provide informed consent g y Exclusion criteria for caregivers include: (1) age < 18 years old, (2) unable to read or comprehend English, and (3) unable to provide informed consent Study design: Multicenter, randomized, assessor-blind, parallel-groups, pragmatic, hybrid type 1 effec- tiveness-implementation trial involving patients from three oncology practices in the United States Target sample size: 160 patient-caregiver dyads (total 320 participants) Target sample size: 160 patient-caregiver dyads (total 320 participants) Pending: Participants are not yet being recruited or enrolled at any site Pending: Participants are not yet being recruited or enrolled at any site Outcome Name: Advance care planning g Metric/method of measurement: Self-reported or documented Advance Directive or a documented discussion of care preferences between the patient and caregiver or healthcare team Timepoints Baseline 2 months 6 months Outcome Name: Quality of life Metric/method of measurement: Quality of Life measured by the Functional Assessment of Chronic Illness Therapy - Palliative Care Outcome Name: Quality of life Metric/method of measurement: Quality of Life measured by the Functional Assessment of Chronic Illness Therapy - Palliative Care Plan to share IPD: Yes Plan description: We plan to make the full protocol, deidentified participant-level data, and the statistical code available from the corresponding author on reasonable request Siddiqi et al. Intervention group – DeCIDE PC intervention BMC Palliative Care (2023) 22:139 Page 8 of 15 Fig 1 Study design Fig. 2 Trial flow diagram Fig. 2 Trial flow diagram Page 9 of 15 Siddiqi et al. BMC Palliative Care (2023) 22:139 Siddiqi et al. BMC Palliative Care (2023) 22:139 Siddiqi et al. BMC Palliative Care (2023) 22:139 to assess knowledge gain, perceived competence, and sat- isfaction with the training. CHWs will also undergo lon- gitudinal training throughout the study period. We will also onboard a Palliative Care Liaison at each enrollment site, who will be a member of the palliative care team and will serve as a resource and source of support for the CHW throughout the study. Finally, CHWs will have access to up-to-date palliative care content from national organizations, such as the Center to Advance Palliative Care (CAPC). Strategies to improve adherence to interventions Strategies to improve adherence to interventions To improve adherence to the intervention and survey completion, participants in each dyad will receive mon- etary compensation ($50 per participant, $100 per dyad) upon completion of the 6-month survey. If the patient passes away prior to administration of the 6-month sur- vey, the patient’s caregiver or family will receive the mon- etary compensation. Provisions for post‑trial care We do not anticipate that participants will suffer harm from trial participation, and therefore do not anticipate the need for additional compensation or ancillary care. Patients will continue to receive oncological standard of care following study completion. Comparator group – enhanced standard of care Participants in the comparison group will receive stand- ard of care in accordance with national and local guide- lines for management of their disease. Due to the many known benefits of palliative care for patients with advanced cancer, a brochure on palliative care and its ser- vice offerings will also be provided to patients within the enhanced standard of care pathway at the time of enroll- ment. Additionally, patients may be referred to specialty palliative care at any time throughout the study period at their oncologist’s discretion. Relevant concomitant care permitted or prohibited during the trial Patients will be permitted to receive oncologic care through- out the trial. Oncologists may refer patients in the enhanced standard of care group to speciality palliative care, however, to prevent crossover between groups throughout the trial, cannot refer patients to CHW support. Participants will be prohibited from participating in concomitant clinical trials in palliative care. Outcomesh BMC Palliative Care (2023) 22:139 Page 10 of 15 Table 4 Timetable for data collection Table 4 Timetable for data collection a ACP Advance care planning b FACIT-Pal Functional Assessment of Chronic Illness Therapy - Palliative Care [27] c QOC Quality of Communication Questionnaire [29] d ESAS Edmonton Symptom Assessment Scale [30] e CES-D Center for Epidemiologic Studies Depression Scale [31] f PMH/PSQ-MD Princess Margaret Hospital Satisfaction with Doctor Questionnaire [32, 33] g FAMCARE Family Satisfaction with Advanced Cancer Care [34] h PRAPARE Protocol for Responding to & Assessing Patients’ Assets, Risks & Experiences [35] STUDY PERIOD Enrollment Allocation Baseline (t0) 2 months (t1) 6 months (t2) Eligibility screen X Informed consent X Allocation X Demographic information X Medical comorbidities X Performance status X Disease characteristics X ACPa X X X FACIT-Palb X X X QOCc X X X Hospice care utilization X X X ESASd X X X CES-De X X X PMH/PSQ-MDf X X X FAMCAREg X X X Palliative and hospice care utilization X X X Resource utilization X X X PRAPAREh X X X a ACP Advance care planning b FACIT-Pal Functional Assessment of Chronic Illness Therapy - Palliative Care [27] c QOC Quality of Communication Questionnaire [29] d ESAS Edmonton Symptom Assessment Scale [30] e CES-D Center for Epidemiologic Studies Depression Scale [31] f PMH/PSQ-MD Princess Margaret Hospital Satisfaction with Doctor Questionnaire [32, 33] g FAMCARE Family Satisfaction with Advanced Cancer Care [34] h PRAPARE Protocol for Responding to & Assessing Patients’ Assets, Risks & Experiences [35] Edmonton Symptom Assessment Scale (ESAS) [30] and the Center for Epidemiologic Studies Depression Scale (CES-D) [31]. Advance Directive, such as a living will (LW) or durable power of attorney (DPOA), or a documented discussion of care preferences between the patient and caregiver or the patient and the healthcare team. ACP has been widely used and validated in palliative care studies [25, 26]. Our principal secondary outcome is QOL, meas- ured by the Functional Assessment of Chronic Illness Therapy - Palliative Care (FACIT-Pal) [27]. FACIT-Pal contains five subscales, including physical, social/fam- ily, emotional, and functional wellbeing, as well as pal- liative care. Participants will be provided with a list of statements and asked to rate each statement on a scale of 0 (not at all) to 4 (very much) based on their recol- lection of the last 7 days. Participant timelineh The timetable for data collection is presented in Table 4. Research coordinators from each enrollment site will screen potential participants for eligibility, obtain informed consent, enroll patients and their caregivers, and perform random allocation. They will then conduct EMR review and administer surveys at baseline (t0), 2 months (t1), and 6 months (t2). Outcomesh The primary, secondary, and exploratory outcomes are summarized in Table 3. The primary outcome for this study is ACP, defined as a self-reported or documented We will discontinue the study intervention at the partici- pant’s request. Table 3 Overview of primary, secondary, and exploratory outcome measures Outcome measure Description Primary outcome Advance care planning Defined as a self-reported or documented Advance Directive, such as a living will (LW) or durable power of attorney (DPOA), or a documented discussion of care preferences between the patient and caregiver or healthcare team Secondary outcomes Quality of life Functional Assessment of Chronic Illness Therapy - Palliative Care (FACIT-Pal) [27] Quality of communication Quality of Communication (QOC) Questionnaire [29] Hospice care utilization Utilization of hospice care within 14 days of death (Yes/No) Patient symptoms Edmonton Symptom Assessment Scale (ESAS) [30] Center for Epidemiologic Studies Depression Scale (CES-D) [31] Exploratory outcomes Patient physician communication Princess Margaret Hospital Satisfaction with Doctor Questionnaire (PMH/PSQ-MD) [32, 33] Caregiver satisfaction Family Satisfaction with Advanced Cancer Care (FAMCARE) [34] Palliative and hospice care utilization Palliative care consulted (Yes/No), hospice care referral made (Yes/No), length of stay in hospice care (days), hospice care withdrawal (Yes/No) Resource utilization Length of stay in hospital (days), length of stay in the Intensive Care Unit (days), visits to the Emergency Depart- ment (number of visits), readmission(s) (Yes/No), timing of readmissions (days since discharge), time spent with CHW (hours), cost (dollars) Social determinants of health Protocol for Responding to & Assessing Patients’ Assets, Risks, & Experiences (PRAPARE) [35] Table 3 Overview of primary, secondary, and exploratory outcome measures Defined as a self-reported or documented Advance Directive, such as a living will (LW) or durable power of attorney (DPOA), or a documented discussion of care preferences between the patient and caregiver or healthcare team Protocol for Responding to & Assessing Patients’ Assets, Risks, & Experiences (PRAPARE) [35] Siddiqi et al. Outcomesh The subscale scores will then be added to generate a total score, with higher scores reflecting a higher QOL. FACIT-Pal has also undergone extensive validation in palliative care studies [27, 28]. Other secondary outcomes include (1) Quality of Com- munication (QOC), measured by the QOC Question- naire [29]; (2) hospice care utilization within 14 days of death; and (3) patient symptoms, measured by the Data collection Data collection will be the responsibility of the research coordinators with oversight from the site principal inves- tigators. Investigators will be responsible for ensuring the accuracy, completeness, legibility, and timeliness of the data collection. Baseline demographics, medical history, performance status, and disease characteristics will be obtained from EMR review. The remaining data will be gathered from EMR review and surveys administered at baseline (within 1 week of enrollment), 2 months, and 6 months. Research coordinators will administer all surveys by telephone and record responses in the central REDCap database, stored on password protected computers on servers within Johns Hopkins University [37]. Allocation Patient-caregiver dyads will be randomized in a 1:1 ratio into one of two groups, the DeCIDE PC intervention group or the enhanced standard of care group. To yield balanced groups, the random allocation sequence will be computer-generated with a block size of 6. Randomi- zation will be stratified by cancer stage (AJCC stage III or IV). Allocation will be entered into the Research Elec- tronic Data Capture (REDCap) database by a unblinded statistician at the central study site who is not directly involved in the study design, study conduct, or par- ticipant enrollment. Randomization will be performed immediately after enrollment by unblinded research coordinators through REDCap. Data managementh The lead principal investigator and central study team will have access to the data sets from all three enroll- ment sites. The site principal investigators and research coordinators will only have access to their own site’s data. All electronic data will be stored on password protected computers on servers within Johns Hop- kins University. All data collected using paper forms will be stored in a locked filing cabinet within an office assigned to the study team. Study data will be retained in a deidentified manner for 5 years following study completion. Sample size We calculated our sample size to ensure sufficient power to detect a clinically meaningful difference in both our primary outcome (ACP) and our principal secondary outcome (QOL) between the study arms. With an alpha of 0.05 and power of 90%, we estimate a sample size of 56 participant per study arm to detect a 30 percentage point difference in ACP at 6 months, based on effect sizes Siddiqi et al. BMC Palliative Care (2023) 22:139 Siddiqi et al. BMC Palliative Care (2023) 22:139 Page 11 of 15 not be blinded. Additionally, the research coordinators and program manager will not be blinded to treatment allocation. We do not anticipate the need for unblinding, however, any inadvertent unblinding will be reported. from previous studies and conservatively assuming that 30% of patients in the enhanced standard care arm will have ACP [26, 36]. After accounting for a 20% attrition rate over the study period, the adjusted final sample size based on our primary outcome is 70 participants per study arm (140 participants total). The sample size cal- culation based on our principal secondary outcome of QOL, measured by FACIT-Pal, is based on a recent study, which found that palliative care was associated with a standardized mean difference (SMD) in QOL of 0.46 (95% CI 0.08–0.83) [28]. With an alpha of 0.05 and power of 80%, and after accounting for a 20% attrition rate, we estimate a sample size of 80 participants per study arm (160 participants total) to detect a SMD in QOL of 0.5. To ensure adequate power for both outcomes, we will plan to enroll 160 participants. We will seek to recruit a caregiver alongside each patient as a patient-caregiver dyad, but a lack of caregiver participation will not pre- clude a patient’s enrollment. Our total potential sample size for the trial is therefore 320 participants, including 160 patients and 160 caregivers. Recruitmenth To promote participant retention and complete fol- low-up, research coordinators will make every effort to remain in contact with study participants throughout their time on the study. If a participant is not reachable within 2 weeks of the baseline, 2-month, and 6-month surveys, the research coordinators will attempt to regain contact through three telephone calls, scheduled visits for clinical care, and if necessary, a letter to the partici- pant’s last known mailing address. These attempts will be documented in the participant’s medical record and study file. If issues with retention occur, these will be brought to the CABs to aid in addressing and correcting these issues. The three enrollment sites have well established cancer centers, albeit different patient volumes. Based on the patient volumes at the three enrollment sites, we antici- pate that 40% of the patient-caregiver dyads (65 dyads) will be recruited from Johns Hopkins Hospital, 35% (55 dyads) from University of Alabama at Birmingham Hos- pital, and the remaining 25% (40 dyads) from Tidal- Health Peninsula Regional. Study recruitment will occur over 2–4 years. The CAB from each enrollment site will provide insight and advice to guide ongoing recruitment. Data safety monitoring boardh The DSMB is comprised of three independent health services researchers, a community health worker with related expertise, and an unblinded statistician who is not involved in study design or conduct. The DSMB will function independently from the research team through- out the trial to ensure that all study procedures are being performed in accordance with the study protocol. Meet- ings will be arranged via video conferencing to review protocols, procedures, and concerns related to research integrity. A charter has been established by DSMB mem- bers to guide its governance. Confidentiality The principal investigators and lead statistician will be blinded to participant randomization. Due to the nature of the intervention, trial participants and oncologists will We will assign each participant with a study ID, which will be stored securely on REDCap and will only be Page 12 of 15 Siddiqi et al. BMC Palliative Care (2023) 22:139 Page 12 of 15 accessible to the study team. All stored data will be deidentified. to be within 5%. Mixed-effects models can account for missing data under the assumption of missing at random (MAR), where it is assumed that missing scores depend on patient covariates in the model as well as the scores at previous time points. In addi- tion, depending on the proportion of losses to follow-up, we will employ a different strategy for sen- sitivity analyses. If we observe > 5% missing data at the patient-level and an assumption of MAR is plau- sible, we will perform multiple imputation to address partial data. Missing outcome data will be imputed, and the treatment effect will be estimated under dif- ferent scenarios to enable comparison of different sensitivity analyses. Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use This is not applicable to the study. Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use This is not applicable to the study. Plans to give access to the full protocol, participant‑level data and statistical codei We plan to make the full protocol, deidentified partici- pant-level data, and the statistical code available from the corresponding author on reasonable request. Oversight and monitoringh The coordinating center for this trial is Johns Hopkins University, School of Medicine, Department of Sur- gery, and Division of Surgical Oncology. The trial steer- ing committee meets weekly and is comprised of the lead principal investigator, program manager, research coordinator, and lead CHW, as well as a senior research program coordinator and the clinical research program administrator within the Department of Surgery at Johns Hopkins University. Statistical analysis plan Intention-to-treat analysis will be used with each patient to be included in the group to which they were assigned at randomization regardless of adherence to the intervention or crossover. Descriptive statistics will be performed and presented as means with standard deviations, medians with ranges, or frequencies with proportions, overall and by study arm. To estimate the treatment effect on completion of ACP, we will use a logistic regression model with study arm as the pri- mary predictor and enrollment site and cancer stage (AJCC stage III or IV) as the covariates. The exponen- tiated coefficient for the study arm will estimate the odds ratio of ACP comparing the intervention arm to the enhanced standard of care arm. Any other covari- ates that are differentially distributed by study arm will also be included in the model for adjustment. To esti- mate the treatment effect on QOL, we will use gener- alized mixed-effects linear regression modeling with a random intercept and robust variance estimate. We will use longitudinal generalized mixed-effects mod- eling methods for all other secondary outcomes. We will report the effect size, standard error, and 95% con- fidence interval for the estimate of the treatment effects at 6 months. Plans to give access to the full protocol, participant‑level data and statistical code We plan to make the full protocol, deidentified partici- pant-level data, and the statistical code available from the corresponding author on reasonable request. Interim analyses We do not anticipate significant safety issues associ- ated with the study intervention or participation in the study. However, interim reports of enrollment, outcome completion and safety data will be prepared for the Data and Safety Monitoring Board (DSMB) on a pre-deter- mined schedule. No formal interim analyses for efficacy or futility are planned unless requested by the DSMB. Additional analyses We do not have any additional analyses planned. Frequency and plans for auditing trial conduct performed through multilevel analysis of intervention delivery and qualitative interviews with key stakeholders who were involved in implementation of the interven- tion. We believe this will add insight and context to the summative findings from the effectiveness trial and will contribute to future dissemination efforts. Each enrollment site will perform internal quality control of study conduct, data collection, documentation, and completion. Quality control at the central study site will include the following procedures: (1) the research coordina- tor will review documentation of the consenting pro- cess and completion of the consent documents; (2) the program manager and lead principal investigator will assess data quality on a monthly basis by conducting a random case review of 5% of all clinical data collected that month to assess for missing or incomplete data and excessive variability; (3) the study team will moni- tor consistent delivery of the study intervention at each enrollment site throughout the trial through monthly meetings and feedback from the lead CHW; (4) the study team will review protocol deviations on an ongo- ing basis and will implement corrective actions when the quantity or nature of deviations are deemed to be at a level of concern. f We expect the DeCIDE PC intervention to improve integration of palliative care, reduce barriers to care, enhance linkage to resources, and improve palliative care outcomes for African American patients with advanced cancer. If found effective, DeCIDE PC may be a trans- formative model with the potential to guide large-scale adoption of promising strategies to improve palliative care use and will serve as a model for similar action in other academic and community oncology centers. Frequency and plans for auditing trial conduct Abbreviations QOL Quality of life ASCO American Society of Clinical Oncology SDOH Social determinants of health CHW Community health worker CHWs Community health workers DeCIDE PC Community Health Worker Intervention for Disparities in Pallia- tive Care ACP Advance care planning CAB Community Advisory Board CFIR Consolidated Framework for Implementation Research RE-AIM Reach, Effectiveness, Adoption, Implementation, Maintenance SPIRIT Standard Protocol Items: Recommendations for Interventional Trials AJCC American Joint Committee on Cancer EMR Electronic medical record CAPC Center to Advance Palliative Care LW Living will DPOA Durable power of attorney FACIT-Pal Functional Assessment of Chronic Illness Therapy - Palliative Care QOC Quality of Communication ESAS Edmonton Symptom Assessment Scale CES-D Center for Epidemiologic Studies Depression Scale SMD Standardized mean difference REDCap Research Electronic Data Capture MAR Missing at random DSMB Data and Safety Monitoring Board IRB Institutional review board Authors’ contributions CRediT authorship contribution statement: AS: Methodology, Writing- Original Draft, Project administration. OM: Methodology, Writing-Original Draft, Project administration. AW: Methodology, Writing-Review & Editing, Project administration. TM: Writing-Review & Editing, Project administration. SF: Writing-Review & Editing. JO: Conceptualization, Methodology, Writing- Review & Editing. GY: Conceptualization, Methodology, Writing-Review & Editing. LAC: Conceptualization, Methodology, Writing-Review & Editing. KMF: Conceptualization, Methodology, Writing-Review & Editing. TJS: Conceptu- alization, Methodology, Writing-Review & Editing. JSK: Conceptualization, Methodology, Writing-Review & Editing. KLC: Conceptualization, Methodology, Writing-Review & Editing. RJ: Conceptualization, Methodology, Writing-Review & Editing. RE: Conceptualization, Methodology, Writing-Review & Editing. FMJ: Conceptualization, Methodology, Writing-Original Draft, Writing-Review & Editing, Supervision, Funding acquisition. Plans for communicating important protocol amendments to relevant partiesii Any significant modifications to the study protocol that could impact study conduct, potential benefits or harms to patients and their caregivers, or participant safety will require a formal protocol amendment. Protocol amendments will be agreed upon by investigators from all enrollment sites and approved by the institutional review board (IRB) at Johns Hopkins University prior to implementation. Protocol non‑adherence and missing data This study does not involve any invasive procedures and as such, there are minimal safety concerns for study par- ticipants. Participants will be provided with the lead principal investigator’s contact information and will be advised report any adverse events or potential harms directly to the principal investigator. We expect missing values to be minimal due to the ability to retrieve the outcome data even for patients who pass away or withdraw from the study through chart review and caregiver-provided information. Missing data on the baseline covariates are expected Page 13 of 15 Page 13 of 15 Siddiqi et al. BMC Palliative Care (2023) 22:139 Dissemination planshi The study findings will be presented to the CABs at each enrollment site. We will consider hosting a symposium for all stakeholders, including patients, caregivers, clini- cians and other frontline clinical workers, health system leaders, community organizations, advocacy organiza- tions, as well as payors and policymakers. The study findings will also be presented at national and interna- tional conferences and a manuscript outlining the study findings will be submitted to a high-impact journal for publication. Author details 1 14. Ballard M, Montgomery P. Systematic review of interventions for improv- ing the performance of community health workers in low-income and middle-income countries. BMJ Open. 2017;7:e014216. 1 Department of Surgery, Johns Hopkins University School of Medicine, 600 N Wolfe Street, Baltimore, MD 21287, USA. 2 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, 615 N Wolfe Street, Baltimore, MD 21205, USA. 3 Department of Health, Behavior and Society, Johns Hopkins Bloomberg School of Public Health, 624 N Broadway, Baltimore, MD 21205, USA. 4 Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, 615 N Wolfe Street, Baltimore, MD 21205, USA. 5 Johns Hopkins University School of Nursing, 525 N Wolfe Street, Baltimore, MD 21205, USA. 6 Department of Medicine, Johns Hopkins University School of Medicine, 2024 East Monument Street, Suite 2‑515, Baltimore, MD 21287, USA. 7 Department of Medicine, Tufts University School of Medicine, 800 Washington Street, Boston, MA 02111, USA. 8 Department of Medicine, Johns Hopkins University School of Medicine, 600 N Wolfe Street, Baltimore, MD 21287, USA. 9 Department of Medicine, University of Colorado School of Medicine, 12401 E 17th Ave, Aurora, CO 80045, USA. 10 Department of Medicine, School of Medicine, University of Colorado Anschutz Medical Campus, 13001 E 17th Place, Aurora, CO 80045, USA. 11 Richard A. Hen- son Research Institute, TidalHealth Peninsula Regional, 100 East Carroll Street, Salisbury, MD 21801, USA. 12 Department of Medicine, University of Alabama at Birmingham, 933 19th Street S, Birmingham, AL 35205, USA. 13 Division of Sur- gical Oncology, Department of Surgery, Johns Hopkins University, 600 N Wolfe Street, Blalock 606, Baltimore, MD 21287, USA. 15. Gibbons MC, Tyus NC. Systematic review of U.S.-based randomized con- trolled trials using community health workers. Prog Community Health Partnersh Res Educ Action. 2007;1:371–81. 16. Cooper LA, Hill MN, Powe NR. Designing and evaluating interventions to eliminate racial and ethnic disparities in health care. J Gen Intern Med. 2002;17:477–86. 17. Raj A, Ko N, Battaglia TA, Chabner BA, Moy B. Patient navigation for underserved patients diagnosed with breast cancer. Oncologist. 2012;17:1027–31. 18. Chen LA, Santos S, Jandorf L, Christie J, Castillo A, Winkel G, et al. A Pro- gram to enhance completion of screening colonoscopy among Urban minorities. Clin Gastroenterol Hepatol. 2008;6:443–50. 19. Wells KJ, Battaglia TA, Dudley DJ, Garcia R, Greene A, Calhoun E, et al. Patient navigation: State of the art or is it science? Cancer. 2008;113:1999–2010. 20. Funding 6. Mack JW, Paulk ME, Viswanath K, Prigerson HG. Racial disparities in the outcomes of communication on medical care received near death. Arch Intern Med. 2010;170:1533–40. This study is funded by the National Cancer Institute (NCI), grant number: 1R01CA252101-01A1. 7. Loggers ET, Maciejewski PK, Paulk E, DeSanto-Madeya S, Nilsson M, Viswa- nath K, et al. Racial differences in predictors of intensive end-of-life care in patients with advanced cancer. J Clin Oncol Off J Am Soc Clin Oncol. 2009;27:5559–64. Author details 1 Sedhom R, Nudotor R, Freund KM, Smith TJ, Cooper LA, Owczarzak JT, et al. Can community health workers increase palliative care use for Afri- can American patients? A Pilot Study JCO Oncol Pract. 2021;17:e158–67. 21. Johnston FM, Neiman JH, Parmley LE, Conca-Cheng A, Freund KM, Concannon TW, et al. Stakeholder perspectives on the use of community health workers to improve palliative care use by African Americans with cancer. J Palliat Med. 2019;22:302–6. Received: 9 August 2023 Accepted: 24 August 2023 Received: 9 August 2023 Accepted: 24 August 2023 22. Chan A-W, Tetzlaff JM, Altman DG, Laupacis A, Gøtzsche PC, Krleža-Jerić K, et al. SPIRIT 2013 statement: defining standard protocol items for clinical trials. Ann Intern Med. 2013;158:200. 23. Hunt BR, Allgood KL, Kanoon JM, Benjamins MR. Keys to the successful implementation of community-based outreach and navigation: lessons from a breast health navigation program. J Cancer Educ. 2017;32:175–82. Declarations 9. Loggers ET, Maciejewski PK, Jimenez R, Nilsson M, Paulk E, Stieglitz H, et al. Predictors of intensive end-of-life and hospice care in latino and white advanced cancer patients. J Palliat Med. 2013;16:1249–54. Availability of data and materials The full protocol will be available from the corresponding author on reason- able request. This manuscript does not contain participant-level data. 8. Johnson KS. Racial and ethnic disparities in palliative care. J Palliat Med. 2013;16:1329–34. p g The authors declare no competing interests. The authors declare no competing interests. Authors’ information Not applicable. with cancer: impact of terminal illness acknowledgment, religious- ness, and treatment preferences. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26:4131–7. with cancer: impact of terminal illness acknowledgment, religious- ness, and treatment preferences. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26:4131–7. with cancer: impact of terminal illness acknowledgment, religious- ness, and treatment preferences. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26:4131–7. with cancer: impact of terminal illness acknowledgment, religious- ness, and treatment preferences. J Clin Oncol Off J Am Soc Clin Oncol. 2008;26:4131–7. Consent for publication Not applicable. g 12. Kamal AH, Bull J, Wolf SP, Portman D, Strand J, Johnson KS. Unmet needs of African Americans and whites at the time of palliative care consulta- tion. Am J Hosp Palliat Med. 2017;34:461–5. Ethics approval and consent to participate This study protocol was approved by the IRB at all three enrollment sites (Johns Hopkins Hospital, University of Alabama at Birmingham Hospital, and TidalHealth Peninsula Regional). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent will be obtained from all subjects prior to study participation. 10. Northouse LL, Katapodi MC, Song L, Zhang L, Mood DW. Interventions with Family Caregivers of Cancer Patients: Meta-Analysis of Randomized Trials. CA Cancer J Clin. 2010;:n/a-n/a. 11. O’Mara AM, St. Germain D, Ferrell B, Bornemann T. Challenges to and lessons learned from conducting palliative care research. J Pain Symptom Manage. 2009;37:387–94. Competing interests 13. Demiris G, Parker Oliver D, Capurro D, Wittenberg-Lyles E. Implementa- tion science: implications for intervention research in hospice and pallia- tive care. Gerontologist. 2014;54:163–71. 3. Higginson IJ, Evans CJ. What is the evidence that palliative care teams improve outcomes for cancer patients and their families? Cancer J Sud- bury Mass. 2010;16:423–35. Discussionh The DeCIDE PC intervention utilizes CHWs as pallia- tive care team members with the goal of enhancing pal- liative care outcomes and reducing disparities for African American patients with advanced cancer. Upon com- pletion of the clinical trial, we will evaluate the imple- mentation of the DeCIDE PC intervention to identify facilitators and barriers to implementation. This will be Page 14 of 15 Siddiqi et al. BMC Palliative Care (2023) 22:139 References 1. Lee KC, Walling AM, Senglaub SS, Kelley AS, Cooper Z. Defining seri- ous illness among adult surgical patients. J Pain Symptom Manag. 2019;58:844–850.e2. 24. Hunt BR, Allgood K, Sproles C, Whitman S. Metrics for the systematic evaluation of community-based outreach. J Cancer Educ. 2013;28:633–8. 2. Ferrell BR, Temel JS, Temin S, Alesi ER, Balboni TA, Basch EM, et al. Integra- tion of palliative care into standard oncology care: American society of clinical oncology clinical practice guideline update. J Clin Oncol. 2017;35:96–112. 25. Patel MI, Sundaram V, Desai M, Periyakoil VS, Kahn JS, Bhattacharya J, et al. Effect of a lay health worker intervention on goals-of-care documenta- tion and on health care use, costs, and satisfaction among patients with cancer: a randomized clinical trial. JAMA Oncol. 2018;4:1359–66. 3. Higginson IJ, Evans CJ. What is the evidence that palliative care teams improve outcomes for cancer patients and their families? Cancer J Sud- bury Mass. 2010;16:423–35. 26. Patel MI, Kapphahn K, Dewland M, Aguilar V, Sanchez B, Sisay E, et al. Effect of a community health worker intervention on acute care use, advance care planning, and patient-reported outcomes among adults with advanced stages of cancer: a randomized clinical trial. JAMA Oncol. 2022. https://doi.org/10.1001/jamaoncol.2022.1997. 4. Hannon B, Swami N, Rodin G, Pope A, Zimmermann C. Experiences of patients and caregivers with early palliative care: a qualitative study. Pal- liat Med. 2017;31:72–81. 4. Hannon B, Swami N, Rodin G, Pope A, Zimmermann C. Experiences of patients and caregivers with early palliative care: a qualitative study. Pal- liat Med. 2017;31:72–81. 27. Lyons KD, Bakitas M, Hegel MT, Hanscom B, Hull J, Ahles TA. Reliability and Validity of the Functional Assessment of Chronic Illness Therapy-Palliative Care (FACIT-Pal) scale. J Pain Symptom Manage. 2009;37:23–32. 5. Smith AK, McCarthy EP, Paulk E, Balboni TA, Maciejewski PK, Block SD, et al. Racial and ethnic differences in advance care planning among patients 5. Smith AK, McCarthy EP, Paulk E, Balboni TA, Maciejewski PK, Block SD, et al. Racial and ethnic differences in advance care planning among patients Page 15 of 15 Siddiqi et al. BMC Palliative Care (2023) 22:139 Siddiqi et al. BMC Palliative Care (2023) 22:139 28. Kavalieratos D, Corbelli J, Zhang D, Dionne-Odom JN, Ernecoff NC, Han- mer J, et al. Association between palliative care and patient and caregiver outcomes: a systematic review and meta-analysis. JAMA. 2016;316:2104. 29. Engelberg R, Downey L, Curtis JR. References Psychometric characteristics of a qual- ity of communication questionnaire assessing communication about end-of-life care. J Palliat Med. 2006;9:1086–98. 30. Subbiah IM, Charone MM, Roszik J, Haider A, Vidal M, Wong A, et al. Asso- ciation of edmonton symptom assessment system global distress score with overall survival in patients with advanced cancer. JAMA Netw Open. 2021;4:e2117295. 31. Hann D, Winter K, Jacobsen P. Measurement of depressive symptoms in cancer patients. J Psychosom Res. 1999;46:437–43. 32. Landen CN, Younger NO, Collins Sharp BA, Underwood PB. Cancer patients’ satisfaction with physicians: Princess Margaret hospital satisfaction with doctor questionnaire results. Am J Obstet Gynecol. 2003;188:1177–9. 33. Loblaw DA, Bezjak A, Bunston† T. Development and Testing of a Visit- Specific Patient Satisfaction Questionnaire: the Princess Margaret Hospital Satisfaction With Doctor Questionnaire. JCO. 1999;17:1931. 34. Lo C, Burman D, Hales S, Swami N, Rodin G, Zimmermann C. The FAMCARE-patient scale: measuring satisfaction with care of outpatients with advanced cancer. Eur J Cancer. 2009;45:3182–8. 35. Weir RC, Proser M, Jester M, Li V, Hood-Ronick CM, Gurewich D. Collecting social determinants of health data in the clinical setting: findings from national PRAPARE implementation. J Health Care Poor Underserved. 2020;31:1018–35. 36. Patel MI, Khateeb S, Coker T. Association of a lay health worker-led intervention on goals of care, quality of life, and clinical trial participation among low-income and minority adults with cancer. JCO Oncol Pract. 2021;17:e1753–62. 37. Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic data capture (REDCap)—a metadata-driven methodology and workflow process for providing translational research informatics sup- port. J Biomed Inform. 2009;42:377–81. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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